Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

PubMed Central

Singh, Mayank; Hunt, Clayton R.; Pandita, Raj K.; Kumar, Rakesh; Yang, Chin-Rang; Horikoshi, Nobuo; Bachoo, Robert; Serag, Sara; Story, Michael D.; Shay, Jerry W.; Powell, Simon N.; Gupta, Arun; Jeffery, Jessie; Pandita, Shruti; Chen, Benjamin P. C.; Deckbar, Dorothee; Löbrich, Markus; Yang, Qin; Khanna, Kum Kum; Worman, Howard J.

2013-01-01

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair. PMID:23319047

Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

PubMed Central

Sasaki, Kaori; Ose, Toyoyuki; Okamoto, Naoaki; Maenaka, Katsumi; Tanaka, Taku; Masai, Hisao; Saito, Mihoko; Shirai, Tsuyoshi; Kohda, Daisuke

2007-01-01

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3′ end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3′-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3′-OH is essential for the 3′-terminal recognition. In contrast, the direct interaction with 3′-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3′ end. Thus, the N-terminal domain of PriA recognizes the 3′-end base in a base-non-selective manner, in addition to the deoxyribose and 5′-side phosphodiester group, of the 3′-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA. PMID:17464287

DNA Replication Origins and Fork Progression at Mammalian Telomeres

PubMed Central

Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

2017-01-01

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest.

PubMed

Kehrli, Keffy; Phelps, Michael; Lazarchuk, Pavlo; Chen, Eleanor; Monnat, Ray; Sidorova, Julia M

2016-11-18

The WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN- or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1-3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

PubMed Central

George, Tresa; Wen, Qin; Griffiths, Richard; Ganesh, Anil; Meuth, Mark; Sanders, Cyril M.

2009-01-01

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity. PMID:19700773

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

PubMed

Toledo, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Claudia; Larsen, Dorthe Helena; Povlsen, Lou Klitgaard; Bekker-Jensen, Simon; Mailand, Niels; Bartek, Jiri; Lukas, Jiri

2013-11-21

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

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

Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

PubMed Central

Recolin, Bénédicte; Van Der Laan, Siem; Maiorano, Domenico

2012-01-01

Uncoupling between DNA polymerases and helicase activities at replication forks, induced by diverse DNA lesions or replication inhibitors, generate long stretches of primed single-stranded DNA that is implicated in activation of the S-phase checkpoint. It is currently unclear whether nucleation of the essential replication factor RPA onto this substrate stimulates the ATR-dependent checkpoint response independently of its role in DNA synthesis. Using Xenopus egg extracts to investigate the role of RPA recruitment at uncoupled forks in checkpoint activation we have surprisingly found that in conditions in which DNA synthesis occurs, RPA accumulation at forks stalled by either replication stress or UV irradiation is dispensable for Chk1 phosphorylation. In contrast, when both replication fork uncoupling and RPA hyperloading are suppressed, Chk1 phosphorylation is inhibited. Moreover, we show that extracts containing reduced levels of RPA accumulate ssDNA and induce spontaneous, caffeine-sensitive, Chk1 phosphorylation in S-phase. These results strongly suggest that disturbance of enzymatic activities of replication forks, rather than RPA hyperloading at stalled forks, is a critical determinant of ATR activation. PMID:22187152

Cancer therapy and replication stress: forks on the road to perdition.

PubMed

Kotsantis, Panagiotis; Jones, Rebecca M; Higgs, Martin R; Petermann, Eva

2015-01-01

Deregulated DNA replication occurs in cancer where it contributes to genomic instability. This process is a target of cytotoxic therapies. Chemotherapies exploit high DNA replication in cancer cells by modifying the DNA template or by inhibiting vital enzymatic activities that lead to slowing or stalling replication fork progression. Stalled replication forks can be converted into toxic DNA double-strand breaks resulting in cell death, i.e., replication stress. While likely crucial for many cancer treatments, replication stress is poorly understood due to its complexity. While we still know relatively little about the role of replication stress in cancer therapy, technical advances in recent years have shed new light on the effect that cancer therapeutics have on replication forks and the molecular mechanisms that lead from obstructed fork progression to cell death. This chapter will give an overview of our current understanding of replication stress in the context of cancer therapy. © 2015 Elsevier Inc. All rights reserved.

Molecular basis for PrimPol recruitment to replication forks by RPA.

PubMed

Guilliam, Thomas A; Brissett, Nigel C; Ehlinger, Aaron; Keen, Benjamin A; Kolesar, Peter; Taylor, Elaine M; Bailey, Laura J; Lindsay, Howard D; Chazin, Walter J; Doherty, Aidan J

2017-05-23

DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

β2-spectrin depletion impairs DNA damage repair

PubMed Central

Horikoshi, Nobuo; Pandita, Raj K.; Mujoo, Kalpana; Hambarde, Shashank; Sharma, Dharmendra; Mattoo, Abid R.; Chakraborty, Sharmistha; Charaka, Vijaya; Hunt, Clayton R.; Pandita, Tej K.

2016-01-01

β2-Spectrin (β2SP/SPTBN1, gene SPTBN1) is a key TGF-β/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-β signaling and can contribute to liver cancer development. Here we report that cells deficient in β2-Spectrin (β2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), β2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, β2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in β2SP deficient cells. We propose that β2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination. PMID:27248179

The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

PubMed

Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie

2011-06-01

Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.

PubMed

Takahashi, Daisuke; Sato, Koichi; Hirayama, Emiko; Takata, Minoru; Kurumizaka, Hitoshi

2015-09-01

Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

PubMed

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

2013-12-15

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

Systematic identification of fragile sites via genome-wide location analysis of γ-H2AX

PubMed Central

Szilard, Rachel K.; Jacques, Pierre-Étienne; Laramée, Louise; Cheng, Benjamin; Galicia, Sarah; Bataille, Alain R.; Yeung, ManTek; Mendez, Megan; Bergeron, Maxime; Robert, François; Durocher, Daniel

2011-01-01

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication fork stalling results in histone H2A phosphorylation to yield γ-H2A (yeast γ-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)- dependent manner. Here, we describe the genome-wide location analysis of γ-H2A as a strategy to identify loci prone to engage the Mec1 and Tel1 pathways. Remarkably, γ-H2A enrichment overlaps with loci prone to replication fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for γ-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of γ-H2A at these loci. Finally, our work indicates that high resolution mapping of γ-H2AX is a fruitful route to map fragile sites in eukaryotic genomes. PMID:20139982

FANC Pathway Promotes UV-Induced Stalled Replication Forks Recovery by Acting Both Upstream and Downstream Polη and Rev1

PubMed Central

Renaud, Emilie; Rosselli, Filippo

2013-01-01

To cope with ultraviolet C (UVC)-stalled replication forks and restart DNA synthesis, cells either undergo DNA translesion synthesis (TLS) by specialised DNA polymerases or tolerate the lesions using homologous recombination (HR)-based mechanisms. To gain insight into how cells manage UVC-induced stalled replication forks, we analysed the molecular crosstalk between the TLS DNA polymerases Polη and Rev1, the double-strand break repair (DSB)-associated protein MDC1 and the FANC pathway. We describe three novel functional interactions that occur in response to UVC-induced DNA lesions. First, Polη and Rev1, whose optimal expression and/or relocalisation depend on the FANC core complex, act upstream of FANCD2 and are required for the proper relocalisation of monoubiquitinylated FANCD2 (Ub-FANCD2) to subnuclear foci. Second, during S-phase, Ub-FANCD2 and MDC1 relocalise to UVC-damaged nuclear areas or foci simultaneously but independently of each other. Third, Ub-FANCD2 and MDC1 are independently required for optimal BRCA1 relocalisation. While RPA32 phosphorylation (p-RPA32) and RPA foci formation were reduced in parallel with increasing levels of H2AX phosphorylation and MDC1 foci in UVC-irradiated FANC pathway-depleted cells, MDC1 depletion was associated with increased UVC-induced Ub-FANCD2 and FANCD2 foci as well as p-RPA32 levels and p-RPA32 foci. On the basis of the previous observations, we propose that the FANC pathway participates in the rescue of UVC-stalled replication forks in association with TLS by maintaining the integrity of ssDNA regions and by preserving genome stability and preventing the formation of DSBs, the resolution of which would require the intervention of MDC1. PMID:23365640

RPA Mediates Recruitment of MRX to Forks and Double-Strand Breaks to Hold Sister Chromatids Together.

PubMed

Seeber, Andrew; Hegnauer, Anna Maria; Hustedt, Nicole; Deshpande, Ishan; Poli, Jérôme; Eglinger, Jan; Pasero, Philippe; Gut, Heinz; Shinohara, Miki; Hopfner, Karl-Peter; Shimada, Kenji; Gasser, Susan M

2016-12-01

The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks. Copyright © 2016 Elsevier Inc. All rights reserved.

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition

NASA Astrophysics Data System (ADS)

Gill, Martin R.; Harun, Siti Norain; Halder, Swagata; Boghozian, Ramon A.; Ramadan, Kristijan; Ahmad, Haslina; Vallis, Katherine A.

2016-08-01

Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

PubMed Central

Tran, Phuoc T.; Fey, Julien P.; Erdeniz, Naz; Gellon, Lionel; Boiteux, Serge; Liskay, R. Michael

2007-01-01

Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR. PMID:17602897

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition.

PubMed

Gill, Martin R; Harun, Siti Norain; Halder, Swagata; Boghozian, Ramon A; Ramadan, Kristijan; Ahmad, Haslina; Vallis, Katherine A

2016-08-25

Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)](2+) (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)](2+) before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response.

PubMed

Mori, Tetsuya; Nakamura, Tatsuro; Okazaki, Naoto; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

2012-01-01

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

R-loop-mediated genomic instability is caused by impairment of replication fork progression

PubMed Central

Gan, Wenjian; Guan, Zhishuang; Liu, Jie; Gui, Ting; Shen, Keng; Manley, James L.; Li, Xialu

2011-01-01

Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans. PMID:21979917

Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

PubMed

Hilton, Benjamin A; Liu, Ji; Cartwright, Brian M; Liu, Yiyong; Breitman, Maya; Wang, Youjie; Jones, Rowdy; Tang, Hui; Rusinol, Antonio; Musich, Phillip R; Zou, Yue

2017-09-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin). We previously reported that XPA mislocalized to the progerin-induced DNA double-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation, DNA damage responses, and early replication arrest in HGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNA at replication forks. Our results suggest that although PCNA is much more competitive than XPA in binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPA-replication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in HGPS.-Hilton, B. A., Liu, J., Cartwright, B. M., Liu, Y., Breitman, M., Wang, Y., Jones, R., Tang, H., Rusinol, A., Musich, P. R., Zou, Y. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes. © FASEB.

Human Adipose‐Derived Stem Cells Expanded Under Ambient Oxygen Concentration Accumulate Oxidative DNA Lesions and Experience Procarcinogenic DNA Replication Stress

PubMed Central

Renoud, Marie‐Laure; Hoede, Claire; Gonzalez, Ignacio; Jones, Natalie; Longy, Michel; Sensebé, Luc; Cazaux, Christophe

2016-01-01

Abstract Adipose‐derived stem cells (ADSCs) have led to growing interest in cell‐based therapy because they can be easily harvested from an abundant tissue. ADSCs must be expanded in vitro before transplantation. This essential step causes concerns about the safety of adult stem cells in terms of potential transformation. Tumorigenesis is driven in its earliest step by DNA replication stress, which is characterized by the accumulation of stalled DNA replication forks and activation of the DNA damage response. Thus, to evaluate the safety of ADSCs during ex vivo expansion, we monitored DNA replication under atmospheric (21%) or physiologic (1%) oxygen concentration. Here, by combining immunofluorescence and DNA combing, we show that ADSCs cultured under 21% oxygen accumulate endogenous oxidative DNA lesions, which interfere with DNA replication by increasing fork stalling events, thereby leading to incomplete DNA replication and fork collapse. Moreover, we found by RNA sequencing (RNA‐seq) that culture of ADSCs under atmospheric oxygen concentration leads to misexpression of cell cycle and DNA replication genes, which could contribute to DNA replication stress. Finally, analysis of acquired small nucleotide polymorphism shows that expansion of ADSCs under 21% oxygen induces a mutational bias toward deleterious transversions. Overall, our results suggest that expanding ADSCs at a low oxygen concentration could reduce the risk for DNA replication stress‐associated transformation, as occurs in neoplastic tissues. Stem Cells Translational Medicine 2017;6:68–76 PMID:28170194

MOF Suppresses Replication Stress and Contributes to Resolution of Stalled Replication Forks.

PubMed

Singh, Dharmendra Kumar; Pandita, Raj K; Singh, Mayank; Chakraborty, Sharmistha; Hambarde, Shashank; Ramnarain, Deepti; Charaka, Vijaya; Ahmed, Kazi Mokim; Hunt, Clayton R; Pandita, Tej K

2018-03-15

The human MOF (hMOF) protein belongs to the MYST family of histone acetyltransferases and plays a critical role in transcription and the DNA damage response. MOF is essential for cell proliferation; however, its role during replication and replicative stress is unknown. Here we demonstrate that cells depleted of MOF and under replicative stress induced by cisplatin, hydroxyurea, or camptothecin have reduced survival, a higher frequency of S-phase-specific chromosome damage, and increased R-loop formation. MOF depletion decreased replication fork speed and, when combined with replicative stress, also increased stalled replication forks as well as new origin firing. MOF interacted with PCNA, a key coordinator of replication and repair machinery at replication forks, and affected its ubiquitination and recruitment to the DNA damage site. Depletion of MOF, therefore, compromised the DNA damage repair response as evidenced by decreased Mre11, RPA70, Rad51, and PCNA focus formation, reduced DNA end resection, and decreased CHK1 phosphorylation in cells after exposure to hydroxyurea or cisplatin. These results support the argument that MOF plays an important role in suppressing replication stress induced by genotoxic agents at several stages during the DNA damage response. Copyright © 2018 American Society for Microbiology.

ATR suppresses endogenous DNA damage and allows completion of homologous recombination repair.

PubMed

Brown, Adam D; Sager, Brian W; Gorthi, Aparna; Tonapi, Sonal S; Brown, Eric J; Bishop, Alexander J R

2014-01-01

DNA replication fork stalling or collapse that arises from endogenous damage poses a serious threat to genome stability, but cells invoke an intricate signaling cascade referred to as the DNA damage response (DDR) to prevent such damage. The gene product ataxia telangiectasia and Rad3-related (ATR) responds primarily to replication stress by regulating cell cycle checkpoint control, yet it's role in DNA repair, particularly homologous recombination (HR), remains unclear. This is of particular interest since HR is one way in which replication restart can occur in the presence of a stalled or collapsed fork. Hypomorphic mutations in human ATR cause the rare autosomal-recessive disease Seckel syndrome, and complete loss of Atr in mice leads to embryonic lethality. We recently adapted the in vivo murine pink-eyed unstable (pun) assay for measuring HR frequency to be able to investigate the role of essential genes on HR using a conditional Cre/loxP system. Our system allows for the unique opportunity to test the effect of ATR loss on HR in somatic cells under physiological conditions. Using this system, we provide evidence that retinal pigment epithelium (RPE) cells lacking ATR have decreased density with abnormal morphology, a decreased frequency of HR and an increased level of chromosomal damage.

NEK8 Links the ATR-regulated Replication Stress Response and S-phase CDK Activity to Renal Ciliopathies

PubMed Central

Choi, Hyo Jei Claudia; Lin, Jia-Ren; Vannier, Jean-Baptiste; Slaats, Gisela G.; Kile, Andrew C.; Paulsen, Renee D.; Manning, Danielle K.; Beier, David R.; Giles, Rachel H.; Boulton, Simon J.; Cimprich, Karlene A.

2013-01-01

Summary Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) which further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders. PMID:23973373

Replication Stress: A Lifetime of Epigenetic Change

PubMed Central

Khurana, Simran; Oberdoerffer, Philipp

2015-01-01

DNA replication is essential for cell division. Challenges to the progression of DNA polymerase can result in replication stress, promoting the stalling and ultimately collapse of replication forks. The latter involves the formation of DNA double-strand breaks (DSBs) and has been linked to both genome instability and irreversible cell cycle arrest (senescence). Recent technological advances have elucidated many of the factors that contribute to the sensing and repair of stalled or broken replication forks. In addition to bona fide repair factors, these efforts highlight a range of chromatin-associated changes at and near sites of replication stress, suggesting defects in epigenome maintenance as a potential outcome of aberrant DNA replication. Here, we will summarize recent insight into replication stress-induced chromatin-reorganization and will speculate on possible adverse effects for gene expression, nuclear integrity and, ultimately, cell function. PMID:26378584

Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

PubMed

Morafraile, Esther C; Diffley, John F X; Tercero, José Antonio; Segurado, Mónica

2015-01-20

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

PubMed

Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

2017-11-03

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

PubMed Central

Melnik, Andre; Wilson-Zbinden, Caroline; Schellhaas, René; Kastner, Lisa; Piwko, Wojciech; Dees, Martina; Picotti, Paola; Maric, Marija; Labib, Karim; Luke, Brian; Peter, Matthias

2016-01-01

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks. PMID:26849847

RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

PubMed

Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

2016-12-19

ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distinct functions of human RecQ helicases during DNA replication.

PubMed

Urban, Vaclav; Dobrovolna, Jana; Janscak, Pavel

2017-06-01

DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. RecQ helicases promote recovery of replication forks being stalled due to different replication roadblocks of either exogenous or endogenous source. They prevent generation of aberrant replication fork structures and replication fork collapse, and are involved in proper checkpoint signaling. The essential role of human RecQ helicases in the genome maintenance during DNA replication is underlined by association of defects in their function with cancer predisposition. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

PubMed

Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

2013-08-15

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea.

PubMed

Shuttleworth, Gillian; Fogg, Mark J; Kurpiewski, Michael R; Jen-Jacobson, Linda; Connolly, Bernard A

2004-03-26

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.

NSC30049 inhibits Chk1 pathway in 5-FU-resistant CRC bulk and stem cell populations.

PubMed

Narayan, Satya; Jaiswal, Aruna S; Sharma, Ritika; Nawab, Akbar; Duckworth, Lizette Vila; Law, Brian K; Zajac-Kaye, Maria; George, Thomas J; Sharma, Jay; Sharma, Arun K; Hromas, Robert A

2017-08-22

The 5-fluorouracil (5-FU) treatment induces DNA damage and stalling of DNA replication forks. These stalled replication forks then collapse to form one sided double-strand breaks, leading to apoptosis. However, colorectal cancer (CRC) stem cells rapidly repair the stalled/collapsed replication forks and overcome treatment effects. Recent evidence suggests a critical role of checkpoint kinase 1 (Chk1) in preventing the replicative stress. Therefore, Chk1 kinase has been a target for developing mono or combination therapeutic agents. In the present study, we have identified a novel orphan molecule NSC30049 (NSC49L) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC heterogeneous bulk and FOLFOX-resistant cell lines in culture with minimal effect on normal colonic epithelial cells. It also inhibits the sphere forming activity of CRC stem cells, and decreases the expression levels of mRNAs of CRC stem cell marker genes. Results showed that NSC49L induces 5-FU-mediated S-phase cell cycle arrest due to increased load of DNA damage and increased γ-H2AX staining as a mechanism of cytotoxicity. The pharmacokinetic analysis showed a higher bioavailability of this compound, however, with a short plasma half-life. The drug is highly tolerated by animals with no pathological aberrations. Furthermore, NSC49L showed very potent activity in a HDTX model of CRC stem cell tumors either alone or in combination with 5-FU. Thus, NSC49L as a single agent or combined with 5-FU can be developed as a therapeutic agent by targeting the Chk1 pathway in 5-FU-resistant CRC heterogeneous bulk and CRC stem cell populations.

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

NASA Astrophysics Data System (ADS)

Min, Wookee; Bruhn, Christopher; Grigaravicius, Paulius; Zhou, Zhong-Wei; Li, Fu; Krüger, Anja; Siddeek, Bénazir; Greulich, Karl-Otto; Popp, Oliver; Meisezahl, Chris; Calkhoven, Cornelis F.; Bürkle, Alexander; Xu, Xingzhi; Wang, Zhao-Qi

2013-12-01

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

The Location of the Bacterial Origin of Replication is Critical for Initial Ciproflaxcin Antibiotic Resistance

NASA Astrophysics Data System (ADS)

Bos, Julia; Nehring, Ralph; Cruz, Diane; Austin, Doug; Rosenberg, Susan; Austin, Robert

By using E. coli cells in which the unique origin of replication has been moved to a ectopic chromosome location distant from the native one, we probe how perturbation of gene order near the origin of replication impacts genome stability and survival under genomic attack. We find that when challenged with sub-inhibitory doses of ciprofloxacin, an antibiotic that generates replication fork stalling, cells with the ectopic origin show significant fitness loss. We show that genes functionally relevant to the cipro-induced stress response are largely located near the native origin, even in distantly related species. We show that while cipro induces increased copy number of genes proximal to the origin of replication as a direct consequence of replication fork stalling, gene copy number variation was reduced near the ectopic origin. Altered gene dosage in cells with an ectopic origin resulted in impaired replication fork repair and chromosome instability. We propose that gene distribution in the origin region acts as a fundamental first line of defense when the integrity of the genome is threatened and that genes proximal to the origin of replication serve as a mechanism of genetic innovation and a driving force of genome evolution in the presence of genotoxic antibiotics. Lewis Sigler Institute for Integrative Genomics and the Physics Department at Princeton University.

Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells.

PubMed

Renaud, Emilie; Barascu, Aurelia; Rosselli, Filippo

2016-01-29

To rescue collapsed replication forks cells utilize homologous recombination (HR)-mediated mechanisms to avoid the induction of gross chromosomal abnormalities that would be generated by non-homologous end joining (NHEJ). Using DNA interstrand crosslinks as a replication barrier, we investigated how the Fanconi anemia (FA) pathway promotes HR at stalled replication forks. FA pathway inactivation results in Fanconi anemia, which is associated with a predisposition to cancer. FANCD2 monoubiquitination and assembly in subnuclear foci appear to be involved in TIP60 relocalization to the chromatin to acetylates histone H4K16 and prevents the binding of 53BP1 to its docking site, H4K20Me2. Thus, FA pathway loss-of-function results in accumulation of 53BP1, RIF1 and RAP80 at damaged chromatin, which impair DNA resection at stalled replication fork-associated DNA breaks and impede HR. Consequently, DNA repair in FA cells proceeds through the NHEJ pathway, which is likely responsible for the accumulation of chromosome abnormalities. We demonstrate that the inhibition of NHEJ or deacetylase activity rescue HR in FA cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX

PubMed Central

Clynes, David; Jelinska, Clare; Xella, Barbara; Ayyub, Helena; Scott, Caroline; Mitson, Matthew; Taylor, Stephen; Higgs, Douglas R.; Gibbons, Richard J.

2015-01-01

Fifteen per cent of cancers maintain telomere length independently of telomerase by the homologous recombination (HR)-associated alternative lengthening of telomeres (ALT) pathway. A unifying feature of these tumours are mutations in ATRX. Here we show that expression of ectopic ATRX triggers a suppression of the pathway and telomere shortening. Importantly ATRX-mediated ALT suppression is dependent on the histone chaperone DAXX. Re-expression of ATRX is associated with a reduction in replication fork stalling, a known trigger for HR and loss of MRN from telomeres. A G-quadruplex stabilizer partially reverses the effect of ATRX, inferring ATRX may normally facilitate replication through these sequences that, if they persist, promote ALT. We propose that defective telomere chromatinization through loss of ATRX promotes the persistence of aberrant DNA secondary structures, which in turn present a barrier to DNA replication, leading to replication fork stalling, collapse, HR and subsequent recombination-mediated telomere synthesis in ALT cancers. PMID:26143912

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

Investigating the Role of Helicobacter pylori PriA Protein.

PubMed

Singh, Aparna; Blaskovic, Dusan; Joo, Jungsoo; Yang, Zhen; Jackson, Sharon H; Coleman, William G; Yan, Ming

2016-08-01

In bacteria, PriA protein, a conserved DEXH-type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA binding property allows it to recognize and stabilize stalled forks and the structures derived from them. PriA plays a very critical role in replication fork stabilization and DNA repair in E. coli and N. gonorrhoeae. In our in vivo expression technology screen, priA gene was induced in vivo when Helicobacter pylori infects mouse stomach. We decided to elucidate the role of H. pylori PriA protein in survival in mouse stomach, survival in gastric epithelial cells and macrophage cells, DNA repair, acid stress, and oxidative stress. The priA null mutant strain was unable to colonize mice stomach mucosa after long-term infections. Mouse colonization was observed after 1 week of infection, but the levels were much lower than the wild-type HpSS1 strain. PriA protein was found to be important for intracellular survival of epithelial cell-/macrophage cell-ingested H. pylori. Also, a priA null mutant was more sensitive to DNA-damaging agents and was much more sensitive to acid and oxidative stress as compared to the wild-type strain. These data suggest that the PriA protein is needed for survival and persistence of H. pylori in mice stomach mucosa. © 2016 John Wiley & Sons Ltd.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

PubMed Central

García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

2016-01-01

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis

PubMed Central

Bergoglio, Valérie; Boyer, Anne-Sophie; Walsh, Erin; Naim, Valeria; Legube, Gaëlle; Lee, Marietta Y.W.T.; Rey, Laurie; Rosselli, Filippo; Cazaux, Christophe; Eckert, Kristin A.

2013-01-01

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation–induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role. We demonstrated that Pol η accumulated at CFSs upon partial replication stress and could efficiently replicate non-B DNA sequences within CFSs. Pol η deficiency led to persistence of checkpoint-blind under-replicated CFS regions in mitosis, detectable as FANCD2-associated chromosomal sites that were transmitted to daughter cells in 53BP1-shielded nuclear bodies. Expression of a catalytically inactive mutant of Pol η increased replication fork stalling and activated the replication checkpoint. These data are consistent with the requirement of Pol η–dependent DNA synthesis during S phase at replication forks stalled in CFS regions to suppress CFS instability by preventing checkpoint-blind under-replicated DNA in mitosis. PMID:23609533

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

PubMed Central

Acevedo, Julyana; Yan, Shan; Michael, W. Matthew

2016-01-01

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

PubMed

Feeney, Laura; Muñoz, Ivan M; Lachaud, Christophe; Toth, Rachel; Appleton, Paul L; Schindler, Detlev; Rouse, John

2017-06-01

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

BLM and the FANC proteins collaborate in a common pathway in response to stalled replication forks

PubMed Central

Pichierri, Pietro; Franchitto, Annapaola; Rosselli, Filippo

2004-01-01

Fanconi anaemia (FA) and Bloom syndrome (BS) are autosomal recessive diseases characterised by chromosome fragility and cancer proneness. Here, we report that BLM and the FA pathway are activated in response to both crosslinked DNA and replication fork stall. We provide evidence that BLM and FANCD2 colocalise and co-immunoprecipitate following treatment with either DNA crosslinkers or agents inducing replication arrest. We also find that the FA core complex is necessary for BLM phosphorylation and assembly in nuclear foci in response to crosslinked DNA. Moreover, we show that knock-down of the MRE11 complex, whose function is also under the control of the FA core complex, enhances cellular and chromosomal sensitivity to DNA interstrand crosslinks in BS cells. These findings suggest the existence of a functional link between BLM and the FA pathway and that BLM and the MRE11 complex are in two separated branches of a pathway resulting in S-phase checkpoint activation, chromosome integrity and cell survival in response to crosslinked DNA. PMID:15257300

hMSH5 Facilitates the Repair of Camptothecin-induced Double-strand Breaks through an Interaction with FANCJ*

PubMed Central

Xu, Yang; Wu, Xiling; Her, Chengtao

2015-01-01

Replication stress from stalled or collapsed replication forks is a major challenge to genomic integrity. The anticancer agent camptothecin (CPT) is a DNA topoisomerase I inhibitor that causes fork collapse and double-strand breaks amid DNA replication. Here we report that hMSH5 promotes cell survival in response to CPT-induced DNA damage. Cells deficient in hMSH5 show elevated CPT-induced γ-H2AX and RPA2 foci with concomitant reduction of Rad51 foci, indicative of impaired homologous recombination. In addition, CPT-treated hMSH5-deficient cells exhibit aberrant activation of Chk1 and Chk2 kinases and therefore abnormal cell cycle progression. Furthermore, the hMSH5-FANCJ chromatin recruitment underlies the effects of hMSH5 on homologous recombination and Chk1 activation. Intriguingly, FANCJ depletion desensitizes hMSH5-deficient cells to CPT-elicited cell killing. Collectively, our data point to the existence of a functional interplay between hMSH5 and FANCJ in double-strand break repair induced by replication stress. PMID:26055704

Disintegration of Nascent Replication Bubbles during Thymine Starvation Triggers RecA- and RecBCD-dependent Replication Origin Destruction*

PubMed Central

Kuong, Kawai J.; Kuzminov, Andrei

2012-01-01

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA+ cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction. PMID:22621921

The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity*

PubMed Central

Manhart, Carol M.; McHenry, Charles S.

2013-01-01

The PriA protein serves as an initiator for the restart of DNA replication on stalled replication forks and as a checkpoint protein that prevents the replicase from advancing in a strand displacement reaction on forks that do not contain a functional replicative helicase. We have developed a primosomal protein-dependent fluorescence resonance energy transfer (FRET) assay using a minimal fork substrate composed of synthetic oligonucleotides. We demonstrate that a self-loading reaction, which proceeds at high helicase concentrations, occurs by threading of a preassembled helicase over free 5′-ends, an event that can be blocked by attaching a steric block to the 5′-end or coating DNA with single-stranded DNA binding protein. The specificity of PriA for replication forks is regulated by its intrinsic ATPase. ATPase-defective PriA K230R shows a strong preference for substrates that contain no gap between the leading strand and the duplex portion of the fork, as demonstrated previously. Wild-type PriA prefers substrates with larger gaps, showing maximal activity on substrates on which PriA K230R is inactive. We demonstrate that PriA blocks replicase function on forks by blocking its binding. PMID:23264623

TopBP1-mediated DNA processing during mitosis.

PubMed

Gallina, Irene; Christiansen, Signe Korbo; Pedersen, Rune Troelsgaard; Lisby, Michael; Oestergaard, Vibe H

2016-01-01

Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

TopoIIα prevents telomere fragility and formation of ultra thin DNA bridges during mitosis through TRF1-dependent binding to telomeres.

PubMed

d'Alcontres, Martina Stagno; Palacios, Jose Alejandro; Mejias, Diego; Blasco, Maria A

2014-01-01

Telomeres are repetitive nucleoprotein structures at the ends of chromosomes. Like most genomic regions consisting of repetitive DNA, telomeres are fragile sites prone to replication fork stalling and generation of chromosomal instability. In particular, abrogation of the TRF1 telomere binding protein leads to stalled replication forks and aberrant telomere structures known as "multitelomeric signals". Here, we report that TRF1 deficiency also leads to the formation of "ultra-fine bridges" (UFB) during mitosis, and to an increased time to complete mitosis mediated by the spindle assembly checkpoint proteins (SAC). We find that topoisomerase IIα (TopoIIα), an enzyme essential for resolution of DNA replication intermediates, binds telomeres in a TRF1-mediated manner. Indeed, similar to TRF1 abrogation, TopoIIα downregulation leads to telomere fragility and UFB, suggesting that these phenotypes are due to decreased TopoIIα at telomeres. We find that SAC proteins bind telomeres in vivo, and that this is disrupted upon TRF1 deletion. These findings suggest that TRF1 links TopoIIα and SAC proteins in a pathway that ensures correct telomere replication and mitotic segregation, unveiling how TRF1 protects from telomere fragility and mitotic defects.

Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

PubMed

Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

2015-05-22

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

PubMed Central

Zucca, Elisa; Bertoletti, Federica; Wimmer, Ursula; Ferrari, Elena; Mazzini, Giuliano; Khoronenkova, Svetlana; Grosse, Nicole; van Loon, Barbara; Dianov, Grigory; Hübscher, Ulrich; Maga, Giovanni

2013-01-01

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells. PMID:23118481

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

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

Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

DOE PAGES

Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; ...

2015-09-08

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

PubMed

Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

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

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.

The expanding polymerase universe.

PubMed

Goodman, M F; Tippin, B

2000-11-01

Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

Fork stalling and template switching as a mechanism for polyalanine tract expansion affecting the DYC mutant of HOXD13, a new murine model of synpolydactyly.

PubMed

Cocquempot, Olivier; Brault, Véronique; Babinet, Charles; Herault, Yann

2009-09-01

Polyalanine expansion diseases are proposed to result from unequal crossover of sister chromatids that increases the number of repeats. In this report we suggest an alternative mechanism we put forward while we investigated a new spontaneous mutant that we named "Dyc" for "Digit in Y and Carpe" phenotype. Phenotypic analysis revealed an abnormal limb patterning similar to that of the human inherited congenital disease synpolydactyly (SPD) and to the mouse mutant model Spdh. Both human SPD and mouse Spdh mutations affect the Hoxd13 gene within a 15-residue polyalanine-encoding repeat in the first exon of the gene, leading to a dominant negative HOXD13. Genetic analysis of the Dyc mutant revealed a trinucleotide expansion in the polyalanine-encoding region of the Hoxd13 gene resulting in a 7-alanine expansion. However, unlike the Spdh mutation, this expansion cannot result from a simple duplication of a short segment. Instead, we propose the fork stalling and template switching (FosTeS) described for generation of nonrecurrent genomic rearrangements as a possible mechanism for the Dyc polyalanine extension, as well as for other polyalanine expansions described in the literature and that could not be explained by unequal crossing over.

Fork Stalling and Template Switching As a Mechanism for Polyalanine Tract Expansion Affecting the DYC Mutant of HOXD13, a New Murine Model of Synpolydactyly

PubMed Central

Cocquempot, Olivier; Brault, Véronique; Babinet, Charles; Herault, Yann

2009-01-01

Polyalanine expansion diseases are proposed to result from unequal crossover of sister chromatids that increases the number of repeats. In this report we suggest an alternative mechanism we put forward while we investigated a new spontaneous mutant that we named “Dyc” for “Digit in Y and Carpe” phenotype. Phenotypic analysis revealed an abnormal limb patterning similar to that of the human inherited congenital disease synpolydactyly (SPD) and to the mouse mutant model Spdh. Both human SPD and mouse Spdh mutations affect the Hoxd13 gene within a 15-residue polyalanine-encoding repeat in the first exon of the gene, leading to a dominant negative HOXD13. Genetic analysis of the Dyc mutant revealed a trinucleotide expansion in the polyalanine-encoding region of the Hoxd13 gene resulting in a 7-alanine expansion. However, unlike the Spdh mutation, this expansion cannot result from a simple duplication of a short segment. Instead, we propose the fork stalling and template switching (FosTeS) described for generation of nonrecurrent genomic rearrangements as a possible mechanism for the Dyc polyalanine extension, as well as for other polyalanine expansions described in the literature and that could not be explained by unequal crossing over. PMID:19546318

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

Physical interaction between replication protein A (RPA) and MRN: involvement of RPA2 phosphorylation and the N-terminus of RPA1.

PubMed

Oakley, Greg G; Tillison, Kristin; Opiyo, Stephen A; Glanzer, Jason G; Horn, Jeffrey M; Patrick, Steve M

2009-08-11

Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2, and RPA3 subunits that binds to single-stranded DNA (ssDNA) with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11-RAD50-NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-double-stranded DNA (dsDNA) junctions or breaks, and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic weakens the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates interactions of RPA with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 shows that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage.

Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability

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

Trego, Kelly S.; Groesser, Torsten; Davalos, Albert R.

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. In this paper, we identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatinmore » binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. Finally, these unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.« less

Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability

DOE PAGES

Trego, Kelly S.; Groesser, Torsten; Davalos, Albert R.; ...

2016-01-28

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. In this paper, we identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatinmore » binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. Finally, these unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.« less

Transcription and Recombination: When RNA Meets DNA

PubMed Central

Aguilera, Andrés; Gaillard, Hélène

2014-01-01

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

Novel SNP array analysis and exome sequencing detect a homozygous exon 7 deletion of MEGF10 causing early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD)

PubMed Central

Pierson, Tyler Mark; Markello, Thomas; Accardi, John; Wolfe, Lynne; Adams, David; Sincan, Murat; Tarazi, Noor M.; Fajardo, Karin Fuentes; Cherukuri, Praveen F.; Bajraktari, Ilda; Meilleur, Katy G.; Donkervoort, Sandra; Jain, Mina; Hu, Ying; Lehky, Tanya J.; Cruz, Pedro; Mullikin, James C.; Bonnemann, Carsten; Gahl, William A.; Boerkoel, Cornelius F.; Tifft, Cynthia J.

2013-01-01

Early-onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD) is a myopathic disorder associated with mutations in MEGF10. By novel analysis of SNP array hybridization and exome sequence coverage, we diagnosed a 10-year old girl with EMARDD following identification of a novel homozygous deletion of exon 7 in MEGF10. In contrast to previously reported EMARDD patients, her weakness was more prominent proximally than distally, and involved her legs more than her arms. MRI of her pelvis and thighs showed muscle atrophy and fatty replacement. Ultrasound of several muscle groups revealed dense homogenous increases in echogenicity. Cloning and sequencing of the deletion breakpoint identified features suggesting the mutation arose by fork stalling and template switching. These findings constitute the first genomic deletion causing EMARDD, expand the clinical phenotype, and provide new insight into the pattern and histology of its muscular pathology. PMID:23453856

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

PubMed

Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

2015-09-01

Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Architecture and DNA Recognition Elements of the Fanconi Anemia FANCM-FAAP24 Complex

PubMed Central

Coulthard, Rachel; Deans, Andrew J.; Swuec, Paolo; Bowles, Maureen; Costa, Alessandro; West, Stephen C.; McDonald, Neil Q.

2013-01-01

Summary Fanconi anemia (FA) is a disorder associated with a failure in DNA repair. FANCM (defective in FA complementation group M) and its partner FAAP24 target other FA proteins to sites of DNA damage. FANCM-FAAP24 is related to XPF/MUS81 endonucleases but lacks endonucleolytic activity. We report a structure of an FANCM C-terminal fragment (FANCMCTD) bound to FAAP24 and DNA. This S-shaped structure reveals the FANCM (HhH)2 domain is buried, whereas the FAAP24 (HhH)2 domain engages DNA. We identify a second DNA contact and a metal center within the FANCM pseudo-nuclease domain and demonstrate that mutations in either region impair double-stranded DNA binding in vitro and FANCM-FAAP24 function in vivo. We show the FANCM translocase domain lies in proximity to FANCMCTD by electron microscopy and that binding fork DNA structures stimulate its ATPase activity. This suggests a tracking model for FANCM-FAAP24 until an encounter with a stalled replication fork triggers ATPase-mediated fork remodeling. PMID:23932590

Transcription and recombination: when RNA meets DNA.

PubMed

Aguilera, Andrés; Gaillard, Hélène

2014-08-01

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Characterization of RACK7 as a Novel Factor Involved in BRCA1 Mutation Mediated Breast Cancer

DTIC Science & Technology

2012-10-01

Characterization of RACK7 as a Novel Factor Involved in BRCA1 Mutation Mediated Breast Cancer Inder Verma, Quan Zhu, Martin Preyer, and Amy Rommel Salk...samples from 5 more human BRCA1 mutant tumors. We continue to collect more human BRCA1-mutant tumors from Dr. Petra Nederlof at the Netherland Cancer...damage pathway. Abstract: "The Novel Zinc-finger Protein ZMYND8 Is Involved in the Stabilization of Stalled Replication Forks", Martin Preyer and

Function of ZFAND3 in the DNA Damage Response

DTIC Science & Technology

2013-06-01

Department of Defense Breast Cancer Program Era of Hope Conference August 2011 iv. Sirbu BM, Couch FB, Feigerle JT, Bhaskara S, Hiebert SW, Cortez D...Analysis of protein dynamics at active, stalled and collapsed replication forks; Vanderbilt Institute of Chemical and Physical Biology August 2011...BRIP1 MED16 FANCD2 COMT TONSL FANCI CUL2 TRRAP MDC1 DMD UNG PDS5B DNPH1 WRN POLE FANCI RFC1 INCENP RPA1 JMJD6 SART3 KIAA1598 BLM SMARCAD1 NBAS BRIP1

The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation

PubMed Central

Galick, Heather A.; Marsden, Carolyn G.; Kathe, Scott; Dragon, Julie A.; Volk, Lindsay; Nemec, Antonia A.; Wallace, Susan S.; Prakash, Aishwarya; Doublié, Sylvie; Sweasy, Joann B.

2017-01-01

Base excision repair (BER) is a key genome maintenance pathway. The NEIL1 DNA glycosylase recognizes oxidized bases, and likely removes damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene is a rare human germline variant that encodes the NEIL1 G83D protein, which is devoid of DNA glycosylase activity. Here we show that expression of G83D NEIL1 in MCF10A immortalized but non-transformed mammary epithelial cells leads to replication fork stress. Upon treatment with hydrogen peroxide, we observe increased levels of stalled replication forks in cells expressing G83D NEIL1 versus cells expressing the wild-type (WT) protein. Double-strand breaks (DSBs) arise in G83D-expressing cells during the S and G2/M phases of the cell cycle. Interestingly, these breaks result in genomic instability in the form of high levels of chromosomal aberrations and micronuclei. Cells expressing G83D also grow in an anchorage independent manner, suggesting that the genomic instability results in a carcinogenic phenotype. Our results are consistent with the idea that an inability to remove oxidative damage in an efficient manner at the replication fork leads to genomic instability and mutagenesis. We suggest that individuals who harbor the G83D NEIL1 variant face an increased risk for human cancer. PMID:29156764

Tipin functions in the protection against topoisomerase I inhibitor.

PubMed

Hosono, Yoshifumi; Abe, Takuya; Higuchi, Masato; Kajii, Kosa; Sakuraba, Shuichi; Tada, Shusuke; Enomoto, Takemi; Seki, Masayuki

2014-04-18

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.

Human Fanconi Anemia Complementation Group A Protein Stimulates the 5’ Flap Endonuclease Activity of FEN1

PubMed Central

Qian, Liangyue; Yuan, Fenghua; Rodriguez-Tello, Paola; Padgaonkar, Suyog; Zhang, Yanbin

2013-01-01

In eukaryotic cells, Flap endonuclease 1 (FEN1) is a major structure-specific endonuclease that processes 5’ flapped structures during maturation of lagging strand DNA synthesis, long patch base excision repair, and rescue of stalled replication forks. Here we report that fanconi anemia complementation group A protein (FANCA), a protein that recognizes 5’ flap structures and is involved in DNA repair and maintenance of replication forks, constantly stimulates FEN1-mediated incision of both DNA and RNA flaps. Kinetic analyses indicate that FANCA stimulates FEN1 by increasing the turnover rate of FEN1 and altering its substrate affinity. More importantly, six pathogenic FANCA mutants are significantly less efficient than the wild-type at stimulating FEN1 endonuclease activity, implicating that regulation of FEN1 by FANCA contributes to the maintenance of genomic stability. PMID:24349332

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PubMed Central

Mimmler, Maximilian; Peter, Simon; Kraus, Alexander; Stroh, Svenja; Nikolova, Teodora; Seiwert, Nina; Hasselwander, Solveig; Neitzel, Carina; Haub, Jessica; Monien, Bernhard H.; Nicken, Petra; Steinberg, Pablo; Shay, Jerry W.; Kaina, Bernd; Fahrer, Jörg

2016-01-01

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs. PMID:27599846

Archaeal replicative primases can perform translesion DNA synthesis.

PubMed

Jozwiakowski, Stanislaw K; Borazjani Gholami, Farimah; Doherty, Aidan J

2015-02-17

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2'-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

PubMed Central

Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

2014-01-01

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

Caenorhabditis elegans HIM-18/SLX-4 interacts with SLX-1 and XPF-1 and maintains genomic integrity in the germline by processing recombination intermediates.

PubMed

Saito, Takamune T; Youds, Jillian L; Boulton, Simon J; Colaiácovo, Monica P

2009-11-01

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR-mediated repair at stalled replication forks. A reduction in crossover recombination frequencies-accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction-support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.

Caenorhabditis elegans HIM-18/SLX-4 Interacts with SLX-1 and XPF-1 and Maintains Genomic Integrity in the Germline by Processing Recombination Intermediates

PubMed Central

Saito, Takamune T.; Youds, Jillian L.; Boulton, Simon J.; Colaiácovo, Monica P.

2009-01-01

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR–mediated repair at stalled replication forks. A reduction in crossover recombination frequencies—accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction—support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline. PMID:19936019

CRISPR adaptation biases explain preference for acquisition of foreign DNA

PubMed Central

Yosef, Ido; Auster, Oren; Manor, Miriam; Amitai, Gil; Edgar, Rotem; Qimron, Udi; Sorek, Rotem

2015-01-01

In the process of CRISPR adaptation, short pieces of DNA (“spacers”) are acquired from foreign elements and integrated into the CRISPR array. It so far remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of “self” is mediated by the RecBCD dsDNA break repair complex. Our results suggest that in E. coli, acquisition of new spacers depends on RecBCD-mediated processing of dsDNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers from both high copy plasmids and phages. PMID:25874675

Chromosome demise in the wake of ligase-deficient replication.

PubMed

Kouzminova, Elena A; Kuzminov, Andrei

2012-06-01

Bacterial DNA ligases, NAD⁺-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. © 2012 Blackwell Publishing Ltd.

When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro.

PubMed

Pipathsouk, Anne; Belotserkovskii, Boris P; Hanawalt, Philip C

2017-02-01

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest. Copyright © 2016 Elsevier B.V. All rights reserved.

Stochastic Endogenous Replication Stress Causes ATR-Triggered Fluctuations in CDK2 Activity that Dynamically Adjust Global DNA Synthesis Rates.

PubMed

Daigh, Leighton H; Liu, Chad; Chung, Mingyu; Cimprich, Karlene A; Meyer, Tobias

2018-06-04

Faithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged S phase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout S phase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events. Copyright © 2018. Published by Elsevier Inc.

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

PubMed Central

Huang, Shar-yin N.; Murai, Junko; Dalla Rosa, Ilaria; Dexheimer, Thomas S.; Naumova, Alena; Gmeiner, William H.; Pommier, Yves

2013-01-01

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs. PMID:23775789

BRCA1 is Required for Post-replication Repair After UV-induced DNA Damage

PubMed Central

Pathania, Shailja; Nguyen, Jenna; Hill, Sarah J.; Scully, Ralph; Feunteun, Jean; Livingston, David M.

2011-01-01

BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent, but nucleotide excision repair- independent manner. More specifically, at UV- stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and post- replicative repair. These BRCA1 functions differ from those required for DSBR. PMID:21963239

Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site.

PubMed

Letessier, Anne; Millot, Gaël A; Koundrioukoff, Stéphane; Lachagès, Anne-Marie; Vogt, Nicolas; Hansen, R Scott; Malfoy, Bernard; Brison, Olivier; Debatisse, Michelle

2011-02-03

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

PubMed Central

Schwab, Rebekka A.V.; Niedzwiedz, Wojciech

2011-01-01

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope. PMID:22064662

DNA damage tolerance in hematopoietic stem and progenitor cells in mice

PubMed Central

Pilzecker, Bas; Buoninfante, Olimpia Alessandra; van den Berk, Paul; Lancini, Cesare; Song, Ji-Ying; Citterio, Elisabetta

2017-01-01

DNA damage tolerance (DDT) enables bypassing of DNA lesions during replication, thereby preventing fork stalling, replication stress, and secondary DNA damage related to fork stalling. Three modes of DDT have been documented: translesion synthesis (TLS), template switching (TS), and repriming. TLS and TS depend on site-specific PCNA K164 monoubiquitination and polyubiquitination, respectively. To investigate the role of DDT in maintaining hematopoietic stem cells (HSCs) and progenitors, we used PcnaK164R/K164R mice as a unique DDT-defective mouse model. Analysis of the composition of HSCs and HSC-derived multipotent progenitors (MPPs) revealed a significantly reduced number of HSCs, likely owing to increased differentiation of HSCs toward myeloid/erythroid-associated MPP2s. This skewing came at the expense of the number of lymphoid-primed MPP4s, which appeared to be compensated for by increased MPP4 proliferation. Furthermore, defective DDT decreased the numbers of MPP-derived common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP) cells, accompanied by increased cell cycle arrest in CMPs. The HSC and MPP phenotypes are reminiscent of premature aging and stressed hematopoiesis, and indeed progressed with age and were exacerbated on cisplatin exposure. Bone marrow transplantations revealed a strong cell intrinsic defect of DDT-deficient HSCs in reconstituting lethally irradiated mice and a strong competitive disadvantage when cotransplanted with wild-type HSCs. These findings indicate a critical role of DDT in maintaining HSCs and progenitor cells, and in preventing premature aging. PMID:28761001

Acute inactivation of the replicative helicase in human cells triggers MCM8–9-dependent DNA synthesis

PubMed Central

Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy; Bhowmick, Rahul; Hickson, Ian D.; Kanemaki, Masato T.

2017-01-01

DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8–9 complex, a paralog of the MCM2–7 replicative helicase. We show that MCM8–9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8–9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8–9 as an alternative replicative helicase. PMID:28487407

Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication.

PubMed

Morrow, Carl A; Nguyen, Michael O; Fower, Andrew; Wong, Io Nam; Osman, Fekret; Bryer, Claire; Whitby, Matthew C

2017-06-06

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

PubMed

Schalbetter, Stephanie A; Mansoubi, Sahar; Chambers, Anna L; Downs, Jessica A; Baxter, Jonathan

2015-08-18

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

Mutant POLG2 Disrupts DNA Polymerase γ Subunits and Causes Progressive External Ophthalmoplegia

PubMed Central

Longley, Matthew J.; Clark, Susanna; Yu Wai Man, Cynthia; Hudson, Gavin; Durham, Steve E.; Taylor, Robert W.; Nightingale, Simon; Turnbull, Douglass M.; Copeland, William C.; Chinnery, Patrick F.

2006-01-01

DNA polymerase γ (pol γ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol γ (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G→A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol γ, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)–deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID:16685652

Fanconi anemia: causes and consequences of genetic instability.

PubMed

Kalb, R; Neveling, K; Nanda, I; Schindler, D; Hoehn, H

2006-01-01

Fanconi anemia (FA) is a rare recessive disease that reflects the cellular and phenotypic consequences of genetic instability: growth retardation, congenital malformations, bone marrow failure, high risk of neoplasia, and premature aging. At the cellular level, manifestations of genetic instability include chromosomal breakage, cell cycle disturbance, and increased somatic mutation rates. FA cells are exquisitely sensitive towards oxygen and alkylating drugs such as mitomycin C or diepoxybutane, pointing to a function of FA genes in the defense against reactive oxygen species and other DNA damaging agents. FA is caused by biallelic mutations in at least 12 different genes which appear to function in the maintenance of genomic stability. Eight of the FA proteins form a nuclear core complex with a catalytic function involving ubiquitination of the central FANCD2 protein. The posttranslational modification of FANCD2 promotes its accumulation in nuclear foci, together with known DNA maintenance proteins such as BRCA1, BRCA2, and the RAD51 recombinase. Biallelic mutations in BRCA2 cause a severe FA-like phenotype, as do biallelic mutations in FANCD2. In fact, only leaky or hypomorphic mutations in this central group of FA genes appear to be compatible with life birth and survival. The newly discovered FANCJ (= BRIP1) and FANCM (= Hef ) genes correspond to known DNA-maintenance genes (helicase resp. helicase-associated endonuclease for fork-structured DNA). These genes provide the most convincing evidence to date of a direct involvement of FA genes in DNA repair functions associated with the resolution of DNA crosslinks and stalled replication forks. Even though genetic instability caused by mutational inactivation of the FANC genes has detrimental effects for the majority of FA patients, around 20% of patients appear to benefit from genetic instability since genetic instability also increases the chance of somatic reversion of their constitutional mutations. Intragenic crossover, gene conversion, back mutation and compensating mutations in cis have all been observed in revertant, and, consequently, mosaic FA-patients, leading to improved bone marrow function. There probably is no other experiment of nature in our species in which causes and consequences of genetic instability, including the role of reactive oxygen species, can be better documented and explored than in FA.

Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

PubMed

Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

2014-01-01

DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. © 2013 John Wiley & Sons Ltd.

Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

PubMed

Berniak, K; Rybak, P; Bernas, T; Zarębski, M; Biela, E; Zhao, H; Darzynkiewicz, Z; Dobrucki, J W

2013-10-01

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

hSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases.

PubMed

Ashton, Nicholas W; Paquet, Nicolas; Shirran, Sally L; Bolderson, Emma; Kariawasam, Ruvini; Touma, Christine; Fallahbaghery, Azadeh; Gamsjaeger, Roland; Cubeddu, Liza; Botting, Catherine; Pollock, Pamela M; O'Byrne, Kenneth J; Richard, Derek J

2017-06-01

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

PRP19 Transforms into a Sensor of RPA-ssDNA after DNA Damage and Drives ATR Activation via a Ubiquitin-Mediated Circuitry

PubMed Central

Maréchal, Alexandre; Wu, Ching-Shyi; Yazinski, Stephanie A.; Nguyen, Hai Dang; Liu, Shizhou; Jiménez, Amanda E.; Jin, Jianping; Zou, Lee

2014-01-01

Summary PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). While the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 binds RPA directly and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ATR kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, the recovery of stalled replication forks, and the progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR. PMID:24332808

Common Chemical Inductors of Replication Stress:  Focus on Cell-Based Studies.

PubMed

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-02-21

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.

Common Chemical Inductors of Replication Stress: Focus on Cell-Based Studies

PubMed Central

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-01-01

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses. PMID:28230817

Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1.

PubMed

Boddy, M N; Lopez-Girona, A; Shanahan, P; Interthal, H; Heyer, W D; Russell, P

2000-12-01

Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.

Rotating stall simulation for axial and centrifugal compressors

NASA Astrophysics Data System (ADS)

Halawa, Taher; Gadala, Mohamed S.

2017-05-01

This study presents a numerical simulation of the rotating stall phenomenon in axial and centrifugal compressors with detailed descriptions of stall precursors and its development with time. Results showed that the vaneless region of the centrifugal compressor is the most critical location affected by stall. It was found that the tip leakage flow and the back flow impingement are the main cause of the stall development at the impeller exit area for centrifugal compressors. The results of the axial compressor simulations indicated that the early separated flow combined with the tip leakage flow can block the impeller passages during stall.

Kinetic modeling predicts a stimulatory role for ribosome collisions at elongation stall sites in bacteria

PubMed Central

Ferrin, Michael A; Subramaniam, Arvind R

2017-01-01

Ribosome stalling on mRNAs can decrease protein expression. To decipher ribosome kinetics at stall sites, we induced ribosome stalling at specific codons by starving the bacterium Escherichia coli for the cognate amino acid. We measured protein synthesis rates from a reporter library of over 100 variants that encoded systematic perturbations of translation initiation rate, the number of stall sites, and the distance between stall sites. Our measurements are quantitatively inconsistent with two widely-used kinetic models for stalled ribosomes: ribosome traffic jams that block initiation, and abortive (premature) termination of stalled ribosomes. Rather, our measurements support a model in which collision with a trailing ribosome causes abortive termination of the stalled ribosome. In our computational analysis, ribosome collisions selectively stimulate abortive termination without fine-tuning of kinetic rate parameters at ribosome stall sites. We propose that ribosome collisions serve as a robust timer for translational quality control pathways to recognize stalled ribosomes. DOI: http://dx.doi.org/10.7554/eLife.23629.001 PMID:28498106

The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

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

Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.

XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks andmore » telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.« less

14 CFR 23.251 - Vibration and buffeting.

Code of Federal Regulations, 2012 CFR

2012-01-01

... interfere with the satisfactory control of the airplane or cause excessive fatigue to the flight crew. Stall... flight condition, including configuration changes during cruise, severe enough to interfere with the satisfactory control of the airplane or cause excessive fatigue to the flight crew. Stall warning buffeting...

PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry.

PubMed

Maréchal, Alexandre; Li, Ju-Mei; Ji, Xiao Ye; Wu, Ching-Shyi; Yazinski, Stephanie A; Nguyen, Hai Dang; Liu, Shizhou; Jiménez, Amanda E; Jin, Jianping; Zou, Lee

2014-01-23

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR. Copyright © 2014 Elsevier Inc. All rights reserved.

PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

PubMed

Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao Huang, Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

2009-07-01

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

Inferring Causes of Biological Impairment in the Clear Fork Watershed, West Virginia (Final)

EPA Science Inventory

EPA announced the availability of the final report, Inferring Causes of Biological Impairment in the Clear Fork Watershed, West Virginia. This study illustrates a causal assessment in a watershed using the US EPA stressor identification process described on the www.epa....

Enhancing BEM simulations of a stalled wind turbine using a 3D correction model

NASA Astrophysics Data System (ADS)

Bangga, Galih; Hutomo, Go; Syawitri, Taurista; Kusumadewi, Tri; Oktavia, Winda; Sabila, Ahmad; Setiadi, Herlambang; Faisal, Muhamad; Hendranata, Yongki; Lastomo, Dwi; Putra, Louis; Kristiadi, Stefanus; Bumi, Ilmi

2018-03-01

Nowadays wind turbine rotors are usually employed with pitch control mechanisms to avoid deep stall conditions. Despite that, wind turbines often operate under pitch fault situation causing massive flow separation to occur. Pure Blade Element Momentum (BEM) approaches are not designed for this situation and inaccurate load predictions are already expected. In the present studies, BEM predictions are improved through the inclusion of a stall delay model for a wind turbine rotor operating under pitch fault situation of -2.3° towards stall. The accuracy of the stall delay model is assessed by comparing the results with available Computational Fluid Dynamics (CFD) simulations data.

How bumps on whale flippers delay stall: an aerodynamic model.

PubMed

van Nierop, Ernst A; Alben, Silas; Brenner, Michael P

2008-02-08

Wind tunnel experiments have shown that bumps on the leading edge of model humpback whale flippers cause them to "stall" (i.e., lose lift dramatically) more gradually and at a higher angle of attack. Here we develop an aerodynamic model which explains the observed increase in stall angle. The model predicts that as the amplitude of the bumps is increased, the lift curve flattens out, leading to potentially desirable control properties. We find that stall delay is insensitive to the wavelength of the bumps, in accordance with experimental observations.

Experimental Investigation of Rotating Stall in a Research Multistage Axial Compressor

NASA Technical Reports Server (NTRS)

Lepicovsky, Jan; Braunscheidel, Edward P.; Welch, Gerard E.

2007-01-01

A collection of experimental data acquired in the NASA low-speed multistage axial compressor while operated in rotating stall is presented in this paper. The compressor was instrumented with high-response wall pressure modules and a static pressure disc probe for in-flow measurement, and a split-fiber probe for simultaneous measurements of velocity magnitude and flow direction. The data acquired to-date have indicated that a single fully developed stall cell rotates about the flow annulus at 50.6% of the rotor speed. The stall phenomenon is substantially periodic at a fixed frequency of 8.29 Hz. It was determined that the rotating stall cell extends throughout the entire compressor, primarily in the axial direction. Spanwise distributions of the instantaneous absolute flow angle, axial and tangential velocity components, and static pressure acquired behind the first rotor are presented in the form of contour plots to visualize different patterns in the outer (midspan to casing) and inner (hub to mid-span) flow annuli during rotating stall. In most of the cases observed, the rotating stall started with a single cell. On occasion, rotating stall started with two emerging stall cells. The root cause of the variable stall cell count is unknown, but is not attributed to operating procedures.

Preliminary Tests of a Buffet Stall-Warning Device on a 1/5-Scale Model of the Republic XP-84 Airplane

NASA Technical Reports Server (NTRS)

Tucker, Warren A.; Comisarow, Paul

1946-01-01

During the first flight tests of the Republic XP-84 airplane it was discovered that there was a complete lack of stall warning. A short series of development tests of a suitable stall-warning device for the airplane was therefore made on a 1/5-scale model in the Langley 300 MPH 7- by 10-foot tunnel. Two similar stall-warning devices, each designed to produce early root stall which would provide a buffet warning, were tested. It appeared that either device would give a satisfactory buffet warning in the flap-up configuration, at the cost of an increase of 8 or 10 miles per hour in minimum speed. Although neither device seemed to give a true buffet warning in the flaps-down configuration, it appeared that either device would improve the flaps-down stalling characteristics by lessening the severity of the stall and by maintaining better control at the stall. The flaps-down minimum-speed increase caused by the devices was only 1 or 2 miles per hour.

Control of airplanes at low speeds

NASA Technical Reports Server (NTRS)

Wood, R Mckinnon

1923-01-01

Loss of control over the orientation of an airplane as the incidence approaches and enters the region of stalled flight is a prolific cause of serious accidents. This report discusses methods of landing at slow speeds approaching stall.

Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

PubMed

Tian, Yanyan; Shen, Xi; Wang, Rui; Klages-Mundt, Naeh L; Lynn, Erica J; Martin, Sara K; Ye, Yin; Gao, Min; Chen, Junjie; Schlacher, Katharina; Li, Lei

2017-12-08

In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

PubMed Central

Fredsøe, Jacob; Nielsen, Ida; Pedersen, Jakob Madsen; Bentsen, Iben Bach; Lisby, Michael; Bjergbaek, Lotte; Andersen, Anni H

2015-01-01

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time. PMID:26630413

PCNA-coupled p21 degradation after DNA damage: The exception that confirms the rule?

PubMed

Soria, Gastón; Gottifredi, Vanesa

2010-04-04

While many are the examples of DNA damaging treatments that induce p21 accumulation, the conception of p21 upregulation as the universal response to genotoxic stress has come to an end. Compelling evidences have demonstrated the existence of converging signals that negatively regulate p21 bellow basal levels when replication forks are blocked. Moreover, conclusive reports identified the E3-ligase CRL4(CDT2) (CUL4-DDB1-CDT2) as the enzymatic complex that promotes p21 proteolysis when treatments such as UV irradiation trigger replication fork stress. A pre-requisite for CRL4(CDT2)-driven proteolysis is the interaction of p21 with PCNA. Interestingly as well, CRL4(CDT2)-dependent proteolysis is not limited to p21 and affects other PCNA partners, including the specialized DNA polymerase eta (pol eta). These recent discoveries are particularly intriguing since the UV-induced degradation of p21 has been shown to be required for efficient pol eta recruitment to DNA lesions. Herein we review the findings that lead to the identification of the molecular mechanism that triggers damage-induced PCNA-coupled protein proteolysis. We propose a novel model in which CRL4(CDT2)-dependent protein degradation facilitates a sequential and dynamic exchange between PIP box bearing proteins at stall forks during Translesion DNA synthesis (TLS). Moreover, given the tight spatiotemporal control that CRL4(CDT2)-driven proteolysis is able to confer to PCNA-regulated processes, we discuss the impact that this degradation mechanism might have in other molecular switches associated with the repair of damaged DNA. 2010 Elsevier B.V. All rights reserved.

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.

Replication, checkpoint suppression and structure of centromeric DNA

PubMed Central

Romeo, Francesco; Costanzo, Vincenzo

2016-01-01

ABSTRACT Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells. PMID:27893298

New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

PubMed

Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

2015-05-01

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory proteins guiding distinct BER sub-pathways.

System-wide Analysis of SUMOylation Dynamics in Response to Replication Stress Reveals Novel Small Ubiquitin-like Modified Target Proteins and Acceptor Lysines Relevant for Genome Stability*

PubMed Central

Xiao, Zhenyu; Chang, Jer-Gung; Hendriks, Ivo A.; Sigurðsson, Jón Otti; Olsen, Jesper V.; Vertegaal, Alfred C.O.

2015-01-01

Genotoxic agents can cause replication fork stalling in dividing cells because of DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 h of hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and two proteins were down-regulated for SUMOylation, whereas after 24 h, 35 proteins were up-regulated for SUMOylation, and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1000 SUMO-2 acceptor lysines in target proteins. The methodology is generic and is widely applicable in the ubiquitin field. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1, and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the aim of dealing with the induced DNA damage. PMID:25755297

Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele-Specific Expression

PubMed Central

Giorgio, Elisa; Rolyan, Harshvardhan; Kropp, Laura; Chakka, Anish Baswanth; Yatsenko, Svetlana; Gregorio, Eleonora Di; Lacerenza, Daniela; Vaula, Giovanna; Talarico, Flavia; Mandich, Paola; Toro, Camilo; Pierre, Eleonore Eymard; Labauge, Pierre; Capellari, Sabina; Cortelli, Pietro; Vairo, Filippo Pinto; Miguel, Diego; Stubbolo, Danielle; Marques, Lourenco Charles; Gahl, William; Boespflug-Tanguy, Odile; Melberg, Atle; Hassin-Baer, Sharon; Cohen, Oren S; Pjontek, Rastislav; Grau, Armin; Klopstock, Thomas; Fogel, Brent; Meijer, Inge; Rouleau, Guy; Bouchard, Jean-Pierre L; Ganapathiraju, Madhavi; Vanderver, Adeline; Dahl, Niklas; Hobson, Grace; Brusco, Alfredo; Brussino, Alessandro; Padiath, Quasar Saleem

2013-01-01

ABSTRACT Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels. PMID:23649844

The ubiquitin family meets the Fanconi anemia proteins.

PubMed

Renaudin, Xavier; Koch Lerner, Leticia; Menck, Carlos Frederico Martins; Rosselli, Filippo

2016-01-01

Fanconi anaemia (FA) is a hereditary disorder characterized by bone marrow failure, developmental defects, predisposition to cancer and chromosomal abnormalities. FA is caused by biallelic mutations that inactivate genes encoding proteins involved in replication stress-associated DNA damage responses. The 20 FANC proteins identified to date constitute the FANC pathway. A key event in this pathway involves the monoubiquitination of the FANCD2-FANCI heterodimer by the collective action of at least 10 different proteins assembled in the FANC core complex. The FANC core complex-mediated monoubiquitination of FANCD2-FANCI is essential to assemble the heterodimer in subnuclear, chromatin-associated, foci and to regulate the process of DNA repair as well as the rescue of stalled replication forks. Several recent works have demonstrated that the activity of the FANC pathway is linked to several other protein post-translational modifications from the ubiquitin-like family, including SUMO and NEDD8. These modifications are related to DNA damage responses but may also affect other cellular functions potentially related to the clinical phenotypes of the syndrome. This review summarizes the interplay between the ubiquitin and ubiquitin-like proteins and the FANC proteins that constitute a major pathway for the surveillance of the genomic integrity and addresses the implications of their interactions in maintaining genome stability. Copyright © 2016 Elsevier B.V. All rights reserved.

Numerical investigation of the unsteady tip leakage flow and rotating stall inception in a transonic compressor

NASA Astrophysics Data System (ADS)

Zhang, Yanfeng; Lu, Xingen; Chu, Wuli; Zhu, Junqiang

2010-08-01

It is well known that tip leakage flow has a strong effect on the compressor performance and stability. This paper reports on a numerical investigation of detailed flow structures in an isolated transonic compressor rotor-NASA Rotor 37 at near stall and stalled conditions aimed at improving understanding of changes in 3D tip leakage flow structures with rotating stall inception. Steady and unsteady 3D Navier-Stokes analyses were conducted to investigate flow structures in the same rotor. For steady analysis, the predicted results agree well with the experimental data for the estimation of compressor rotor global performance. For unsteady flow analysis, the unsteady flow nature caused by the breakdown of the tip leakage vortex in blade tip region in the transonic compressor rotor at near stall condition has been captured with a single blade passage. On the other hand, the time-accurate unsteady computations of multi-blade passage at near stall condition indicate that the unsteady breakdown of the tip leakage vortex triggered the short length-scale — spike type rotating stall inception at blade tip region. It was the forward spillage of the tip leakage flow at blade leading edge resulting in the spike stall inception. As the mass flow ratio is decreased, the rotating stall cell was further developed in the blade passage.

Analysis of Low-Speed Stall Aerodynamics of a Swept Wing with Laminar-Flow Glove

NASA Technical Reports Server (NTRS)

Bui, Trong T.

2014-01-01

Reynolds-Averaged Navier-Stokes (RANS) computational fluid dynamics (CFD) analysis was conducted to study the low-speed stall aerodynamics of a GIII aircraft's swept wing modified with a laminar-flow wing glove. The stall aerodynamics of the gloved wing were analyzed and compared with the unmodified wing for the flight speed of 120 knots and altitude of 2300 ft above mean sea level (MSL). The Star-CCM+ polyhedral unstructured CFD code was first validated for wing stall predictions using the wing-body geometry from the First American Institute of Aeronautics and Astronautics (AIAA) CFD High-Lift Prediction Workshop. It was found that the Star-CCM+ CFD code can produce results that are within the scattering of other CFD codes considered at the workshop. In particular, the Star-CCM+ CFD code was able to predict wing stall for the AIAA wing-body geometry to within 1 degree of angle of attack as compared to benchmark wind-tunnel test data. Current results show that the addition of the laminar-flow wing glove causes the gloved wing to stall much earlier than the unmodified wing. Furthermore, the gloved wing has a different stall characteristic than the clean wing, with no sharp lift drop-off at stall for the gloved wing.

Analysis of Low Speed Stall Aerodynamics of a Swept Wing with Laminar Flow Glove

NASA Technical Reports Server (NTRS)

Bui, Trong T.

2014-01-01

Reynolds-Averaged Navier-Stokes (RANS) computational fluid dynamics (CFD) analysis was conducted to study the low-speed stall aerodynamics of a GIII aircraft's swept wing modified with a laminar-flow wing glove. The stall aerodynamics of the gloved wing were analyzed and compared with the unmodified wing for the flight speed of 120 knots and altitude of 2300 ft above mean sea level (MSL). The Star-CCM+ polyhedral unstructured CFD code was first validated for wing stall predictions using the wing-body geometry from the First American Institute of Aeronautics and Astronautics (AIAA) CFD High-Lift Prediction Workshop. It was found that the Star-CCM+ CFD code can produce results that are within the scattering of other CFD codes considered at the workshop. In particular, the Star-CCM+ CFD code was able to predict wing stall for the AIAA wing-body geometry to within 1 degree of angle of attack as compared to benchmark wind-tunnel test data. Current results show that the addition of the laminar-flow wing glove causes the gloved wing to stall much earlier than the unmodified wing. Furthermore, the gloved wing has a different stall characteristic than the clean wing, with no sharp lift drop-off at stall for the gloved wing.

A time-varying subjective quality model for mobile streaming videos with stalling events

NASA Astrophysics Data System (ADS)

Ghadiyaram, Deepti; Pan, Janice; Bovik, Alan C.

2015-09-01

Over-the-top mobile video streaming is invariably influenced by volatile network conditions which cause playback interruptions (stalling events), thereby impairing users' quality of experience (QoE). Developing models that can accurately predict users' QoE could enable the more efficient design of quality-control protocols for video streaming networks that reduce network operational costs while still delivering high-quality video content to the customers. Existing objective models that predict QoE are based on global video features, such as the number of stall events and their lengths, and are trained and validated on a small pool of ad hoc video datasets, most of which are not publicly available. The model we propose in this work goes beyond previous models as it also accounts for the fundamental effect that a viewer's recent level of satisfaction or dissatisfaction has on their overall viewing experience. In other words, the proposed model accounts for and adapts to the recency, or hysteresis effect caused by a stall event in addition to accounting for the lengths, frequency of occurrence, and the positions of stall events - factors that interact in a complex way to affect a user's QoE. On the recently introduced LIVE-Avvasi Mobile Video Database, which consists of 180 distorted videos of varied content that are afflicted solely with over 25 unique realistic stalling events, we trained and validated our model to accurately predict the QoE, attaining standout QoE prediction performance.

Genome-wide alterations of the DNA replication program during tumor progression

NASA Astrophysics Data System (ADS)

Arneodo, A.; Goldar, A.; Argoul, F.; Hyrien, O.; Audit, B.

2016-08-01

Oncogenic stress is a major driving force in the early stages of cancer development. Recent experimental findings reveal that, in precancerous lesions and cancers, activated oncogenes may induce stalling and dissociation of DNA replication forks resulting in DNA damage. Replication timing is emerging as an important epigenetic feature that recapitulates several genomic, epigenetic and functional specificities of even closely related cell types. There is increasing evidence that chromosome rearrangements, the hallmark of many cancer genomes, are intimately associated with the DNA replication program and that epigenetic replication timing changes often precede chromosomic rearrangements. The recent development of a novel methodology to map replication fork polarity using deep sequencing of Okazaki fragments has provided new and complementary genome-wide replication profiling data. We review the results of a wavelet-based multi-scale analysis of genomic and epigenetic data including replication profiles along human chromosomes. These results provide new insight into the spatio-temporal replication program and its dynamics during differentiation. Here our goal is to bring to cancer research, the experimental protocols and computational methodologies for replication program profiling, and also the modeling of the spatio-temporal replication program. To illustrate our purpose, we report very preliminary results obtained for the chronic myelogeneous leukemia, the archetype model of cancer. Finally, we discuss promising perspectives on using genome-wide DNA replication profiling as a novel efficient tool for cancer diagnosis, prognosis and personalized treatment.

SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

PubMed Central

Meyer, Katherine; Harper, J. Wade; Colaiácovo, Monica P.

2012-01-01

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4–dependent endonuclease activity toward replication forks, 5′-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs. PMID:22927825

Unsteady behavior of leading-edge vortex and diffuser stall in a centrifugal compressor with vaned diffuser

NASA Astrophysics Data System (ADS)

Fujisawa, Nobumichi; Hara, Shotaro; Ohta, Yutaka

2016-02-01

The characteristics of a rotating stall of an impeller and diffuser and the evolution of a vortex generated at the diffuser leading-edge (i.e., the leading-edge vortex (LEV)) in a centrifugal compressor were investigated by experiments and numerical analysis. The results of the experiments revealed that both the impeller and diffuser rotating stalls occurred at 55 and 25 Hz during off-design flow operation. For both, stall cells existed only on the shroud side of the flow passages, which is very close to the source location of the LEV. According to the CFD results, the LEV is made up of multiple vortices. The LEV is a combination of a separated vortex near the leading- edge and a longitudinal vortex generated by the extended tip-leakage flow from the impeller. Therefore, the LEV is generated by the accumulation of vorticity caused by the velocity gradient of the impeller discharge flow. In partial-flow operation, the spanwise extent and the position of the LEV origin are temporarily transmuted. The LEV develops with a drop in the velocity in the diffuser passage and forms a significant blockage within the diffuser passage. Therefore, the LEV may be regarded as being one of the causes of a diffuser stall in a centrifugal compressor.

Laser velocimeter measurements of dynamic stall. [conducted in the Ames two foot wind tunnel

NASA Technical Reports Server (NTRS)

Owen, F. K.

1984-01-01

Laser velocimeter measurements were made during the study of a two-dimensional NACA 0012 airfoil undergoing conditions of dynamic stall. The measurements, which were obtained in the Ames 2 foot wind tunnel at reduced frequencies of 0.12 and 1.2, show significant flow field hysteresis around the static stall angle. Comparisons were also made with dual-plate interferograms and good agreement was found for the attached flow cases. For separated flow, characteristic vortex shedding caused poor agreement and significantly increased the measured Reynolds shear stresses.

A model for the selective amplification of spatially coherent waves in a centrifugal compressor on the verge of rotating stall

NASA Technical Reports Server (NTRS)

Lawless, Patrick B.; Fleeter, Sanford

1993-01-01

A simple model for the stability zones of a low speed centrifugal compressor is developed, with the goal of understanding the driving mechanism for the changes in stalling behavior predicted for, and observed in, the Purdue Low Speed Centrifugal Research Compressor Facility. To this end, earlier analyses of rotating stall suppression in centrifugal compressors are presented in a reduced form that preserves the essential parameters of the model that affect the stalling behavior of the compressor. The model is then used to illuminate the relationship between compressor geometry, expected mode shape, and regions of amplification for weak waves which are indicative of the susceptibility of the system to rotating stall. The results demonstrate that increasing the stagger angle of the diffuser vanes, and consequently the diffusion path length, results in the compressor moving towards a condition where higher-order spatial modes are excited during stall initiation. Similarly, flow acceleration in the diffuser section caused by an increase in the number of diffuser vanes also results in the excitation of higher modes.

Task II: Three-dimensional Rotating Stall Inception and Effects of Rotating Tip Clearance Asymmetry in Axial Compressors

NASA Technical Reports Server (NTRS)

Suder, Kenneth (Technical Monitor); Tan, Choon-Sooi

2003-01-01

The effects of two types of flow non-uniformity on stall inception behavior were assessed with linearized stability analyses of two compressor flow models. Response to rotating tip clearance asymmetries induced by a whirling rotor shaft or rotor height variations were investigated with a two-dimensional flow model. A 3-D compressor model was also developed to study the stability of both full-span and part-span rotating stall modes in annular geometries with radial flow variations. The studies focussed on (1) understanding what compressor designs were sensitive to these types of circumferential and spanwise flow non-uniformities, and (2) situations where 2-D stability theories were inadequate because of 3-D flow effects. Rotating tip clearance non-uniformity caused the greatest performance loss for shafts whirling at the rotating stall frequency. A whirling shaft displacement of 1 percent chord caused the stalling mass flow to rise by as much as 10 percent and the peak pressure rise to decrease by 6 percent. These changes were an order of magnitude larger than for equivalent-sized stationary or rotor-locked clearance asymmetries. Spanwise flow non-uniformity always destabilized the compressor, so that 2-D models over-predicted that stall margin compared to 3-D theory. The difference increased for compressors with larger spanwise variations of characteristic slope and reduced characteristic curvature near the peak. Differences between 2-D and 3-D stall point predictions were generally unacceptable (2 - 4 percent of flow coefficient) for single-stage configurations, but were less than 1 percent for multistage compressors. 2-D analyses predicted the wrong stall mode for specific cases of radial inlet flow distortion, mismatching and annulus area contraction, where higher-order radial modes led to stall. The stability behavior of flows with circumferential or radial non-uniformity was unified through a single stability criterion. The stall point for both cases was set by the integral around the annulus of the pressure rise characteristic slope, weighted by the amplitude of the mode shape. For the case of steady circumferential variations, this criterion reduced to the integrated mean slope (IMS) condition associated with steady inlet distortions. The rotating tip clearance asymmetry model was also used to demonstrate the feasibility of actively controlling the shaft position to suppress rotating stall. In axisymmetric mean flow, this method only stabilized the first harmonic mode, increasing the operating range until surge or higher harmonic modes became unstable.

Stream bank and sediment movement associated with 2008 flooding, South Fork Iowa River

USDA-ARS?s Scientific Manuscript database

Stream bank erosion can cause substantial damage to riparian systems and impact the use of water downstream. Risks of bank erosion increase during extreme flood events, and frequencies of extreme events may be increasing under changing climate. We assessed bank erosion within the South Fork Iowa Riv...

40 CFR 86.1336-84 - Engine starting, restarting, and shutdown.

Code of Federal Regulations, 2012 CFR

2012-07-01

.... (4) If a failure to start occurs during the hot start portion of the test and is caused by engine... stalling. (1) If the engine stalls during the initial idle period of either the cold or hot start test, the engine shall be restarted immediately using the appropriate cold or hot starting procedure and the test...

40 CFR 86.1336-84 - Engine starting, restarting, and shutdown.

Code of Federal Regulations, 2013 CFR

2013-07-01

.... (4) If a failure to start occurs during the hot start portion of the test and is caused by engine... stalling. (1) If the engine stalls during the initial idle period of either the cold or hot start test, the engine shall be restarted immediately using the appropriate cold or hot starting procedure and the test...

40 CFR 86.1336-84 - Engine starting, restarting, and shutdown.

Code of Federal Regulations, 2011 CFR

2011-07-01

.... (4) If a failure to start occurs during the hot start portion of the test and is caused by engine... stalling. (1) If the engine stalls during the initial idle period of either the cold or hot start test, the engine shall be restarted immediately using the appropriate cold or hot starting procedure and the test...

The dynamics of genome replication using deep sequencing

PubMed Central

Müller, Carolin A.; Hawkins, Michelle; Retkute, Renata; Malla, Sunir; Wilson, Ray; Blythe, Martin J.; Nakato, Ryuichiro; Komata, Makiko; Shirahige, Katsuhiko; de Moura, Alessandro P.S.; Nieduszynski, Conrad A.

2014-01-01

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology. PMID:24089142

Helicase promotes replication re-initiation from an RNA transcript.

PubMed

Sun, Bo; Singh, Anupam; Sultana, Shemaila; Inman, James T; Patel, Smita S; Wang, Michelle D

2018-06-13

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.

Eukaryotic DNA polymerase ζ

PubMed Central

Makarova, Alena V.; Burgers, Peter M.

2015-01-01

This review focuses on eukaryotic DNA polymerase ζ (Pol ζ), the enzyme responsible for the bulk of mutagenesis in eukaryotic cells in response to DNA damage. Pol ζ is also responsible for a large portion of mutagenesis during normal cell growth, in response to spontaneous damage or to certain DNA structures and other blocks that stall DNA replication forks. Novel insights in mutagenesis have been derived from recent advances in the elucidation of the subunit structure of Pol ζ. The lagging strand DNA polymerase δ shares the small Pol31 and Pol32 subunits with the Rev3-Rev7 core assembly giving a four subunit Pol ζ complex that is the active form in mutagenesis. Furthermore, Pol ζ forms essential interactions with the mutasome assembly factor Rev1 and with proliferating cell nuclear antigen (PCNA). These interactions are modulated by posttranslational modifications such as ubiquitination and phosphorylation that enhance translesion synthesis (TLS) and mutagenesis. PMID:25737057

Longitudinal Stability and Stalling Characteristics of a 1/8.33-Scale Model of the Republic XF-12 Airplane

NASA Technical Reports Server (NTRS)

Pepper, Edward; Foster, Gerald V.

1946-01-01

The XF-12 airplane is a high performance, photo-reconnaissance aircraft designed by the Republic Aviation Corporation for Army Air Forces. A series of tests of a 1/8.33-scale powered model was conducted in the Langley 9-foot pressure tunnel to obtain information relative to the aerodynamic design of the airplane. This report presents the results of tests to determine the static longitudinal stability and stalling characteristics of the model. From this investigation it was indicated that the airplane will possess a positive static margin for all probable flight conditions. The stalling characteristics are considered satisfactory in that the stall initiates near the root section and progresses toward the tips. Early root section stalling occurs, with the flaps retracted and may cause undesirable tail buffeting and erratic elevator control in the normal flight range. From considerations of sinking speed landing flap deflections of 40 degrees may be preferable to 55 degrees of 65 degrees.

Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures

PubMed Central

Kang, Young-Hoon; Munashingha, Palinda Ruvan; Lee, Chul-Hwan; Nguyen, Tuan Anh; Seo, Yeon-Soo

2012-01-01

Saccharomyces cerevisiae Mph1 is a 3–5′ DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3′ to 5′ directionality, Mph1 displaced 3′-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5′-flap strand more efficiently than the 3′ flap strand from double-flap substrates, which is not expected for a 3–5′ DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5′ single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway. PMID:22090425

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light

PubMed Central

Jansen, Jacob G.; Temviriyanukul, Piya; Wit, Niek; Delbos, Frédéric; Reynaud, Claude-Agnès; Jacobs, Heinz; de Wind, Niels

2014-01-01

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects. PMID:25170086

(1)H, (13)C, and (15)N backbone resonance assignments of the full-length 40 kDa S. acidocaldarius Y-family DNA polymerase, dinB homolog.

PubMed

Moro, Sean L; Cocco, Melanie J

2015-10-01

The dinB homolog (Dbh) is a member of the Y-family of translesion DNA polymerases, which are specialized to accurately replicate DNA across from a wide variety of lesions in living cells. Lesioned bases block the progression of high-fidelity polymerases and cause detrimental replication fork stalling; Y-family polymerases can bypass these lesions. The active site of the translesion synthesis polymerase is more open than that of a replicative polymerase; consequently Dbh polymerizes with low fidelity. Bypass polymerases also have low processivity. Short extension past the lesion allows the high-fidelity polymerase to switch back onto the site of replication. Dbh and the other Y-family polymerases have been used as structural models to investigate the mechanisms of DNA polymerization and lesion bypass. Many high-resolution crystal structures of Y-family polymerases have been reported. NMR dynamics studies can complement these structures by providing a measure of protein motions. Here we report the (15)N, (1)H, and (13)C backbone resonance assignments at two temperatures (35 and 50 °C) for Sulfolobus acidocaldarius Dbh polymerase. Backbone resonance assignments have been obtained for 86 % of the residues. The polymerase active site is assigned as well as the majority of residues in each of the four domains.

Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex.

PubMed

Sugahara, R; Mon, H; Lee, J M; Kusakabe, T

2014-04-01

The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals. © 2013 The Royal Entomological Society.

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase.

PubMed

Liu, Na-Nv; Duan, Xiao-Lei; Ai, Xia; Yang, Yan-Tao; Li, Ming; Dou, Shuo-Xing; Rety, Stephane; Deprez, Eric; Xi, Xu-Guang

2015-10-15

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effect of spatial inlet temperature and pressure distortion on turbofan engine stability

NASA Technical Reports Server (NTRS)

Mehalic, Charles M.

1988-01-01

The effects of circumferential and radial inlet temperature distortion, circumferential pressure distortion, and combined temperature and pressure distortion on the stability of an advanced turbofan engine were investigated experimentally at simulated altitude conditions. With circumferential and radial inlet temperature distortion, a flow instability generated by the fan operating near stall caused the high-pressure compressor to surge at, or near, the same time as the fan. The effect of combined distortion was dependent on the relative location of the high-temperature and low-pressure regions; high-pressure compressor stalls occurred when the regions coincided, and fan stalls occurred with the regions separated.

Effect of inlet ingestion of a wing tip vortex on compressor face flow and turbojet stall margin

NASA Technical Reports Server (NTRS)

Mitchell, G. A.

1975-01-01

A two-dimensional inlet was alternately mated to a coldpipe plug assembly and a J85-GE-13 turbojet engine, and placed in a Mach 0.4 stream so as to ingest the tip vortex of a forward mounted wing. Vortex properties were measured just forward of the inlet and at the compressor face. Results show that ingestion of a wing tip vortex by a turbojet engine can cause a large reduction in engine stall margin. The loss in stall compressor pressure ratio was primarily dependent on vortex location and rotational direction and not on total-pressure distortion.

Dynamic Stall Characteristics of Drooped Leading Edge Airfoils

NASA Technical Reports Server (NTRS)

Sankar, Lakshmi N.; Sahin, Mehmet; Gopal, Naveen

2000-01-01

Helicopters in high-speed forward flight usually experience large regions of dynamic stall over the retreating side of the rotor disk. The rapid variations in the lift and pitching moments associated with the stall process can result in vibratory loads, and can cause fatigue and failure of pitch links. In some instances, the large time lag between the aerodynamic forces and the blade motion can trigger stall flutter. A number of techniques for the alleviation of dynamic stall have been proposed and studied by researchers. Passive and active control techniques have both been explored. Passive techniques include the use of high solidity rotors that reduce the lift coefficients of individual blades, leading edge slots and leading edge slats. Active control techniques include steady and unsteady blowing, and dynamically deformable leading edge (DDLE) airfoils. Considerable amount of experimental and numerical data has been collected on the effectiveness of these concepts. One concept that has not received as much attention is the drooped-leading edge airfoil idea. It has been observed in wind tunnel studies and flight tests that drooped leading edge airfoils can have a milder dynamic stall, with a significantly milder load hysteresis. Drooped leading edge airfoils may not, however, be suitable at other conditions, e.g. in hover, or in transonic flow. Work needs to be done on the analysis and design of drooped leading edge airfoils for efficient operation in a variety of flight regimes (hover, dynamic stall, and transonic flow). One concept that is worthy of investigation is the dynamically drooping airfoil, where the leading edge shape is changed roughly once-per-rev to mitigate the dynamic stall.

Effects of rainbow trout fry of a metals-contaminated diet of benthic invertebrates from the Clark Fork River, Montana

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

Woodward, D.F.; Brumbaugh, W.G.; DeLonay, A.J.

1994-01-01

The upper Clark Fork River in northwestern Montana has received mining wastes from the Butte and Anaconda areas since 1880. These wastes have contaminated areas of the river bed and floodplain with tailings and heavy metal sludge, resulting in elevated concentration of metals in surface water, sediments, and biota. Rainbow trout Oncorhynchus mykiss were exposed immediately after hatching for 91 d to cadmium, copper, lead, and zinc in water at concentrations simulating those in Clark Fork River. From exogenous feeding (21 d posthatch) through 91 d, fry were also fed benthic invertebrates from the Clark Fork River that contained elevatedmore » concentrations of arsenic, cadmium, copper, and lead. Evaluations of different combinations of diet and water exposure indicated diet-borne metals were more important than water-borne metals - at the concentrations we tested - in reducing survival and growth of rainbow trout. Whole-body metal concentrations ([mu]g/g, wet weight) at 91 d in fish fed Clark Fork invertebrates without exposure to Clark Fork water were arsenic, 1.4; cadmium, 0.16; and copper, 6.7. These were similar to concentrations found in Clark Fork River fishes. Livers from fish on the high-metals diets exhibited degenerative changes and generally lacked glycogen vacuolation. Indigenous Clark Fork River invertebrates provide a concentrated source of metals for accumulation into young fishes, and probably were the cause of decreased survival and growth of age-0 rainbow trout in our laboratory exposures. 30 refs., 8 figs., 4 tabs.« less

Experimental Investigation of Stall Cells on NACA0015 Airfoils

NASA Astrophysics Data System (ADS)

Dell'Orso, Haley

A particular type of 3-D separation, known as a stall cell, was investigated experimentally on two NACA0015 airfoils with aspect ratios of AR = 4 and 2.67. A parametric map of the angles of attack and Reynolds number conditions under which stall cells form was created using oil flow visualization. It was observed that stalls cells form naturally under specific conditions when the Reynolds number exceeds a critical Reynolds number, Re c ≥ Recrit. Based on the work of Weihs & Katz, the formation of a stall cell requires sufficient 3-dimensionality in the flow field. Next, full and partial span trips (composed of either zig-zag tape or an artificial step) were added to the airfoil and it was found that the introduction of additional 3-dimensional disturbances reduced the value of Recrit. For full-span step trips, where no additional 3-dimensionalities were introduced to the flow field, a stall cell was not formed at conditions where one was otherwise not present. However, a partial step trip did cause the formation of a stall cell (under specific conditions) through the introduction of three dimensionalities associated with the trip's ends. These results confirm that three dimensionalities need to be present in order for a stall cell to form. Flow field data were used to explore stall cell characteristics with and without external trips. Under conditions where a stall cell was present, two recirculation regions (i.e., stall cell foci) were observed, outboard of which flow abruptly reattached due to entrainment by the foci. Within the stall cell, flow was funneled away from the middle of the stall cell and into the associated focus point. In addition, at mid-span, the separated flow rotated about the spanwise direction. Outboard, the structure also began to rotate about the chord-normal direction; near the foci, all rotation occurred about the chord-normal direction. The fluctuating flow field was also considered, and elevated levels of chordwise (u'u'/Uinfinity 2) and spanwise (w¯'w¯'/Uinfinity 2) components of the normal stress were observed when stall cells were present, concentrated near the foci. Finally, a partial-span dynamic oscillating step trip was incorporated into the NACA0015 model with AR = 2.67. Initially, the actuator was driven by a square wave and the transitory behavior of flow field was explored as the trip moved from the extended to the flush position. It was shown that during this motion the flow was temporarily attached before settling into a state where a small cell was present. The intermediate reattachment was due to the natural oscillations of the actuator at its resonant frequency (ƒres = 100 Hz). This result suggested that actuating the trip at a frequency that is associated with the separated shear layer, which also coincided with the resonance frequency of the actuator, might enable mitigation of the stall cell. Therefore, the trip was driven using a sine wave with ƒ = 100 Hz (corresponding to a dimensionless frequency St = 0.35) when the airfoil was set at alpha = 13.4° and U infinity = 55 m/s, and it caused nearly complete reattachment of a 3-D separated region. At alpha = 16°, the size of the stall cell was very large and extended throughout most of the span when the trip was in the flush position; thus, the dynamic motion of the trip only affected the separated flow directly downstream of the actuator, which was reduced in size and magnitude. Phase-averaged data were also acquired, and it was shown that, during the periodic motion of the trip, coherent vortices were formed and advected downstream as they grew in size. This resulted, in a time average sense, in tilting of the flow towards the surface. However, the reattachment was unsteady.

Conical Magnetic Bearings Developed for Active Stall Control in Gas Turbine Engines

NASA Technical Reports Server (NTRS)

Trudell, Jeffrey J.; Kascak, Albert F.; Provenza, Andrew J.; Buccieri, Carl J.

2004-01-01

Active stall control is a current research area at the NASA Glenn Research Center that offers a great benefit in specific fuel consumption by allowing the gas turbine to operate beyond the onset of stall. Magnetic bearings are being investigated as a new method to perform active stall control. This enabling global aviation safety technology would result in improved fuel efficiency and decreased carbon dioxide emissions, as well as improve safety and reliability by eliminating oil-related delays and failures of engine components, which account for 40 percent of the commercial aircraft departure delays. Active stall control works by perturbing the flow in front of the compressor stage such that it cancels the pressure wave, which causes the compressor to go into stall. Radial magnetic bearings are able to whirl the shaft so that variations in blade tip leakage would flow upstream causing a perturbation wave that could cancel the rotating stall cell. Axial or thrust magnetic bearings cannot be used to cancel the surge mode in the compressor because they have a very low bandwidth and thus cannot modulate at a high enough frequency. Frequency response is limited because the thrust runner cannot be laminated. To improve the bandwidth of magnetic thrust bearings, researchers must use laminations to suppress the eddy currents. A conical magnetic bearing can be laminated, resulting in increased bandwidth in the axial direction. In addition, this design can produce both radial and thrust force in a single bearing, simplifying the installation. The proposed solution combines the radial and thrust bearing into one design that can be laminated--a conical magnetic bearing. The new conical magnetic bearing test rig, funded by a Glenn fiscal year 2002 Director's Discretionary Fund, was needed because none of the existing rigs has an axial degree of freedom. The rotor bearing configuration will simulate that of the main shaft on a gas turbine engine. One conical magnetic bearing replaces the ball bearing in front of the compressor, and the second replaces the roller bearing behind the burner. The rig was made operational to 10,000 rpm under Smart Efficient Components funding, and both position and current adaptive vibration control have been demonstrated. Upon program completion, recommendations will be made as to the efficacy of the conical magnetic bearing for active stall control.

Handling qualities related to stall/spin accidents of supersonic fighter aircraft

NASA Technical Reports Server (NTRS)

Anderson, S. B.

1984-01-01

This paper reviews the handling qualities which influence the high angle of attack (AOA) behavior of supersonic fighter aircraft in order to obtain a clearer understanding of the causes of stall/spin accidents. The results show that, because modern fighters suffer more serious consequences when control is lost, good handling qualities are essential for safe operation at high AOA. Relaxed static stability used on some fighter aircraft can result in control problems at high AOA owing to inertia coupling and the difficulty of a recovery from a deep stall. Indications are that the use of departure/spin resistance and an automatic spin prevention system will greatly improve the safety record for modern supersonic fighters.

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

PubMed Central

Hegde, Muralidhar L.; Hegde, Pavana M.; Bellot, Larry J.; Mandal, Santi M.; Hazra, Tapas K.; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E.; Mitra, Sankar

2013-01-01

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function. PMID:23898192

Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication.

PubMed

Ramadan, Kristijan; Halder, Swagata; Wiseman, Katherine; Vaz, Bruno

2017-02-01

Genome amplification (DNA synthesis) is one of the most demanding cellular processes in all proliferative cells. The DNA replication machinery (also known as the replisome) orchestrates genome amplification during S-phase of the cell cycle. Genetic material is particularly vulnerable to various events that can challenge the replisome during its assembly, activation (firing), progression (elongation) and disassembly from chromatin (termination). Any disturbance of the replisome leads to stalling of the DNA replication fork and firing of dormant replication origins, a process known as DNA replication stress. DNA replication stress is considered to be one of the main causes of sporadic cancers and other pathologies related to tissue degeneration and ageing. The mechanisms of replisome assembly and elongation during DNA synthesis are well understood. However, once DNA synthesis is complete, the process of replisome disassembly, and its removal from chromatin, remains unclear. In recent years, a growing body of evidence has alluded to a central role in replisome regulation for the ubiquitin-dependent protein segregase p97, also known as valosin-containing protein (VCP) in metazoans and Cdc48 in lower eukaryotes. By orchestrating the spatiotemporal turnover of the replisome, p97 plays an essential role in DNA replication. In this review, we will summarise our current knowledge about how p97 controls the replisome from replication initiation, to elongation and finally termination. We will also further examine the more recent findings concerning the role of p97 and how mutations in p97 cofactors, also known as adaptors, cause DNA replication stress induced genomic instability that leads to cancer and accelerated ageing. To our knowledge, this is the first comprehensive review concerning the mechanisms involved in the regulation of DNA replication by p97.

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins*

PubMed Central

Crowder, Justin J.; Geigges, Marco; Gibson, Ryan T.; Fults, Eric S.; Buchanan, Bryce W.; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G.; Rubenstein, Eric M.

2015-01-01

Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane. PMID:26055716

Nonlinear control of rotating stall and surge with axisymmetric bleed and air injection on axial flow compressors

NASA Astrophysics Data System (ADS)

Yeung, Chung-Hei (Simon)

The study of compressor instabilities in gas turbine engines has received much attention in recent years. In particular, rotating stall and surge are major causes of problems ranging from component stress and lifespan reduction to engine explosion. In this thesis, modeling and control of rotating stall and surge using bleed valve and air injection is studied and validated on a low speed, single stage, axial compressor at Caltech. Bleed valve control of stall is achieved only when the compressor characteristic is actuated, due to the fast growth rate of the stall cell compared to the rate limit of the valve. Furthermore, experimental results show that the actuator rate requirement for stall control is reduced by a factor of fourteen via compressor characteristic actuation. Analytical expressions based on low order models (2--3 states) and a high fidelity simulation (37 states) tool are developed to estimate the minimum rate requirement of a bleed valve for control of stall. A comparison of the tools to experiments show a good qualitative agreement, with increasing quantitative accuracy as the complexity of the underlying model increases. Air injection control of stall and surge is also investigated. Simultaneous control of stall and surge is achieved using axisymmetric air injection. Three cases with different injector back pressure are studied. Surge control via binary air injection is achieved in all three cases. Simultaneous stall and surge control is achieved for two of the cases, but is not achieved for the lowest authority case. This is consistent with previous results for control of stall with axisymmetric air injection without a plenum attached. Non-axisymmetric air injection control of stall and surge is also studied. Three existing control algorithms found in literature are modeled and analyzed. A three-state model is obtained for each algorithm. For two cases, conditions for linear stability and bifurcation criticality on control of rotating stall are derived and expressed in terms of implementation-oriented variables such as number of injectors. For the third case, bifurcation criticality conditions are not obtained due to complexity, though linear stability property is derived. A theoretical comparison between the three algorithms is made, via the use of low-order models, to investigate pros and cons of the algorithms in the context of operability. The effects of static distortion on the compressor facility at Caltech is characterized experimentally. Results consistent with literature are obtained. Simulations via a high fidelity model (34 states) are also performed and show good qualitative as well as quantitative agreement to experiments. A non-axisymmetric pulsed air injection controller for stall is shown to be robust to static distortion.

Effect of free stall surface on daily activity patterns in dairy cows with relevance to lameness prevalence.

PubMed

Cook, N B; Bennett, T B; Nordlund, K V

2004-09-01

Differences in behavior of nonlame cows, slightly lame cows, and moderately lame cows in 6 free stall barns with sand bedding (SAND) vs. 6 free stall barns with rubber-crumb geotextile mattress surfaces (MAT) were documented in Wisconsin dairy herds. All lactating cows in the 12 herds were observed and given a locomotion score based on a 4-point scale: 1 = nonlame, 2 = slightly lame, 3 = moderately lame, and 4 = severely lame. Herd least square means +/-SE for prevalence of clinical lameness (locomotion scores = 3 and 4) were 11.1 vs. 24.0 +/- 1.7% for herds using SAND vs. MAT surfaces, respectively. Subsets of 10 cows per herd with locomotion scores of 1 to 3 were observed via video cameras for 24-h periods. Cows in MAT herds spent more time standing in free stalls per day than cows in SAND herds. Differences in standing times were 0.73 h/d for cows that were not lame, 2.32 h/d for cows that were slightly lame, and 4.31 h/d for cows that were moderately lame in MAT herds compared with equivalent cows in SAND herds. In MAT herds, the increase in time spent standing in the stall in moderately lame cows was associated with a significant reduction in stall use sessions per day, which impacted daily lying time. Although cause and effect are not clear, these findings have implications for housing, comfort, and care of cows in dairy herds with different types of free stall surfaces.

Genetic spell-checking: gene editing using single-stranded DNA oligonucleotides.

PubMed

Rivera-Torres, Natalia; Kmiec, Eric B

2016-02-01

Single-stranded oligonucleotides (ssODNs) can be used to direct the exchange of a single nucleotide or the repair of a single base within the coding region of a gene in a process that is known, generically, as gene editing. These molecules are composed of either all DNA residues or a mixture of RNA and DNA bases and utilize inherent metabolic functions to execute the genetic alteration within the context of a chromosome. The mechanism of action of gene editing is now being elucidated as well as an understanding of its regulatory circuitry, work that has been particularly important in establishing a foundation for designing effective gene editing strategies in plants. Double-strand DNA breakage and the activation of the DNA damage response pathway play key roles in determining the frequency with which gene editing activity takes place. Cellular regulators respond to such damage and their action impacts the success or failure of a particular nucleotide exchange reaction. A consequence of such activation is the natural slowing of replication fork progression, which naturally creates a more open chromatin configuration, thereby increasing access of the oligonucleotide to the DNA template. Herein, how critical reaction parameters influence the effectiveness of gene editing is discussed. Functional interrelationships between DNA damage, the activation of DNA response pathways and the stalling of replication forks are presented in detail as potential targets for increasing the frequency of gene editing by ssODNs in plants and plant cells. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Prediction of unsteady airfoil flows at large angles of incidence

NASA Technical Reports Server (NTRS)

Cebeci, Tuncer; Jang, H. M.; Chen, H. H.

1992-01-01

The effect of the unsteady motion of an airfoil on its stall behavior is of considerable interest to many practical applications including the blades of helicopter rotors and of axial compressors and turbines. Experiments with oscillating airfoils, for example, have shown that the flow can remain attached for angles of attack greater than those which would cause stall to occur in a stationary system. This result appears to stem from the formation of a vortex close to the surface of the airfoil which continues to provide lift. It is also evident that the onset of dynamic stall depends strongly on the airfoil section, and as a result, great care is required in the development of a calculation method which will accurately predict this behavior.

Understanding DNA Repair in Hyperthermophilic Archaea: Persistent Gaps and Other Reasons to Focus on the Fork

PubMed Central

Grogan, Dennis W.

2015-01-01

Although hyperthermophilic archaea arguably have a great need for efficient DNA repair, they lack members of several DNA repair protein families broadly conserved among bacteria and eukaryotes. Conversely, the putative DNA repair genes that do occur in these archaea often do not generate the expected phenotype when deleted. The prospect that hyperthermophilic archaea have some unique strategies for coping with DNA damage and replication errors has intellectual and technological appeal, but resolving this question will require alternative coping mechanisms to be proposed and tested experimentally. This review evaluates a combination of four enigmatic properties that distinguishes the hyperthermophilic archaea from all other organisms: DNA polymerase stalling at dU, apparent lack of conventional NER, lack of MutSL homologs, and apparent essentiality of homologous recombination proteins. Hypothetical damage-coping strategies that could explain this set of properties may provide new starting points for efforts to define how archaea differ from conventional models of DNA repair and replication fidelity. PMID:26146487

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

PubMed

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

2015-11-02

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

ATM protein is deficient in over 40% of lung adenocarcinomas.

PubMed

Villaruz, Liza C; Jones, Helen; Dacic, Sanja; Abberbock, Shira; Kurland, Brenda F; Stabile, Laura P; Siegfried, Jill M; Conrads, Thomas P; Smith, Neil R; O'Connor, Mark J; Pierce, Andrew J; Bakkenist, Christopher J

2016-09-06

Lung cancer is the leading cause of cancer-related mortality in the USA and worldwide, and of the estimated 1.2 million new cases of lung cancer diagnosed every year, over 30% are lung adenocarcinomas. The backbone of 1st-line systemic therapy in the metastatic setting, in the absence of an actionable oncogenic driver, is platinum-based chemotherapy. ATM and ATR are DNA damage signaling kinases activated at DNA double-strand breaks (DSBs) and stalled and collapsed replication forks, respectively. ATM protein is lost in a number of cancer cell lines and ATR kinase inhibitors synergize with cisplatin to resolve xenograft models of ATM-deficient lung cancer. We therefore sought to determine the frequency of ATM loss in a tissue microarray (TMA) of lung adenocarcinoma. Here we report the validation of a commercial antibody (ab32420) for the identification of ATM by immunohistochemistry and estimate that 61 of 147 (41%, 95% CI 34%-50%) cases of lung adenocarcinoma are negative for ATM protein expression. As a positive control for ATM staining, nuclear ATM protein was identified in stroma and immune infiltrate in all evaluable cases. ATM loss in lung adenocarcinoma was not associated with overall survival. However, our preclinical findings in ATM-deficient cell lines suggest that ATM could be a predictive biomarker for synergy of an ATR kinase inhibitor with standard-of-care cisplatin. This could improve clinical outcome in 100,000's of patients with ATM-deficient lung adenocarcinoma every year.

ATM protein is deficient in over 40% of lung adenocarcinomas

PubMed Central

Villaruz, Liza C.; Jones, Helen; Dacic, Sanja; Abberbock, Shira; Kurland, Brenda F.; Stabile, Laura P.; Siegfried, Jill M.; Conrads, Thomas P.; Smith, Neil R.; O'Connor, Mark J.; Pierce, Andrew J.; Bakkenist, Christopher J.

2016-01-01

Lung cancer is the leading cause of cancer-related mortality in the USA and worldwide, and of the estimated 1.2 million new cases of lung cancer diagnosed every year, over 30% are lung adenocarcinomas. The backbone of 1st-line systemic therapy in the metastatic setting, in the absence of an actionable oncogenic driver, is platinum-based chemotherapy. ATM and ATR are DNA damage signaling kinases activated at DNA double-strand breaks (DSBs) and stalled and collapsed replication forks, respectively. ATM protein is lost in a number of cancer cell lines and ATR kinase inhibitors synergize with cisplatin to resolve xenograft models of ATM-deficient lung cancer. We therefore sought to determine the frequency of ATM loss in a tissue microarray (TMA) of lung adenocarcinoma. Here we report the validation of a commercial antibody (ab32420) for the identification of ATM by immunohistochemistry and estimate that 61 of 147 (41%, 95% CI 34%-50%) cases of lung adenocarcinoma are negative for ATM protein expression. As a positive control for ATM staining, nuclear ATM protein was identified in stroma and immune infiltrate in all evaluable cases. ATM loss in lung adenocarcinoma was not associated with overall survival. However, our preclinical findings in ATM-deficient cell lines suggest that ATM could be a predictive biomarker for synergy of an ATR kinase inhibitor with standard-of-care cisplatin. This could improve clinical outcome in 100,000's of patients with ATM-deficient lung adenocarcinoma every year. PMID:27259260

Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights

PubMed Central

Thompson, Larry H.; Hinz, John M.

2009-01-01

The Fanconi anemia (FA) molecular network consists of 15 “FANC” proteins, of which 13 are associated with mutations in patients with this cancer-prone chromosome instability disorder. Whereas historically the common phenotype associated with FA mutations is marked sensitivity to DNA interstrand crosslinking agents, the literature supports a more global role for FANC proteins in coping with diverse stresses encountered by replicative polymerases. We have attempted to reconcile and integrate numerous observations into a model in which FANC proteins coordinate the following physiological events during DNA crosslink repair: (a) activating a FANCM-ATR-dependent S-phase checkpoint; (b) mediating enzymatic replication-fork breakage and crosslink unhooking; (c) filling the resulting gap by translesion synthesis (TLS) by error-prone polymerase(s); and (d) restoring the resulting one-ended double-strand break by homologous recombination repair (HRR). The FANC core subcomplex (FANCA, B, C, E, F, G, L, FAAP100) promotes TLS for both crosslink and non-crosslink damage such as spontaneous oxidative base damage, UV-C photoproducts, and alkylated bases. TLS likely helps prevent stalled replication forks from breaking, thereby maintaining chromosome continuity. Diverse DNA damages and replication inhibitors result in monoubiquitination of the FANCD2-FANCI complex by the FANCL ubiquitin ligase activity of the core subcomplex upon its recruitment to chromatin by the FANCM-FAAP24 heterodimeric translocase. We speculate that this translocase activity acts as the primary damage sensor and helps remodel blocked replication forks to facilitate checkpoint activation and repair. Monoubiquitination of FANCD2-FANCI is needed for promoting HRR, in which the FANCD1/BRCA2 and FANCN/PALB2 proteins act at an early step. We conclude that the core subcomplex is required for both TLS and HRR occurring separately for non-crosslink damages and for both events during crosslink repair. The FANCJ/BRIP1/BACH1 helicase functions in association with BRCA1 and may remove structural barriers to replication, such as guanine quadruplex structures, and/or assist in crosslink unhooking. PMID:19622404

Band-pass filtering algorithms for adaptive control of compressor pre-stall modes in aircraft gas-turbine engine

NASA Astrophysics Data System (ADS)

Kuznetsova, T. A.

2018-05-01

The methods for increasing gas-turbine aircraft engines' (GTE) adaptive properties to interference based on empowerment of automatic control systems (ACS) are analyzed. The flow pulsation in suction and a discharge line of the compressor, which may cause the stall, are considered as the interference. The algorithmic solution to the problem of GTE pre-stall modes’ control adapted to stability boundary is proposed. The aim of the study is to develop the band-pass filtering algorithms to provide the detection functions of the compressor pre-stall modes for ACS GTE. The characteristic feature of pre-stall effect is the increase of pressure pulsation amplitude over the impeller at the multiples of the rotor’ frequencies. The used method is based on a band-pass filter combining low-pass and high-pass digital filters. The impulse response of the high-pass filter is determined through a known low-pass filter impulse response by spectral inversion. The resulting transfer function of the second order band-pass filter (BPF) corresponds to a stable system. The two circuit implementations of BPF are synthesized. Designed band-pass filtering algorithms were tested in MATLAB environment. Comparative analysis of amplitude-frequency response of proposed implementation allows choosing the BPF scheme providing the best quality of filtration. The BPF reaction to the periodic sinusoidal signal, simulating the experimentally obtained pressure pulsation function in the pre-stall mode, was considered. The results of model experiment demonstrated the effectiveness of applying band-pass filtering algorithms as part of ACS to identify the pre-stall mode of the compressor for detection of pressure fluctuations’ peaks, characterizing the compressor’s approach to the stability boundary.

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation.

PubMed

Artieri, Carlo G; Fraser, Hunter B

2014-12-01

The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling. © 2014 Artieri and Fraser; Published by Cold Spring Harbor Laboratory Press.

Pilot Human Factors in Stall/Spin Accidents of Supersonic Fighter Aircraft

NASA Technical Reports Server (NTRS)

Anderson, S. B.; Enevoldson, E. K.; Nguyen, L. T.

1983-01-01

A study has been made of pilot human factors related to stall/spin accidents of supersonic fighter aircraft. The military specifications for flight at high angles of attack are examined. Several pilot human factors problems related to stall/spin are discussed. These problems include (1) unsatisfactory nonvisual warning cues; (2) the inability of the pilot to quickly determine if the aircraft is spinning out of control, or to recognize the type of spin; (3) the inability of the pilot to decide on and implement the correct spin recovery technique; (4) the inability of the pilot to move, caused by high angular rotation; and (5) the tendency of pilots to wait too long in deciding to abandon the irrecoverable aircraft. Psycho-physiological phenomena influencing pilot's behavior in stall/spin situations include (1) channelization of sensory inputs, (2) limitations in precisely controlling several muscular inputs, (3) inaccurate judgment of elapsed time, and (4) disorientation of vestibulo-ocular inputs. Results are given of pilot responses to all these problems in the F14A, F16/AB, and F/A-18A aircraft. The use of departure spin resistance and automatic spin prevention systems incorporated on recent supersonic fighters are discussed. These systems should help to improve the stall/spin accident record with some compromise in maneuverability.

Study on casing treatment and stator matching on multistage fan

NASA Astrophysics Data System (ADS)

Wu, Chuangliang; Yuan, Wei; Deng, Zhe

2017-10-01

Casing treatments are required for expanding the stall margin of multi-stage high-load turbofans designed with high blade-tip Mach numbers and high leakage flow. In the case of a low mass flow, the casing treatment effectively reduces the blockages caused by the leakage flow and enlarges the stall margin. However, in the case of a high mass flow, the casing treatment affects the overall flow capacity of the fan, the thrust when operating at the high speeds usually required by design-point specifications. Herein, we study a two-stage high-load fan with three-dimensional numerical simulations. We use the simulation results to propose a scheme that enlarges the stall margin of multistage high-load fans without sacrificing the flow capacity when operating with a large mass flow. Furthermore, a circumferential groove casing treatment is used and adjustments are made to the upstream stator angle to match the casing treatment. The stall margin is thus increased to 16.3%, with no reduction in the maximum mass flow rate or the design thrust performance.

Pitching effect on transonic wing stall of a blended flying wing with low aspect ratio

NASA Astrophysics Data System (ADS)

Tao, Yang; Zhao, Zhongliang; Wu, Junqiang; Fan, Zhaolin; Zhang, Yi

2018-05-01

Numerical simulation of the pitching effect on transonic wing stall of a blended flying wing with low aspect ratio was performed using improved delayed detached eddy simulation (IDDES). To capture the discontinuity caused by shock wave, a second-order upwind scheme with Roe’s flux-difference splitting is introduced into the inviscid flux. The artificial dissipation is also turned off in the region where the upwind scheme is applied. To reveal the pitching effect, the implicit approximate-factorization method with sub-iterations and second-order temporal accuracy is employed to avoid the time integration of the unsteady Navier-Stokes equations solved by finite volume method at Arbitrary Lagrange-Euler (ALE) form. The leading edge vortex (LEV) development and LEV circulation of pitch-up wings at a free-stream Mach number M = 0.9 and a Reynolds number Re = 9.6 × 106 is studied. The Q-criterion is used to capture the LEV structure from shear layer. The result shows that a shock wave/vortex interaction is responsible for the vortex breakdown which eventually causes the wing stall. The balance of the vortex strength and axial flow, and the shock strength, is examined to provide an explanation of the sensitivity of the breakdown location. Pitching motion has great influence on shock wave and shock wave/vortex interactions, which can significantly affect the vortex breakdown behavior and wing stall onset of low aspect ratio blended flying wing.

An archival analysis of stall warning system effectiveness during airborne icing encounters

NASA Astrophysics Data System (ADS)

Maris, John Michael

An archival study was conducted to determine the influence of stall warning system performance on aircrew decision-making outcomes during airborne icing encounters. A Conservative Icing Response Bias (CIRB) model was developed to explain the historical variability in aircrew performance in the face of airframe icing. The model combined Bayes' Theorem with Signal Detection Theory (SDT) concepts to yield testable predictions that were evaluated using a Binary Logistic Regression (BLR) multivariate technique applied to two archives: the NASA Aviation Safety Reporting System (ASRS) incident database, and the National Transportation Safety Board (NTSB) accident databases, both covering the period January 1, 1988 to October 2, 2015. The CIRB model predicted that aircrew would experience more incorrect response outcomes in the face of missed stall warnings than with stall warning False Alarms. These predicted outcomes were observed at high significance levels in the final sample of 132 NASA/NTSB cases. The CIRB model had high sensitivity and specificity, and explained 71.5% (Nagelkerke R2) of the variance of aircrew decision-making outcomes during the icing encounters. The reliability and validity metrics derived from this study suggest indicate that the findings are generalizable to the population of U.S. registered turbine-powered aircraft. These findings suggest that icing-related stall events could be reduced if the incidence of stall warning Misses could be minimized. Observed stall warning Misses stemmed from three principal causes: aerodynamic icing effects, which reduced the stall angle-of-attack (AoA) to below the stall warning calibration threshold; tail stalls, which are not monitored by contemporary protection systems; and icing-induced system issues (such as frozen pitot tubes), which compromised stall warning system effectiveness and airframe envelope protections. Each of these sources of missed stall warnings could be addressed by Aerodynamic Performance Monitoring (APM) systems that directly measure the boundary layer airflow adjacent to the affected aerodynamic surfaces, independent of other aircraft stall protection, air data, and AoA systems. In addition to investigating APM systems, measures should also be taken to include the CIRB phenomenon in aircrew training to better prepare crews to cope with airborne icing encounters. The SDT/BLR technique would allow the forecast gains from these improved systems and training processes to be evaluated objectively and quantitatively. The SDT/BLR model developed for this study has broad application outside the realm of airborne icing. The SDT technique has been extensively validated by prior research, and the BLR is a very robust multivariate technique. Combined, they could be applied to evaluate high order constructs (such as stall awareness for this study), in complex and dynamic environments. The union of SDT and BLR reduces the modeling complexities for each variable into the four binary SDT categories of Hit, Miss, False Alarm, and Correct Rejection, which is the optimum format for the BLR. Despite this reductionist approach to complex situations, the method has demonstrated very high statistical and practical significance, as well as excellent predictive power, when applied to the airborne icing scenario.

Iced Aircraft Flight Data for Flight Simulator Validation

NASA Technical Reports Server (NTRS)

Ratvasky, Thomas P.; Blankenship, Kurt; Rieke, William; Brinker, David J.

2003-01-01

NASA is developing and validating technology to incorporate aircraft icing effects into a flight training device concept demonstrator. Flight simulation models of a DHC-6 Twin Otter were developed from wind tunnel data using a subscale, complete aircraft model with and without simulated ice, and from previously acquired flight data. The validation of the simulation models required additional aircraft response time histories of the airplane configured with simulated ice similar to the subscale model testing. Therefore, a flight test was conducted using the NASA Twin Otter Icing Research Aircraft. Over 500 maneuvers of various types were conducted in this flight test. The validation data consisted of aircraft state parameters, pilot inputs, propulsion, weight, center of gravity, and moments of inertia with the airplane configured with different amounts of simulated ice. Emphasis was made to acquire data at wing stall and tailplane stall since these events are of primary interest to model accurately in the flight training device. Analyses of several datasets are described regarding wing and tailplane stall. Key findings from these analyses are that the simulated wing ice shapes significantly reduced the C , max, while the simulated tail ice caused elevator control force anomalies and tailplane stall when flaps were deflected 30 deg or greater. This effectively reduced the safe operating margins between iced wing and iced tail stall as flap deflection and thrust were increased. This flight test demonstrated that the critical aspects to be modeled in the icing effects flight training device include: iced wing and tail stall speeds, flap and thrust effects, control forces, and control effectiveness.

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

PubMed Central

Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

Evaluation of range and distortion tolerance for high Mach number transonic fan stages. Task 2: Performance of a 1500-foot-per-second tip speed transonic fan stage with variable geometry inlet guide vanes and stator

NASA Technical Reports Server (NTRS)

Bilwakesh, K. R.; Koch, C. C.; Prince, D. C.

1972-01-01

A 0.5 hub/tip radius ratio compressor stage consisting of a 1500 ft/sec tip speed rotor, a variable camber inlet guide vane and a variable stagger stator was designed and tested with undistorted inlet flow, flow with tip radial distortion, and flow with 90 degrees, one-per-rev, circumferential distortion. At the design speed and design IGV and stator setting the design stage pressure ratio was achieved at a weight within 1% of the design flow. Analytical results on rotor tip shock structure, deviation angle and part-span shroud losses at different operating conditions are presented. The variable geometry blading enabled efficient operation with adequate stall margin at the design condition and at 70% speed. Closing the inlet guide vanes to 40 degrees changed the speed-versus-weight flow relationship along the stall line and thus provided the flexibility of operation at off-design conditions. Inlet flow distortion caused considerable losses in peak efficiency, efficiency on a constant throttle line through design pressure ratio at design speed, stall pressure ratio, and stall margin at the 0 degrees IGV setting and high rotative speeds. The use of the 40 degrees inlet guide vane setting enabled partial recovery of the stall margin over the standard constant throttle line.

Numerical analysis of rotating stall instabilities of a pump- turbine in pump mode

NASA Astrophysics Data System (ADS)

Xia, L. S.; Cheng, Y. G.; Zhang, X. X.; Yang, J. D.

2014-03-01

Rotating stall may occur at part load flow of a pump-turbine in pump mode. Unstable flow structures developing under stall condition can lead to a sudden drop of efficiency, high dynamic load and even cavitation. CFD simulations on a pump-turbine model in pump mode were carried out to reveal the onset and developed mechanisms of these unstable flow phenomena at part load. The simulation results of energy-discharge and efficiency characteristics are in good agreement with those obtained by experiments. The more deviate from design conditions with decreasing flow rate, the more flow separations within the vanes. Under specific conditions, four stationary separation zones begin to progress on the circumference, rotating at a fraction of the impeller rotation rate. Rotating stalls lead to the flow in the vane diffuser channels alternating between outward jet flow and blockage. Strong jets impact the spiral casing wall causing high pressure pulsations. Severe separations of the stall cells disturb the flow inducing periodical large amplitude pressure fluctuations, of which the intensity at different span wise of the guide vanes is different. The enforced rotating nonuniform pressure distributions on the circumference lead to dynamic uniform forces on the impeller and guide vanes. The results show that the CFD simulations are capable to gain the complicated flow structure information for analysing the unstable characteristics of the pump mode at part load.

Study of Convective Flow Effects in Endwall Casing Treatments in Transonic Compressor Rotors

NASA Technical Reports Server (NTRS)

Hah, Chunill; Mueller, Martin W.; Schiffer, Heinz-Peter

2012-01-01

The unsteady convective flow effects in a transonic compressor rotor with a circumferential-groove casing treatment are investigated in this paper. Experimental results show that the circumferential-groove casing treatment increases the compressor stall margin by almost 50% for the current transonic compressor rotor. Steady flow simulation of the current casing treatment, however, yields only a 15% gain in stall margin. The flow field at near-stall operation is highly unsteady due to several self-induced flow phenomena. These include shock oscillation, vortex shedding at the trailing edge, and interaction between the passage shock and the tip clearance vortex. The primary focus of the current investigation is to assess the effects of flow unsteadiness and unsteady flow convection on the circumferential-groove casing treatment. Unsteady Reynolds-averaged Navier-Stokes (URANS) and Large Eddy Simulation (LES) techniques were applied in addition to steady Reynolds-averaged Navier-Stokes (RANS) to simulate the flow field at near-stall operation and to determine changes in stall margin. The current investigation reveals that unsteady flow effects are as important as steady flow effects on the performance of the circumferential grooves casing treatment in extending the stall margin of the current transonic compressor rotor. The primary unsteady flow mechanism is unsteady flow injection from the grooves into the main flow near the casing. Flows moving into and out of the grooves are caused due to local pressure difference near the grooves. As the pressure field becomes transient due to self-induced flow oscillation, flow injection from the grooves also becomes unsteady. The unsteady flow simulation shows that this unsteady flow injection from the grooves is substantial and contributes significantly to extending the compressor stall margin. Unsteady flows into and out of the grooves have as large a role as steady flows in the circumferential grooves. While the circumferential-groove casing treatment seems to be a steady flow device, unsteady flow effects should be included to accurately assess its performance as the flow is transient at near-stall operation.

FANCJ promotes DNA synthesis through G-quadruplex structures

PubMed Central

Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

2014-01-01

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes. PMID:25193968

Complex mosaic CDKL5 deletion with two distinct mutant alleles in a 4-year-old girl.

PubMed

Boutry-Kryza, Nadia; Ville, Dorothée; Labalme, Audrey; Calender, Alain; Dupont, Jean-Michel; Touraine, Renaud; Edery, Patrick; des Portes, Vincent; Sanlaville, Damien; Lesca, Gaetan

2014-08-01

Mutations of the CDKL5 gene cause early epileptic encephalopathy. Patients manifest refractory epilepsy, beginning before the age of 3 months, which is associated with severe psychomotor delay and features that overlap with Rett syndrome. We report here a patient with mosaicism for CDKL5 exonic deletion, with the presence of two mutant alleles. The affected 4-year-old girl presented with infantile spasms, beginning at the age of 9 months, but subsequent progression of the disease was consistent with the classical CDKL5-related phenotype. A deletion of exons 17 and 18 was suspected on the basis of Multiplex Ligation Probe Amplification analysis, but unexpected results for cDNA analysis, which showed the presence of an abnormal transcript with the deletion of exon 18 only, led us to suspect that two distinct events might have occurred. We used custom array-CGH to determine the size and breakpoints of these deletions. Exon 18 was deleted from one of the abnormal alleles, and exon 17 was deleted from the other. A Fork Stalling and Template Switching (FoSTeS) mechanism was proposed to explain the two events, given the presence of regions of microhomology at the breakpoints. We propose here an original involvement of the FoSTeS mechanism to explain the co-occurrence of these two events in the CDKL5 gene in a single patient. This patient highlights the difficulties involved in the detection of such abnormalities, particularly when they occur in a mosaic state and involve two distinct mutational events in a single gene. © 2014 Wiley Periodicals, Inc.

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair*

PubMed Central

Bélanger, François; Angers, Jean-Philippe; Fortier, Émile; Hammond-Martel, Ian; Costantino, Santiago; Drobetsky, Elliot; Wurtele, Hugo

2016-01-01

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1–3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response. PMID:26578521

Stall Flutter Control of a Smart Blade Section Undergoing Asymmetric Limit Oscillations

DOE PAGES

Li, Nailu; Balas, Mark J.; Nikoueeyan, Pourya; ...

2016-01-01

Stall flutter is an aeroelastic phenomenon resulting in unwanted oscillatory loads on the blade, such as wind turbine blade, helicopter rotor blade, and other flexible wing blades. While the stall flutter and related aeroelastic control have been studied theoretically and experimentally, microtab control of asymmetric limit cycle oscillations (LCOs) in stall flutter cases has not been generally investigated. This paper presents an aeroservoelastic model to study the microtab control of the blade section undergoing moderate stall flutter and deep stall flutter separately. The effects of different dynamic stall conditions and the consequent asymmetric LCOs for both stall cases are simulatedmore » and analyzed. Then, for the design of the stall flutter controller, the potential sensor signal for the stall flutter, the microtab control capability of the stall flutter, and the control algorithm for the stall flutter are studied. Lastly, the improvement and the superiority of the proposed adaptive stall flutter controller are shown by comparison with a simple stall flutter controller.« less

Factors affecting stall use for different freestall bases.

PubMed

Wagner-Storch, A M; Palmer, R W; Kammel, D W

2003-06-01

The objective of this study was to compare stall use (stall occupancy and cow position) by barn side for factors affecting stall use. A closed circuit television system recorded stall use four times per day for a 9-mo period starting May 9, 2001. Six factors were analyzed: stall base, distance to water, stall location within stall base section, stall location within barn, inside barn temperature, and length of time cows were exposed to stall bases. Two barn sides with different stocking densities were analyzed: low (66%), with cows milked by robotic milker; and high (100%), with cows milked 2X in parlor. Six stall base types were tested: two mattresses, a waterbed, a rubber mat, concrete, and sand (high side only). The base types were grouped 3 to 7 stalls/section and randomly placed in each row. Cows spent more time in mattress-based stalls, but the highest percentage lying was in sand-based stalls. The following significant stall occupancy percentages were found: sand had the highest percentage of cows lying on the high stocking density side (69%), followed by mattress type 1 (65%) > mattress type 2 (57%) > waterbed (45%) > rubber mat (33%) > concrete (23%). Mattress type 1 had the highest percentage stalls occupied (88%), followed by mattress type 2 (84%) > sand (79%) > soft rubber mat (65%) > waterbed (62%) > concrete (39%). On the low stocking rate side, mattress type 1 had the highest percentage cows lying (45%) and occupied (59.6%), followed by mattress type 2 > waterbed > soft rubber mat > concrete. Cow lying and stalls occupied percentages were highest for stalls 1) not at the end of a section, and 2) on the outside row, and varied by base type for time cows exposed to stalls and inside barn temperature. Lying and occupied percentages were different for different mattress types. The percentage of stalls with cows standing was higher for mat and mattress-based stalls. Results show mattress type 1 and sand to be superior and rubber mats and concrete inferior stall bases.

Future Jet Technologies. Part B. F-35 Future Risks v. JS-Education of Pilots & Engineers

NASA Astrophysics Data System (ADS)

Gal-Or, Benjamin

2011-09-01

Design of “Next-Generation” airframes based on supermarket-jet-engine-components is nowadays passé. A novel integration methodology [Gal-Or, “Editorial-Review, Part A”, 2011, Gal-Or, “Vectored Propulsion, Supermaneuverability and Robot Aircraft”, Springer Verlag, Gal-Or, Int'l. J. of Thermal and Fluid Sciences 7: 1-6, 1998, “Introduction”, 2011] is nowadays in. For advanced fighter aircraft it begins with JS-based powerplant, which takes up to three times longer to mature vis-à-vis the airframe, unless “committee's design” enforces a dormant catastrophe. Jet Steering (JS) or Thrust Vectoring Flight Control, is a classified, integrated engine-airframe technology aimed at maximizing post-stall-maneuverability, flight safety, efficiency and flight envelopes of manned and unmanned air vehicles, especially in the “impossible-to-fly”, post-stall flight domains where the 100+ years old, stall-spin-limited, Conventional Flight Control fails. Worldwide success in adopting the post-stall, JS-revolution, opens a new era in aviation, with unprecedented design variables identified here for a critical review of F-35 future risks v. future fleets of jet-steered, pilotless vehicles, like the X-47B/C. From the educational point of view, it is also instructive to comprehend the causes of long, intensive opposition to adopt post-stall, JS ideas. A review of such debates may also curb a future opposition to adopt more advanced, JS-based technologies, tests, strategies, tactics and missions within the evolving air, marine and land applications of JS. Most important, re-education of pilots and engineers requires adding post-stall, JS-based studies to curriculum & R&D.

77 FR 50668 - Proposed Flood Elevation Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-22

... County, Texas, and Incorporated Areas. Specifically, it addresses the flooding sources: Black Fork Creek, Black Fork Creek Tributary BF-1, Black Fork Creek Tributary BF-M-1, Black Fork Creek Tributary D, Black Fork Creek Tributary D-1, Black Fork Creek Tributary D-2, Black Fork Creek Tributary D-3, Butler Creek...

Dietary effects of metals-contaminated invertebrates from the Coeur d'Alene River, Idaho, on cutthroat trout

USGS Publications Warehouse

Farag, A.M.; Woodward, D.F.; Brumbaugh, W.; Goldstein, J.N.; MacConnell, Elizabeth; Hogstrand, Christer; Barrows, F.T.

1999-01-01

Benthic macroinvertebrates with elevated concentrations of metals were collected from the Coeur d'Alene (CDA) River, Idaho, pasteurized, and fed to cutthroat trout Oncorhynchus clarki in the laboratory from start of feeding until 90 d posthatch. Invertebrates were collected from two sites known to contain elevated concentrations of metals: near Pinehurst in the South Fork of the CDA River and at Cataldo, approximately 5 km below the confluence of the South Fork and the North Fork. Invertebrates collected from a relatively clean site in the North Fork were used as a reference diet. We performed measurements of fish health that indicate reduced fitness of fish fed the South Fork and Cataldo diets. Effects measured were reduced feeding activity, increased number of macrophage aggregates and hyperplasia of cells in the kidney, degeneration of mucosal epithelium in the pyloric caecae, and metallothionein induction. These effects would likely reduce growth and survival of fish in the wild. Vacuolization of glial cells were also observed in fish fed the Cataldo diet. Metals in the water often exacerbated the histological effects observed. Although the invertebrates collected near Cataldo had lower concentrations of arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) than the invertebrates from the South Fork, fish fed the Cataldo diet had equally high or higher concentrations of all metals except as by day 44. The Cataldo diet also caused the most deleterious effects on survival and growth. These findings are especially important for early life stage fish, whose diet consists wholly of benthic macroinvertebrates. Therefore, fish feeding on invertebrates in the CDA River below the Bunker Hill smelting complex are at risk of reduced fitness.

The human intra-S checkpoint response to UVC-induced DNA damage.

PubMed

Kaufmann, William K

2010-05-01

The intra-S checkpoint response to 254 nm light (UVC)-induced DNA damage appears to have dual functions to slow the rate of DNA synthesis and stabilize replication forks that become stalled at sites of UVC-induced photoproducts in DNA. These functions should provide more time for repair of damaged DNA before its replication and thereby reduce the frequencies of mutations and chromosomal aberrations in surviving cells. This review tries to summarize the history of discovery of the checkpoint, the current state of understanding of the biological features of intra-S checkpoint signaling and its mechanisms of action with a focus primarily on intra-S checkpoint responses in human cells. The differences in the intra-S checkpoint responses to UVC and ionizing radiation-induced DNA damage are emphasized. Evidence that [6-4]pyrimidine-pyrimidone photoproducts in DNA trigger the response is discussed and the relationships between cellular responses to UVC and the molecular dose of UVC-induced DNA damage are briefly summarized. The role of the intra-S checkpoint response in protecting against solar radiation carcinogenesis remains to be determined.

Mrc1 Marks Early-Firing Origins and Coordinates Timing and Efficiency of Initiation in Fission Yeast ▿ †

PubMed Central

Hayano, Motoshi; Kanoh, Yutaka; Matsumoto, Seiji; Masai, Hisao

2011-01-01

How early- and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here, we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in the fission yeast Schizosaccharomyces pombe. In mrc1Δ cells (and in swi1Δ cells to some extent), efficiency of firing is stimulated, and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1Δ cells. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that prefiring binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence of this protein. PMID:21518960

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.

The nuclear DEK interactome supports multi-functionality.

PubMed

Smith, Eric A; Krumpelbeck, Eric F; Jegga, Anil G; Prell, Malte; Matrka, Marie M; Kappes, Ferdinand; Greis, Kenneth D; Ali, Abdullah M; Meetei, Amom R; Wells, Susanne I

2018-01-01

DEK is an oncoprotein that is overexpressed in many forms of cancer and participates in numerous cellular pathways. Of these different pathways, relevant interacting partners and functions of DEK are well described in regard to the regulation of chromatin structure, epigenetic marks, and transcription. Most of this understanding was derived by investigating DNA-binding and chromatin processing capabilities of the oncoprotein. To facilitate the generation of mechanism-driven hypotheses regarding DEK activities in underexplored areas, we have developed the first DEK interactome model using tandem-affinity purification and mass spectrometry. With this approach, we identify IMPDH2, DDX21, and RPL7a as novel DEK binding partners, hinting at new roles for the oncogene in de novo nucleotide biosynthesis and ribosome formation. Additionally, a hydroxyurea-specific interaction with replication protein A (RPA) was observed, suggesting that a DEK-RPA complex may form in response to DNA replication fork stalling. Taken together, these findings highlight diverse activities for DEK across cellular pathways and support a model wherein this molecule performs a plethora of functions. © 2017 Wiley Periodicals, Inc.

Human CST has independent functions during telomere duplex replication and C-strand fill-in

PubMed Central

Wang, Feng; Stewart, Jason A.; Kasbek, Christopher; Zhao, Yong; Wright, Woodring E.; Price, Carolyn M.

2012-01-01

Summary Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S-phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide the first direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a novel replication factor that solves diverse replication-associated problems. PMID:23142664

Lesion bypass by S. cerevisiae Pol ζ alone

PubMed Central

Stone, Jana E.; Kumar, Dinesh; Binz, Sara K.; Inase, Aki; Iwai, Shigenori; Chabes, Andrei; Burgers, Peter M.; Kunkel, Thomas A.

2011-01-01

DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27–73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS. PMID:21622032

Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι

PubMed Central

McIntyre, Justyna; Vidal, Antonio E.; McLenigan, Mary P.; Bomar, Martha G.; Curti, Elena; McDonald, John P.; Plosky, Brian S.; Ohashi, Eiji; Woodgate, Roger

2013-01-01

Human DNA polymerases η and ι are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in ‘replication factories’ in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polη-ι interacting region to their respective ubiquitin-binding domains (UBZ in polη and UBM1 and UBM2 in polι), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polη precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with polι. Furthermore, a polι-ubiquitin chimera interacts avidly with both polη and PCNA. Thus, the ubiquitination status of polη, or polι plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS. PMID:23248005

Tolerance of Escherichia coli to Fluoroquinolone Antibiotics Depends on Specific Components of the SOS Response Pathway

PubMed Central

Theodore, Alyssa; Lewis, Kim; Vulić, Marin

2013-01-01

Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called “persisters,” depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance. PMID:24077306

Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of the SOS response pathway.

PubMed

Theodore, Alyssa; Lewis, Kim; Vulic, Marin

2013-12-01

Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called "persisters," depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance.

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa

PubMed Central

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

2015-01-01

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5’ end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5’ but not 3’ end, explaining how Rpa regulates cleavage polarity. DOI: http://dx.doi.org/10.7554/eLife.09832.001 PMID:26491943

Validation of an Actuator Line Model Coupled to a Dynamic Stall Model for Pitching Motions Characteristic to Vertical Axis Turbines

NASA Astrophysics Data System (ADS)

Mendoza, Victor; Bachant, Peter; Wosnik, Martin; Goude, Anders

2016-09-01

Vertical axis wind turbines (VAWT) can be used to extract renewable energy from wind flows. A simpler design, low cost of maintenance, and the ability to accept flow from all directions perpendicular to the rotor axis are some of the most important advantages over conventional horizontal axis wind turbines (HAWT). However, VAWT encounter complex and unsteady fluid dynamics, which present significant modeling challenges. One of the most relevant phenomena is dynamic stall, which is caused by the unsteady variation of angle of attack throughout the blade rotation, and is the focus of the present study. Dynamic stall is usually used as a passive control for VAWT operating conditions, hence the importance of predicting its effects. In this study, a coupled model is implemented with the open-source CFD toolbox OpenFOAM for solving the Navier-Stokes equations, where an actuator line model and dynamic stall model are used to compute the blade loading and body force. Force coefficients obtained from the model are validated with experimental data of pitching airfoil in similar operating conditions as an H-rotor type VAWT. Numerical results show reasonable agreement with experimental data for pitching motion.

Piloted Simulator Evaluation Results of Flight Physics Based Stall Recovery Guidance

NASA Technical Reports Server (NTRS)

Lombaerts, Thomas; Schuet, Stefan; Stepanyan, Vahram; Kaneshige, John; Hardy, Gordon; Shish, Kimberlee; Robinson, Peter

2018-01-01

In recent studies, it has been observed that loss of control in flight is the most frequent primary cause of accidents. A significant share of accidents in this category can be remedied by upset prevention if possible, and by upset recovery if necessary, in this order of priorities. One of the most important upsets to be recovered from is stall. Recent accidents have shown that a correct stall recovery maneuver remains a big challenge in civil aviation, partly due to a lack of pilot training. A possible strategy to support the flight crew in this demanding context is calculating a recovery guidance signal, and showing this signal in an intuitive way on one of the cockpit displays, for example by means of the flight director. Different methods for calculating the recovery signal, one based on fast model predictive control and another using an energy based approach, have been evaluated in four relevant operational scenarios by experienced commercial as well as test pilots in the Vertical Motion Simulator at NASA Ames Research Center. Evaluation results show that this approach could be able to assist the pilots in executing a correct stall recovery maneuver.

Transcription and replication: breaking the rules of the road causes genomic instability.

PubMed

Poveda, Ana Maria; Le Clech, Mikael; Pasero, Philippe

2010-01-01

Replication and transcription machineries progress at high speed on the same DNA template, which inevitably causes traffic accidents. Problems are not only caused by frontal collisions between polymerases, but also by cotranscriptional R-loops. These RNA-DNA hybrids induce genomic instability by blocking fork progression and could be implicated in the development of cancer.

3. View of Clark Fork Vehicle Bridge facing southwest. Bridge ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. View of Clark Fork Vehicle Bridge facing southwest. Bridge from north shore of Clark Fork River. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

4. View of Clark Fork Vehicle Bridge facing northeast. Bridge ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

4. View of Clark Fork Vehicle Bridge facing northeast. Bridge from south shoreof Clark Fork River showing 4 spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

Hydrologic conditions and hazards in the Kennicott River basin, Wrangell-St. Elias National Park Preserve, Alaska

USGS Publications Warehouse

Rickman, R.L.; Rosenkrans, D.S.

1997-01-01

McCarthy, Alaska, is on the Kennicott River, about 1 mile from the terminus of Kennicott Glacier in the Wrangell-St. Elias National Park and Preserve. Most visitors to McCarthy and the park cross the West Fork Kennicott River using a hand-pulled tram and cross the East Fork Kennicott River on a temporary footbridge. Outburst floods from glacier-dammed lakes result in channel erosion, aggradation, and migration of the Kennicott River, which disrupt transportation links, destroy property, and threaten life. Hidden Creek Lake, the largest of six glacier-dammed lakes in the Kennicott River Basin, has annual outbursts that cause the largest floods on the Kennicott River. Outbursts from Hidden Creek Lake occur from early fall to mid-summer, and lake levels at the onset of the outbursts have declined between 1909 and 1995. Criteria for impending outbursts for Hidden Creek Lake include lake stage near or above 3,000 to 3,020 feet, stationary or declining lake stage, evidence of recent calving of large ice blocks from the ice margin, slush ice and small icebergs stranded on the lakeshore, and fresh fractures in the ice-margin region. The lower Kennicott Glacier has thinned and retreated since about 1860. The East and West Fork Kennicott River channels migrated in response to changes in the lower Kennicott Glacier. The largest channel changes occur during outburst floods from Hidden Creek Lake, whereas channel changes from the other glacier-dammed lake outbursts are small. Each year, the West Fork Kennicott River conveys a larger percentage of the Kennicott Glacier drainage than it did the previous year. Outburst floods on the Kennicott River cause the river stage to rise over a period of several hours. Smaller spike peaks have a very rapid stage rise. Potential flood magnitude was estimated by combining known maximum discharges from Hidden Creek Lake and Lake Erie outburst floods with a theoretical large regional flood. Flood hazard areas at the transportation corridor were delineated, and possible future geomorphological changes were hypothesized. McCarthy, Alaska, is on the Kennicott River, about 1 mile from the terminus of Kennicott Glacier in the Wrangell-St. Elias National Park and Preserve. Most visitors to McCarthy and the park cross the West Fork Kennicott River using a hand-pulled tram and cross the East Fork Kennicott River on a temporary footbridge. Outburst floods from glacier-dammed lakes result in channel erosion, aggradation, and migration of the Kennicott River, which disrupt transportation links, destroy property, and threaten life. Hidden Creek Lake, the largest of six glacier-dammed lakes in the Kennicott River Basin, has annual outbursts that cause the largest floods on the Kennicott River. Outbursts from Hidden Creek Lake occur from early fall to mid-summer, and lake levels at the onset of the outbursts have declined between 1909 and 1995. Criteria for impending outbursts for Hidden Creek Lake include lake stage near or above 3,000 to 3,020 feet, stationary or declining lake stage, evidence of recent calving of large ice blocks from the ice margin, slush ice and small icebergs stranded on the lakeshore, and fresh fractures in the ice-margin region. The lower Kennicott Glacier has thinned and retreated since about 1860. The East and West Fork Kennicott River channels migrated in response to changes in the lower Kennicott Glacier. The largest channel changes occur during outburst floods from Hidden Creek Lake, whereas channel changes from the other glacier-dammed lake outbursts are small. Each year, the West Fork Kennicott River conveys a larger percentage of the Kennicott Glacier drainage than it did the previous year. Outburst floods on the Kennicott River cause the river stage to rise over a period of several hours. Smaller spike peaks have a very rapid stage rise. Potential flood magnitude was estimated by combining known maximum discharges from Hidden Creek Lake and Lake Erie outburst floods with

2. View of Clark Fork Vehicle Bridge facing northeast. Bridge ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. View of Clark Fork Vehicle Bridge facing northeast. Bridge from south shore of Clark Fork River showing 4 1/2 spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

7. View of Clark Fork Vehicle Bridge facing northwest. Bridge ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

7. View of Clark Fork Vehicle Bridge facing northwest. Bridge from south shore of Clark Fork River showing 4 1/2 spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

1. View of Clark Fork Vehicle Bridge facing west. Panorama ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. View of Clark Fork Vehicle Bridge facing west. Panorama showing the entire span of bridge from north shore of the Clark Fork River. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

5. View of Clark Fork Vehicle Bridge facing east. Bridge ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. View of Clark Fork Vehicle Bridge facing east. Bridge from south shore of Clark Fork River-southernmost span. 1900-era Northern Pacific Railway Bridge in background. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

Dairy Cows Produce Less Milk and Modify Their Behaviour during the Transition between Tie-Stall to Free-Stall

PubMed Central

Broucek, Jan; Uhrincat, Michal; Mihina, Stefan; Soch, Miloslav; Mrekajova, Andrea; Hanus, Anton

2017-01-01

Simple Summary The purpose of this study was to evaluate the influence of moving cows from the barn with stanchion-stall housing to free-stall housing on their behaviour and production. Cows lay down up to ten hours after removing. The cows in their second lactation and open cows tended to lie sooner after removing than cows in their first lactation and pregnant cows. The times of total lying and rumination were increasing from the first day to the tenth day after removing. Cows produced 23.3% less milk at the first day following the transfer than at the last day prior to moving (23.76 ± 7.20 kg vs. 30.97 ± 7.26 kg, p < 0.001). Loss of milk was gradually reduced and on the 14th day, cows achieved maximum production. The difference was found in milk losses due to the shift between cows in first and second lactation. Abstract Transfer of cattle to an unknown barn may result in a reduction in its welfare. Housing and management practices can result in signs of stress that include a long-term suppression of milk efficiency. The purpose of this study was to evaluate the influence of moving cows from the stanchion-stall housing to free-stall housing on their behaviour and production. The Holstein cows were moved into the new facility with free-stall housing from the old barn with stanchion-stall housing. Cows lay down up to ten hours (596.3 ± 282.7 min) after removing. The cows in their second lactation and open cows tended to lie sooner after removing than cows in their first lactation and pregnant cows. The times of total lying and rumination were increasing from the first day to the tenth day after removing (23.76 ± 7.20 kg vs. 30.97 ± 7.26 kg, p < 0.001). Cows produced 23.3% less milk at the first day following the transfer than at the last day prior to moving (p < 0.001). Loss of milk was gradually reduced and maximum production was achieved on the 14th day. The difference was found in milk losses due to the shift between cows on the first and second lactation (p < 0.01). The results of this study suggest that removing from the tie-stall barn with a pipeline milking system into the barn with free-stall housing and a milking parlour caused a decline in the cows’ milk production. However, when the cows are moved to a better environment, they rapidly adapt to the change. PMID:28273810

Relations between precipitation and daily and monthly mean flows in gaged, unmined and valley-filled watersheds, Ballard Fork, West Virginia, 1999-2001

USGS Publications Warehouse

Messinger, Terence; Paybins, Katherine S.

2003-01-01

Large-scale surface mining using valley fills has changed hydrologic storage and processes in the Ballard Fork Watershed in West Virginia. Total unit flow for the 2-year study period (November 15, 1999?November 14, 2001) on the Unnamed Tributary (extensively mined) (11,700 cubic feet per second per square mile) was almost twice that on Spring Branch (unmined) (6,260 cubic feet per second per square mile), and about 1.75 times that on Ballard Fork (downstream, partly mined) (6,690 cubic feet per second per square mile). Unit flow from the Unnamed Tributary exceeded that from the other two streams for all flows analyzed (5?95 percent duration). Unit flow from Ballard Fork exceeded unit flow from Spring Branch about 80 percent of the time, but was about the same for high flows (less than 20 percent duration). The proportional differences among sites were greatest at low flows. Spring Branch was dry for several days in October and November 2000 and for most of October 2001, and the Unnamed Tributary had flow throughout the study period. The increase in flows from mined parts of the Ballard Fork Watershed appears to result from decreases in evapotranspiration caused by removal of trees and soil during mining. During both years, evapotranspiration from the Spring Branch Watershed greatly exceeded that from the Unnamed Tributary Watershed during May through October, when leaves were open. Evapotranspiration from the Unnamed Tributary Watershed slightly exceeded that from the Spring Branch Watershed in February and March during both years. Evapotranspiration, as a percentage of total rainfall, decreased from the first to the second, drier, year from the Unnamed Tributary Watershed (from 61 percent to 49 percent) but changed little from the Spring Branch (from 77 to 76 percent) and Ballard Fork (73 to 76 percent) Watersheds. Precipitation and flow during the study period at three nearby long-term sites, the U.S. Geological Survey stream-gaging station East Fork Twelvepole Creek near Dunlow, West Virginia, and two National Oceanic Atmospheric Administration rain gages at Madison and Dunlow, West Virginia, were less than long-term annual averages. Relations observed among the three streams in the Ballard Fork Watershed during this study may not represent those in years when annual precipitation and flow are closer to long-term averages.

8. View of Clark Fork Vehicle Bridge facing southwest. Looking ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

8. View of Clark Fork Vehicle Bridge facing southwest. Looking at understructure of northernmost span. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

20. View of Clark Fork Vehicle Bridge facing up. Looking ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

20. View of Clark Fork Vehicle Bridge facing up. Looking at understructure of northernmost span. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

The AFDD International Dynamic Stall Workshop on Correlation of Dynamic Stall Models with 3-D Dynamic Stall Data

NASA Technical Reports Server (NTRS)

Tan, C. M.; Carr, L. W.

1996-01-01

A variety of empirical and computational fluid dynamics two-dimensional (2-D) dynamic stall models were compared to recently obtained three-dimensional (3-D) dynamic stall data in a workshop on modeling of 3-D dynamic stall of an unswept, rectangular wing, of aspect ratio 10. Dynamic stall test data both below and above the static stall angle-of-attack were supplied to the participants, along with a 'blind' case where only the test conditions were supplied in advance, with results being compared to experimental data at the workshop itself. Detailed graphical comparisons are presented in the report, which also includes discussion of the methods and the results. The primary conclusion of the workshop was that the 3-D effects of dynamic stall on the oscillating wing studied in the workshop can be reasonably reproduced by existing semi-empirical models once 2-D dynamic stall data have been obtained. The participants also emphasized the need for improved quantification of 2-D dynamic stall.

Environmental Assessment All-Terrain Vehicle Training Area at Grand Forks AFB, North Dakota

DTIC Science & Technology

2004-03-17

during actual construction, and over the long-term due to reduced storm water quality caused by the increase of exposed soil. The short-term effects...actual construction, and over the long-term due to reduced storm water quality caused by the increase of exposed soil. The short-term effects come

18. View of Clark Fork Vehicle Bridge facing north. Looking ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

18. View of Clark Fork Vehicle Bridge facing north. Looking at north concrete abutment and timber stringers. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

Prevalence of lameness among dairy cattle in Wisconsin as a function of housing type and stall surface.

PubMed

Cook, Nigel B

2003-11-01

To determine the prevalence of lameness as a function of season (summer vs winter), housing type (free stalls vs tie stalls), and stall surface (sand vs any other surface) among lactating dairy cows in Wisconsin. Epidemiologic survey. 3,621 lactating dairy cows in 30 herds. Herds were visited once during the summer and once during the winter, and a locomotion score ranging from 1 (no gait abnormality) to 4 (severe lameness) was assigned to all lactating cows. Cows with a score of 3 or 4 were considered to be clinically lame. Mean +/- SD herd lameness prevalence was 21.1 +/- 10.5% during the summer and 23.9 +/- 10.7% during the winter; these values were significantly different. During the winter, mean prevalence of lameness in free-stall herds with non-sand stall surfaces (33.7%) was significantly higher than prevalences in free-stall herds with sand stall surfaces (21.2%), tie-stall herds with non-sand stall surfaces (21.7%), and tie-stall herds with sand stall surfaces (12.1%). Results suggest that the prevalence of lameness among dairy cattle in Wisconsin is higher than previously thought and that lameness prevalence is associated with season, housing type, and stall surface.

A CFD Database for Airfoils and Wings at Post-Stall Angles of Attack

NASA Technical Reports Server (NTRS)

Petrilli, Justin; Paul, Ryan; Gopalarathnam, Ashok; Frink, Neal T.

2013-01-01

This paper presents selected results from an ongoing effort to develop an aerodynamic database from Reynolds-Averaged Navier-Stokes (RANS) computational analysis of airfoils and wings at stall and post-stall angles of attack. The data obtained from this effort will be used for validation and refinement of a low-order post-stall prediction method developed at NCSU, and to fill existing gaps in high angle of attack data in the literature. Such data could have potential applications in post-stall flight dynamics, helicopter aerodynamics and wind turbine aerodynamics. An overview of the NASA TetrUSS CFD package used for the RANS computational approach is presented. Detailed results for three airfoils are presented to compare their stall and post-stall behavior. The results for finite wings at stall and post-stall conditions focus on the effects of taper-ratio and sweep angle, with particular attention to whether the sectional flows can be approximated using two-dimensional flow over a stalled airfoil. While this approximation seems reasonable for unswept wings even at post-stall conditions, significant spanwise flow on stalled swept wings preclude the use of two-dimensional data to model sectional flows on swept wings. Thus, further effort is needed in low-order aerodynamic modeling of swept wings at stalled conditions.

19. View of Clark Fork Vehicle Bridge facing north. Looking ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

19. View of Clark Fork Vehicle Bridge facing north. Looking at north abutment and underside of northernmost span. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

22. View of Clark Fork Vehicle Bridge facing downwest side. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

22. View of Clark Fork Vehicle Bridge facing down-west side. Looking at road deck and vertical laced channel. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

Flow separation on wind turbines blades

NASA Astrophysics Data System (ADS)

Corten, G. P.

2001-01-01

In the year 2000, 15GW of wind power was installed throughout the world, producing 100PJ of energy annually. This contributes to the total electricity demand by only 0.2%. Both the installed power and the generated energy are increasing by 30% per year world-wide. If the airflow over wind turbine blades could be controlled fully, the generation efficiency and thus the energy production would increase by 9%. Power Control To avoid damage to wind turbines, they are cut out above 10 Beaufort (25 m/s) on the wind speed scale. A turbine could be designed in such a way that it converts as much power as possible in all wind speeds, but then it would have to be to heavy. The high costs of such a design would not be compensated by the extra production in high winds, since such winds are rare. Therefore turbines usually reach maximum power at a much lower wind speed: the rated wind speed, which occurs at about 6 Beaufort (12.5 m/s). Above this rated speed, the power intake is kept constant by a control mechanism. Two different mechanisms are commonly used. Active pitch control, where the blades pitch to vane if the turbine maximum is exceeded or, passive stall control, where the power control is an implicit property of the rotor. Stall Control The flow over airfoils is called "attached" when it flows over the surface from the leading edge to the trailing edge. However, when the angle of attack of the flow exceeds a certain critical angle, the flow does not reach the trailing edge, but leaves the surface at the separation line. Beyond this line the flow direction is reversed, i.e. it flows from the trailing edge backward to the separation line. A blade section extracts much less energy from the flow when it separates. This property is used for stall control. Stall controlled rotors always operate at a constant rotation speed. The angle of attack of the flow incident to the blades is determined by the blade speed and the wind speed. Since the latter is variable, it determines the angle of attack. The art of designing stall rotors is to make the separated area on the blades extend in such a way, that the extracted power remains precisely constant, independent of the wind speed, while the power in the wind at cut-out exceeds the maximum power of the turbine by a factor of 8. Since the stall behaviour is influenced by many parameters, this demand cannot be easily met. However, if it can be met, the advantage of stall control is its passive operation, which is reliable and cheap. Problem Definition In practical application, stall control is not very accurate and many stall-controlled turbines do not meet their specifications. Deviations of the design-power in the order of tens of percent are regular. In the nineties, the aerodynamic research on these deviations focussed on: profile aerodynamics, computational fluid dynamics, rotational effects on separation and pressure measurements on test turbines. However, this did not adequately solve the actual problems with stall turbines. In this thesis, we therefore formulated the following as the essential question: "Does the separated blade area really extend with the wind speed, as we predict?" To find the answer a measurement technique was required, which 1) was applicable on large commercial wind turbines, 2) could follow the dynamic changes of the stall pattern, 3) was not influenced by the centrifugal force and 4) did not disturb the flow. Such a technique was not available, therefore we decided to develop it. Stall Flag Method For this method, a few hundred indicators are fixed to the rotor blades in a special pattern. These indicators, called "stall flags" are patented by the Netherlands Energy Research Foundation (ECN). They have a retro-reflective area which, depending on the flow direction, is or is not covered. A powerful light source in the field up to 500m behind the turbine illuminates the swept rotor area. The uncovered reflectors reflect the light to the source, where a digital video camera records the dynamic stall patterns. The images are analysed by image processing software that we developed. The program extracts the stall pattern, the blade azimuth angles and the rotor speed from the stall flags. It also measures the yaw error and the wind speed from the optical signals of other sensors, which are recorded simultaneously. We subsequently characterise the statistical stall behaviour from the sequences of thousands of analysed images. For example, the delay in the stall angle by vortex generators can be measured within 1° of accuracy from the stall flag signals. Properties of the Stall Flag The new indicators are compared to the classic tufts. Stall flags are pressure driven while tufts are driven by frictional drag, which means that they have more drag. The self-excited motion of tufts, due to the Kelvin-Helmholtz instability, complicates the interpretation and gives more drag. We designed stall flags in such a way that this instability is avoided. An experiment with a 65cm diameter propeller confirms the independence of stall flags from the centrifugal force and that stall flags respond quickly to changes in the flow. We developed an optical model of the method to find an optimum set-up. With the present system, we can take measurements on turbines of all actual diameters. The stall flag responds to separated flow with an optical signal. The contrast of this signal exceeds that of tuft-signals by a factor of at least 1000. To detect the stall flag signal we need a factor of 25 fewer pixels of the CCD chip than is necessary for tufts. Stall flags applied on fast moving objects may show light tracks due to motion blur, which in fact yields even more information. In the case of tuft visualisations, even a slight motion blur is fatal. Principal Results In dealing with the fundamental theory of wind turbines, we found a new aspect of the conversion efficiency of a wind turbine, which also concerns the stall behaviour. Another new aspect concerns the effects of rotation on stall. By using the stall flag method, we were able to clear up two practical problems that seriously threatened the performance of stall turbines. These topics will be described briefly. 1. Inherent Heat Generation The classic result for an actuator disk representing a wind turbine is that the power extracted equals the kinetic power transferred. This is a consequence of disregarding the flow around the disk. When this flow is included, we need to introduce a heat generation term in the energy balance. This has the practical consequence that an actuator disk at the Lanchester-Betz limit transfers 50% more kinetic energy than it extracts. This surplus is dissipated in heat. Using this new argument, together with a classic argument on induction, we see no reason to introduce the concept of edge-forces on the tips of the rotor blades (Van Kuik, 1991). We rather recommend following the ideas of Lanchester (1915) on the edge of the actuator disk and on the wind speed at the disc. We analyse the concept induction, and show that correcting for the aspect ratio, for induced drag and application of Blade Element Momentum Theory all have the same significance for a wind turbine. Such corrections are sometimes made twice (Viterna & Corrigan, 1981). 2. Rotational Effects on Flow Separation In designing wind turbine rotors, one uses the aerodynamic characteristics measured in the wind tunnel on fixed aerodynamic profiles. These characteristics are corrected for the effects of rotation and subsequently used for wind turbine rotors. Such a correction was developed by Snel (1990-1999). This correction is based on boundary layer theory, the validity of which we question in regard to separated flow. We estimated the effects of rotation on flow separation by arguing that the separation layer is thick so the velocity gradients are small and viscosity can be neglected. We add the argument that the chord-wise speed and its derivative normal to the wall is zero at the separation line, which causes the terms with the chord-wise speed or accelerations to disappear. The conclusion is that the chord-wise pressure gradient balances the Coriolis force. By doing so we obtain a simple set of equations that can be solved analytically. Subsequently, our model predicts that the convective term with the radial velocity (vrvr/r) is dominant in the equation for the r-direction, precisely the term that was neglected in Snel's analysis. 3. Multiple Power Levels Several large commercial wind turbines demonstrate drops in maximum power levels up to 45%, under apparently equal conditions. Earlier studies attempting to explain this effect by technical malfunctioning, aerodynamic instabilities and blade contamination effects estimated with computational fluid dynamics, have not yet yielded a plausible result. We formulated many hypotheses, three of which were useful. By taking stall flag measurements and making two other crucial experiments, we could confirm one of those three hypotheses: the insect hypothesis. Insects only fly in low wind, impacting upon the blades at specific locations. In these conditions, the insectual remains are located at positions where roughness has little influence on the profile performance, so that the power is not affected. In high winds however, the flow around the blades has changed. As a result, the positions at which the insects have impacted at low winds are very sensitive to contamination. So the contamination level changes at low wind when insects fly and this level determines the power in high winds when insects do not fly. As a consequence we get discrete power levels in high winds. The other two hypotheses, which did not cause the multiple power levels for the case we studied, gave rise to two new insights. First, we expect the power to depend on the wind direction at sites where the shape of the terrain concentrates the wind. In this case the power level of all turbine types, including pitch regulated ones, will be affected. Second, we infer heuristically that the stalled area on wind turbine blades will adapt continuously to wind variations. Therefore, the occurrence of strong bi-stable stall-hysteresis, which most blade sections demonstrate in the wind tunnel, is lost. This has been confirmed by taking special stall flag measurements. 4. Deviation of Specifications The maximum power of stall controlled wind turbines often shows large systematic deviations from the design. We took stall flag measurements on a rotor, the maximum power of which was 30% too high, so that the turbine had to be cut out far below the designed cut-out wind speed. We immediately observed the blade areas with deviating stall behaviour. Some areas that should have stalled did not and caused the excessive power. We adapted those areas by shifting the vortex generators. In this way we obtained a power curve that met the design much more closely and we realised a production increase of 8%.

Surge-Inception Study in a Two-Spool Turbojet Engine. Revised

NASA Technical Reports Server (NTRS)

Wallner, Lewis E.; Lubick, Robert J.; Saari, Martin J.

1957-01-01

A two-spool turbojet engine was operated in the Lewis altitude wind tunnel to study the inception of compressor surge. In addition to the usual steady-state pressure and temperature measurements, the compressors were extensively instrumented with fast-response interstage pressure transducers. Thus it was possible to obtain maps for both compressors, pressure oscillations during rotating stall, effects of stall on efficiency, and stage-loading curves. In addition, with the transient measurements, it was possible to record interstage pressures and then compute stage performance during accelerations to the stall limit. Rotating stall was found to exist at low speeds in the outer spool. Although the stall arose from poor flow conditions at the inlet-stage blade tips, the low-energy air moved through the machine from the tip at the inlet to the outer spool to the hub at the inlet to the inner spool. This tip stall ultimately resulted in compressor surge in the mid-speed region, and necessitated inter-compressor air bleed. Interstage pressure measurements during acceleration to the compressor stall limit indicated that rotating stall was not a necessary condition for compressor surge and that, at the critical stall point, the circumferential interstage pressure distribution was uniform. The exit-stage group of the inner spool was first t o stall; then, the stages upstream stalled in succession until the inlet stage of the outer spool was stalled. With a sufficiently high fuel rate, the process repeated with a cycle time of about 0.1 second. It was possible to construct reproducible stage stall lines as a function of compressor speed from the stage stall points of several such compressor surges. This transient stall line was checked by computing the stall line from a steady-state stage-loading curve. Good agreement between the stage stall lines was obtained by these two methods.

21. View of Clark Fork Vehicle Bridge facing west. Looking ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

21. View of Clark Fork Vehicle Bridge facing west. Looking at bridge deck, guard rail, juncture of two bridge spans. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

11. View of Clark Fork Vehicle Bridge facing northwest. Southernmost ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

11. View of Clark Fork Vehicle Bridge facing northwest. Southernmost span. Plaque was originally located where striped traffic sign is posted. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

Associations between cow hygiene, hock injuries, and free stall usage on US dairy farms.

PubMed

Lombard, J E; Tucker, C B; von Keyserlingk, M A G; Kopral, C A; Weary, D M

2010-10-01

This cross-sectional study evaluated cow comfort measures in free stall dairies across the United States as part of the National Animal Health Monitoring System's Dairy 2007 study. The study was conducted in 17 states and evaluations were completed between March 5 and September 5, 2007. Assessors recorded hygiene and hock scores, number of cows housed in the pen, the number of cows standing with only the front feet in a stall, standing fully in a stall, and lying in a stall. Facility design measures included bedding type, bedding quantity, stall length and width, presence of a neck rail or brisket locator, and relevant distances from the rear and bed of the stall. Of the 491 operations that completed the cow comfort assessment, 297 had Holstein cows housed in free stalls and were included in this analysis. Negative binomial models were constructed to evaluate the following outcomes: the number of cows that were very dirty, had severe hock injuries, stood with front feet in the stall, stood with all feet in the stall, and were lying in the stall. Hygiene was better on farms that did not tail dock cows compared with those that did (5.7 vs. 8.8% were dirty) and on farms located in the study's west region compared with those located in the east region (5.2 vs. 9.7% were dirty). Severe hock injuries were less common on farms in the west than those in the east (0.5 vs. 4.1%). In addition, severe hock injuries were less common on farms that used dirt as a stall base or sand as bedding compared with farms that did not. A higher percentage of cows was standing with front feet in the stall at higher ambient temperatures (incidence rate ratio=1.016) and as time since feeding increased (incidence rate ratio=1.030). A lower percentage of cows were standing with front feet in the stall when the stalls were shorter and when there were fewer cows per stall. Standing fully in a stall was performed by a higher percentage of cows during the summer than during the spring (13.6 vs. 8.1%), when cows were provided free stalls with rubber mats or mattresses, and as the distance from the rear curb to neck rail increased. A higher percentage of cows were lying in a stall when sand bedding was used, when bedding was added more frequently, and during the spring months. Results of this national survey indicate that tail docking provides no benefit to cow hygiene and that stall base and bedding are key factors influencing hock injuries and stall usage on US free stall dairy farms. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

23. View of Clark Fork Vehicle Bridge facing upwest side. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

23. View of Clark Fork Vehicle Bridge facing up-west side. Looking at structural connection of top chord, vertical laced channel and diagonal bars. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

13. View of Clark Fork Vehicle Bridge facing south. Concrete ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

13. View of Clark Fork Vehicle Bridge facing south. Concrete barrier blocks access. Plaque was originally located where strioed traffic sign is posted at right. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

12. View of Clark Fork Vehicle Bridge facing south. Approach ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. View of Clark Fork Vehicle Bridge facing south. Approach from the north road. Plaque was originally located where striped traffic sign is posted. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

The phenomenon of dynamic stall. [vortex shedding phenomenon on oscillating airfoils

NASA Technical Reports Server (NTRS)

Mccroskey, W. J.

1981-01-01

The general features of dynamic stall on oscillating airfoils are explained in terms of the vortex shedding phenomenon, and the important differences between static stall, light dynamic stall, and deep stall are described. An overview of experimentation and prediction techniques is given.

Analysis of stall flutter of a helicopter radar blade

NASA Technical Reports Server (NTRS)

Crimi, P.

1973-01-01

A study of rotor blade aeroelastic stability was carried out, using an analytic model of a two-dimensional airfoil undergoing dynamic stall and an elastomechanical representation including flapping, flapwise bending and torsional degrees of freedom. Results for a hovering rotor demonstrated that the models used are capable of reproducing both classical and stall flutter. The minimum rotor speed for the occurrence of stall flutter in hover, was found to be determined from coupling between torsion and flapping. Instabilities analogous to both classical and stall flutter were found to occur in forward flight. However, the large stall-related torsional oscillations which commonly limit aircraft forward speed appear to be the response to rapid changes in aerodynamic moment which accompany stall and unstall, rather than the result of an aeroelastic instability. The severity of stall-related instabilities and response was found to depend to some extent on linear stability. Increasing linear stability lessens the susceptibility to stall flutter and reduced the magnitude of the torsional response to stall and unstall.

Common fragile sites (CFS) and extremely large CFS genes are targets for human papillomavirus integrations and chromosome rearrangements in oropharyngeal squamous cell carcinoma.

PubMed

Gao, Ge; Johnson, Sarah H; Vasmatzis, George; Pauley, Christina E; Tombers, Nicole M; Kasperbauer, Jan L; Smith, David I

2017-01-01

Common fragile sites (CFS) are chromosome regions that are prone to form gaps or breaks in response to DNA replication stress. They are often found as hotspots for sister chromatid exchanges, deletions, and amplifications in different cancers. Many of the CFS regions are found to span genes whose genomic sequence is greater than 1 Mb, some of which have been demonstrated to function as important tumor suppressors. CFS regions are also hotspots for human papillomavirus (HPV) integrations in cervical cancer. We used mate-pair sequencing to examine HPV integration events and chromosomal structural variations in 34 oropharyngeal squamous cell carcinoma (OPSCC). We used endpoint PCR and Sanger sequencing to validate each HPV integration event and found HPV integrations preferentially occurred within CFS regions similar to what is observed in cervical cancer. We also found that many of the chromosomal alterations detected also occurred at or near the cytogenetic location of CFSs. Several large genes were also found to be recurrent targets of rearrangements, independent of HPV integrations, including CSMD1 (2.1Mb), LRP1B (1.9Mb), and LARGE1 (0.7Mb). Sanger sequencing revealed that the nucleotide sequences near to identified junction sites contained repetitive and AT-rich sequences that were shown to have the potential to form stem-loop DNA secondary structures that might stall DNA replication fork progression during replication stress. This could then cause increased instability in these regions which could lead to cancer development in human cells. Our findings suggest that CFSs and some specific large genes appear to play important roles in OPSCC. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Homozygous deletion of six genes including corneodesmosin on chromosome 6p21.3 is associated with generalized peeling skin disease.

PubMed

Teye, Kwesi; Hamada, Takahiro; Krol, Rafal P; Numata, Sanae; Ishii, Norito; Matsuda, Mitsuhiro; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

2014-07-01

Peeling skin syndrome (PSS) is a rare autosomal recessive form of ichthyosis showing skin exfoliation. PSS is divided into acral and generalized PSS, and the latter is further classified into non-inflammatory type (PSS type A) and inflammatory type (PSS type B). PSS type B is now called peeling skin disease (PSD). Different loss-of-function mutations in the corneodesmosin (CDSN) gene have been reported to cause PSD. The aim of this study was to determine genetic basis of disease in a 14-year-old Japanese patient with PSD. Immunohistochemical study showed lack of corneodesmosin (CDSN) in the skin, and standard PCR for genomic DNA failed to amplify CDSN product, suggesting CDSN defect. Multiplex ligation-dependent probe amplification and genomic quantitative real-time PCR analyses detected large homozygous deletion of 59,184bp extending from 40.6kb upstream to 13.2kb downstream of CDSN, which included 6 genes (TCF19, CCHCR1, PSORS1C2, PSORS1C1, CDSN and C6orf15). The continuous gene lost did not result in additional clinical features. Inverted repeats with 85% similarity flanking the deletion breakpoint were considered to mediate the deletion by non-homologous end joining or fork stalling and template switching/microhomology-mediated break-induced replication. Parents were clinically unaffected and were heterozygote carriers of the same deletion, which was absent in 284 ethnically matched control alleles. We also developed simple PCR method, which is useful for detection of this deletion. Although 5 other genes were also deleted, homozygous deletion of CDSN was considered to be responsible for this PSD. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Environmental Assessment: Extend Parking Lot Building 807 at Grand Forks AFB, North Dakota

DTIC Science & Technology

2003-07-17

Surface water quality could be degraded, both in the short-term, during actual construction, and over the long-term due to reduced storm water quality caused...over the long-term due to reduced storm water quality caused by the increase of paved area. The short-term effects come from possible erosion

Environmental Assessment: Construct Mass/Mobility Parking Lot at Grand Forks AFB, North Dakota

DTIC Science & Technology

2004-02-13

Water: Surface water quality could be degraded, both in the short-term, during actual construction, and over the long-term due to reduced storm water quality caused...term, during actual construction, and over the long-term due to reduced storm water quality caused by the increase of exposed soil. The short-term

Environmental Assessment On-Base Snowmobile Trail at Grand Forks AFB, North Dakota

DTIC Science & Technology

2004-04-01

Water: Surface water quality could be degraded, both in the short-term, and over the long-term due to reduced storm water quality caused by a potential...Surface water quality could be degraded, both in the short-term, and over the long-term due to reduced storm water quality caused by a potential increase

14. View of Clark Fork Vehicle Bridge facing north. Approach ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

14. View of Clark Fork Vehicle Bridge facing north. Approach from the south. Concrete barrier blocks access. Plaque was originally located where striped traffic sign is posted at right. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

40 CFR 131.33 - Idaho.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Creek, Doe Creek, Duck Creek, East Fork Holy Terror Creek, Fawn Creek, Flume Creek, Fly Creek, Forge... Terror Creek, J Fell Creek, Jacobs Ladder Creek, Lewis Creek, Liberty Creek, Lick Creek, Lime Creek... Creek, South Fork Chamberlain Creek, South Fork Holy Terror Creek, South Fork Norton Creek, South Fork...

40 CFR 131.33 - Idaho.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Creek, Doe Creek, Duck Creek, East Fork Holy Terror Creek, Fawn Creek, Flume Creek, Fly Creek, Forge... Terror Creek, J Fell Creek, Jacobs Ladder Creek, Lewis Creek, Liberty Creek, Lick Creek, Lime Creek... Creek, South Fork Chamberlain Creek, South Fork Holy Terror Creek, South Fork Norton Creek, South Fork...

40 CFR 131.33 - Idaho.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Creek, Doe Creek, Duck Creek, East Fork Holy Terror Creek, Fawn Creek, Flume Creek, Fly Creek, Forge... Terror Creek, J Fell Creek, Jacobs Ladder Creek, Lewis Creek, Liberty Creek, Lick Creek, Lime Creek... Creek, South Fork Chamberlain Creek, South Fork Holy Terror Creek, South Fork Norton Creek, South Fork...

40 CFR 131.33 - Idaho.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Creek, Doe Creek, Duck Creek, East Fork Holy Terror Creek, Fawn Creek, Flume Creek, Fly Creek, Forge... Terror Creek, J Fell Creek, Jacobs Ladder Creek, Lewis Creek, Liberty Creek, Lick Creek, Lime Creek... Creek, South Fork Chamberlain Creek, South Fork Holy Terror Creek, South Fork Norton Creek, South Fork...

40 CFR 131.33 - Idaho.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Creek, Doe Creek, Duck Creek, East Fork Holy Terror Creek, Fawn Creek, Flume Creek, Fly Creek, Forge... Terror Creek, J Fell Creek, Jacobs Ladder Creek, Lewis Creek, Liberty Creek, Lick Creek, Lime Creek... Creek, South Fork Chamberlain Creek, South Fork Holy Terror Creek, South Fork Norton Creek, South Fork...

14 CFR 23.201 - Wings level stall.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Wings level stall. 23.201 Section 23.201... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Flight Stalls § 23.201 Wings level... airplane stalls. (b) The wings level stall characteristics must be demonstrated in flight as follows...

14 CFR 25.207 - Stall warning.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Stall warning. 25.207 Section 25.207... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Stalls § 25.207 Stall warning. (a) Stall warning with... be clear and distinctive to the pilot in straight and turning flight. (b) The warning must be...

14 CFR 23.207 - Stall warning.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Stall warning. 23.207 Section 23.207... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Flight Stalls § 23.207 Stall warning. (a) There must be a clear and distinctive stall warning, with the flaps and landing gear in any...

14 CFR 25.207 - Stall warning.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Stall warning. 25.207 Section 25.207... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Stalls § 25.207 Stall warning. (a) Stall warning with... be clear and distinctive to the pilot in straight and turning flight. (b) The warning must be...

14 CFR 23.207 - Stall warning.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Stall warning. 23.207 Section 23.207... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Flight Stalls § 23.207 Stall warning. (a) There must be a clear and distinctive stall warning, with the flaps and landing gear in any...

14 CFR 23.207 - Stall warning.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Stall warning. 23.207 Section 23.207... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Flight Stalls § 23.207 Stall warning. (a) There must be a clear and distinctive stall warning, with the flaps and landing gear in any...

14 CFR 25.207 - Stall warning.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Stall warning. 25.207 Section 25.207... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Stalls § 25.207 Stall warning. (a) Stall warning with... be clear and distinctive to the pilot in straight and turning flight. (b) The warning must be...

14 CFR 23.207 - Stall warning.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Stall warning. 23.207 Section 23.207... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Flight Stalls § 23.207 Stall warning. (a) There must be a clear and distinctive stall warning, with the flaps and landing gear in any...

14 CFR 25.207 - Stall warning.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Stall warning. 25.207 Section 25.207... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Stalls § 25.207 Stall warning. (a) Stall warning with... be clear and distinctive to the pilot in straight and turning flight. (b) The warning must be...

14 CFR 25.207 - Stall warning.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Stall warning. 25.207 Section 25.207... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Stalls § 25.207 Stall warning. (a) Stall warning with... be clear and distinctive to the pilot in straight and turning flight. (b) The warning must be...

14 CFR 23.207 - Stall warning.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Stall warning. 23.207 Section 23.207... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Flight Stalls § 23.207 Stall warning. (a) There must be a clear and distinctive stall warning, with the flaps and landing gear in any...

Dynamic Stall Patterns

NASA Astrophysics Data System (ADS)

Davidson, Phillip; Babbitt, Ashli; Magstadt, Andrew; Nikoueeyan, Pourya; Naughton, Jonathan; Jonathan Naughton Team

2014-11-01

The performance of helicopter and wind turbine blades is affected by dynamic stall. Dynamic stall has received considerable attention, but it is still difficult to simulate and not fully understood. Over the past seven years, many airfoils for helicopter and wind turbine use ranging from 9.5 to 30% thick have been experimentally tested and simulated while dynamically pitching to further characterize dynamic stall. Tests have been run at chord Reynolds number between 225,000-440,000 for various reduced frequencies, mean angles of attack, and oscillation amplitudes. Characterization of stall has been accomplished using data from previous studies as well as the unsteady pressure and flow-field data available from our own work. Where available, combined surface and flow-field data allow for clear identification of the types of stall observed and the flow structure associated with them. The results indicate that thin airfoil stall, leading edge stall, and trailing edge stall are observed in the oscillating airfoil experiments and simulations. These three main stall types are further divided into subcategories. By improving our understanding of the features of dynamic stall, it is expected that physics-based simulations can be improved. Work supported by DOE and a gift from BP.

14 CFR 25.103 - Stall speed.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Stall speed. 25.103 Section 25.103... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Performance § 25.103 Stall speed. (a) The reference stall speed, VSR, is a calibrated airspeed defined by the applicant. VSR may not be less than a 1-g stall...

14 CFR 25.103 - Stall speed.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Stall speed. 25.103 Section 25.103... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Performance § 25.103 Stall speed. (a) The reference stall speed, VSR, is a calibrated airspeed defined by the applicant. VSR may not be less than a 1-g stall...

24. View of one of the plaques from Clark Fork ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

24. View of one of the plaques from Clark Fork Vehicle Bridge. Presently located at the Bonner County Historical Museum in Sandpoint, Idaho. A plaque was attached at each end of the bridge. Only one remains. - Clark Fork Vehicle Bridge, Spanning Clark Fork River, serves Highway 200, Clark Fork, Bonner County, ID

77 FR 76420 - Final Flood Elevation Determinations

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-28

... Fort Gay, confluence. Unincorporated Areas of Wayne County. At the Tug Fork +575 confluence. Mill Creek (backwater effects from From the Tug Fork +575 Town of Fort Gay. Tug Fork). confluence to approximately 1.1 miles upstream of the Tug Fork confluence. Tug Fork At the Big Sandy River +575 Town of Fort Gay...

Analysis of oscillatory pressure data including dynamic stall effects

NASA Technical Reports Server (NTRS)

Carta, F. O.

1974-01-01

The dynamic stall phenomenon was examined in detail by analyzing an existing set of unsteady pressure data obtained on an airfoil oscillating in pitch. Most of the data were for sinusoidal oscillations which penetrated the stall region in varying degrees, and here the effort was concentrated on the chordwise propagation of pressure waves associated with the dynamic stall. It was found that this phenomenon could be quantified in terms of a pressure wave velocity which is consistently much less than free-stream velocity, and which varies directly with frequency. It was also found that even when the stall region has been deeply penetrated and a substantial dynamic stall occurs during the downstroke, stall recovery near minimum incidence will occur, followed by a potential flow behavior up to stall inception.

Suppression of dynamic stall with a leading-edge slat on a VR-7 airfoil

NASA Technical Reports Server (NTRS)

Mcalister, K. W.; Tung, C.

1993-01-01

The VR-7 airfoil was experimentally studied with and without a leading-edge slat at fixed angles of attack from 0 deg to 30 deg at Re = 200,000 and for unsteady pitching motions described by alpha equals alpha(sub m) + 10 deg(sin(wt)). The models were two dimensional, and the test was performed in a water tunnel at Ames Research Center. The unsteady conditions ranged over Re equals 100,000 to 250,000, k equals 0.001 to 0.2, and alpha(sub m) = 10 deg to 20 deg. Unsteady lift, drag, and pitching-moment measurements were obtained along with fluorescent-dye flow visualizations. The addition of the slat was found to delay the static-drag and static-moment stall by about 5 degrees and to eliminate completely the development of a dynamic-stall vortex during unsteady motions that reached angles as high as 25 degrees. In all of the unsteady cases studied, the slat caused a significant reduction in the force and moment hysteresis amplitudes. The reduced frequency was found to have the greatest effect on the results, whereas the Reynolds number had little effect on the behavior of either the basic or the slatted airfoil. The slat caused a slight drag penalty at low angles of attack, but generally increased the lift/drag ratio when averaged over the full cycle of oscillation.

Comfort zone-design free stalls: do they influence the stall use behavior of lame cows?

PubMed

Cook, N B; Marin, M J; Mentink, R L; Bennett, T B; Schaefer, M J

2008-12-01

The behavior of 59 cows in 4 herds, each with Comfort Zone-design free stalls with dimensions suitable for 700-kg, mature Holstein dairy cows, was filmed for a 48-h period. Comparison was made between nonlame, slightly lame, and moderately lame cows on either rubber-crumb-filled mattress stall surfaces bedded with a small amount of sawdust (2 herds) or a Pack Mat design, which consisted of a rubber-crumb-filled mattress pad installed 5 cm below a raised rear curb, bedded with 5 to 8 cm of sand bedding (2 herds). All other stall design components were similar. Despite adequate resting space and freedom to perform normal rising and lying movements, lame cows on mattresses stood in the stall for >2 h longer than nonlame cows. Although a significant increase in stall standing behavior was observed in lame cows on Pack Mat stalls, the mean (95% confidence interval) standing time in the stall was only 0.7 (0 to 3.0) h/d for nonlame cows and 1.6 (0 to 4.2) h/d for moderately lame cows, which was less than the 2.1 (0 to 4.4), 4.3 (1.6 to 6.9), and 4.9 (2.5 to 7.3) h/d spent standing in the stall for nonlame, slightly lame, and moderately lame cows on mattresses, respectively. This observation supports the hypothesis that it is the nature of the stall surface that dictates changes in stall standing behavior observed in lame cows, rather than other components of stall design. The finding that only 5 to 8 cm of sand over a mattress pad provides most of the benefits of deep sand-bedded stalls, along with other advantages related to stall maintenance and manure handling, gives farmers another useful housing alternative with which to improve cow comfort and well-being.

Detection of Mycobacterium avium subspecies paratuberculosis in tie-stall dairy herds using a standardized environmental sampling technique and targeted pooled samples.

PubMed

Arango-Sabogal, Juan C; Côté, Geneviève; Paré, Julie; Labrecque, Olivia; Roy, Jean-Philippe; Buczinski, Sébastien; Doré, Elizabeth; Fairbrother, Julie H; Bissonnette, Nathalie; Wellemans, Vincent; Fecteau, Gilles

2016-07-01

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease, a chronic contagious enteritis of ruminants that causes major economic losses. Several studies, most involving large free-stall herds, have found environmental sampling to be a suitable method for detecting MAP-infected herds. In eastern Canada, where small tie-stall herds are predominant, certain conditions and management practices may influence the survival and transmission of MAP and recovery (isolation). Our objective was to estimate the performance of a standardized environmental and targeted pooled sampling technique for the detection of MAP-infected tie-stall dairy herds. Twenty-four farms (19 MAP-infected and 5 non-infected) were enrolled, but only 20 were visited twice in the same year, to collect 7 environmental samples and 2 pooled samples (sick cows and cows with poor body condition). Concurrent individual sampling of all adult cows in the herds was also carried out. Isolation of MAP was achieved using the MGIT Para TB culture media and the BACTEC 960 detection system. Overall, MAP was isolated in 7% of the environmental cultures. The sensitivity of the environmental culture was 44% [95% confidence interval (CI): 20% to 70%] when combining results from 2 different herd visits and 32% (95% CI: 13% to 57%) when results from only 1 random herd visit were used. The best sampling strategy was to combine samples from the manure pit, gutter, sick cows, and cows with poor body condition. The standardized environmental sampling technique and the targeted pooled samples presented in this study is an alternative sampling strategy to costly individual cultures for detecting MAP-infected tie-stall dairies. Repeated samplings may improve the detection of MAP-infected herds.

Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response

PubMed Central

Wang, Yucai; Han, Xiao; Wu, Fangming; Leung, Justin W; Lowery, Megan G; Do, Huong; Chen, Junjie; Shi, Chaowei; Tian, Changlin; Li, Lei; Gong, Weimin

2013-01-01

The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation. PMID:23999858

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins*

PubMed Central

Matthews, Lindsay A.; Selvaratnam, Rajeevan; Jones, Darryl R.; Akimoto, Madoka; McConkey, Brendan J.; Melacini, Giuseppe; Duncker, Bernard P.; Guarné, Alba

2014-01-01

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules. PMID:24285546

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.

Crystal structure of the Mus81-Eme1 complex.

PubMed

Chang, Jeong Ho; Kim, Jeong Joo; Choi, Jung Min; Lee, Jung Hoon; Cho, Yunje

2008-04-15

The Mus81-Eme1 complex is a structure-specific endonuclease that plays an important role in rescuing stalled replication forks and resolving the meiotic recombination intermediates in eukaryotes. We have determined the crystal structure of the Mus81-Eme1 complex. Both Mus81 and Eme1 consist of a central nuclease domain, two repeats of the helix-hairpin-helix (HhH) motif at their C-terminal region, and a linker helix. While each domain structure resembles archaeal XPF homologs, the overall structure is significantly different from those due to the structure of a linker helix. We show that a flexible intradomain linker that formed with 36 residues in the nuclease domain of Eme1 is essential for the recognition of DNA. We identified several basic residues lining the outer surface of the active site cleft of Mus81 that are involved in the interaction with a flexible arm of a nicked Holliday junction (HJ). These interactions might contribute to the optimal positioning of the opposite junction across the nick into the catalytic site, which provided the basis for the "nick and counternick" mechanism of Mus81-Eme1 and for the nicked HJ to be the favored in vitro substrate of this enzyme.

Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

PubMed Central

Ahmed, A; Yang, J; Maya-Mendoza, A; Jackson, D A; Ashcroft, M

2011-01-01

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1. PMID:21593792

Human PrimPol activity is enhanced by RPA.

PubMed

Martínez-Jiménez, María I; Lahera, Antonio; Blanco, Luis

2017-04-10

Human PrimPol is a primase belonging to the AEP superfamily with the unique ability to synthesize DNA primers de novo, and a non-processive DNA polymerase able to bypass certain DNA lesions. PrimPol facilitates both mitochondrial and nuclear replication fork progression either acting as a conventional TLS polymerase, or repriming downstream of blocking lesions. In vivo assays have shown that PrimPol is rapidly recruited to sites of DNA damage by interaction with the human replication protein A (RPA). In agreement with previous findings, we show here that the higher affinity of RPA for ssDNA inhibits PrimPol activities in short ssDNA templates. In contrast, once the amount of ssDNA increases up to a length in which both proteins can simultaneously bind ssDNA, as expected during replicative stress conditions, PrimPol and RPA functionally interact, and their binding capacities are mutually enhanced. When using M13 ssDNA as template, RPA stimulated both the primase and polymerase activities of PrimPol, either alone or in synergy with Polε. These new findings supports the existence of a functional PrimPol/RPA association that allows repriming at the exposed ssDNA regions formed in the leading strand upon replicase stalling.

The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA.

PubMed

Torseth, Kathrin; Doseth, Berit; Hagen, Lars; Olaisen, Camilla; Liabakk, Nina-Beate; Græsmann, Heidi; Durandy, Anne; Otterlei, Marit; Krokan, Hans E; Kavli, Bodil; Slupphaug, Geir

2012-06-01

In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Representative Stall Model of Regional Aircraft for Simulator Training Using a Spline Shape Prescriptive Modeling Approach

NASA Astrophysics Data System (ADS)

Zhang, Tony S.

Loss-of-control following aerodynamic stall remains the largest contributor to fatal civil aviation accidents. Aerodynamic models past stall are required to train pilots on stall recovery techniques using ground-based simulators, which are safe, inexpensive, and accessible. A methodology for creating representative stall models, which capture essential stall characteristics, is being developed for classes of twin-turboprop commuter and twin-engine regional jet aircraft. Despite having lower fidelity than type specific stall models generated from wind tunnel, flight test, and/or CFD studies data, these models are configuration adjustable and significantly cheaper to construct for high angle-of-attack regimes. Baseline specific stall models are modified to capture changes in aerodynamic coefficients due to configuration variations from a baseline to a target aircraft. A Shape Prescriptive Modeling approach combining existing theory and data using least-squares splines is used to make coefficient change predictions. Initial results are satisfactory and suggest that representative models are suitable for stall training.

Investigation of nonlinear inviscid and viscous flow effects in the analysis of dynamic stall. [air flow and chordwise pressure distribution on airfoil below stall condition

NASA Technical Reports Server (NTRS)

Crimi, P.

1974-01-01

A method for analyzing unsteady airfoil stall was refined by including nonlinear effects in the representation of the inviscid flow. Certain other aspects of the potential-flow model were reexamined and the effects of varying Reynolds number on stall characteristics were investigated. Refinement of the formulation improved the representation of the flow and chordwise pressure distribution below stall, but substantial quantitative differences between computed and measured results are still evident for sinusoidal pitching through stall. Agreement is substantially improved by assuming the growth rate of the dead-air region at the onset of leading-edge stall is of the order of the component of the free stream normal to the airfoil chordline. The method predicts the expected increase in the resistance to stalling with increasing Reynolds number. Results indicate that a given airfoil can undergo both trailing-edge and leading-edge stall under unsteady conditions.

Stall dimensions and the prevalence of lameness, injury, and cleanliness on 317 tie-stall dairy farms in Ontario

PubMed Central

2005-01-01

Abstract The study objectives were to provide a province-wide description of stall dimensions and the aspects of cattle welfare linked to stall design in the tie-stall industry. Data on stall design; stall dimensions; and the prevalence of lameness, injury, and hind limb and udder cleanliness in lactating dairy cattle were collected from a sample of 317 tie-stall farms across Ontario. The majority of the study farms (90%) had stalls with dimensions (length, width, tie-chain length, and tie rail height) that were less than the current recommendations. This may explain, in part, the prevalence of lameness measured as the prevalence of back arch (3.2%) and severe hind claw rotation (23%), hock lesions (44%), neck lesions (3.8%), broken tails (3%), dirty hind limbs (23%), and dirty udders (4.6%). Veterinarians and producers may use this information to compare farms with the industry averages and target areas in need of improvement. PMID:16454382

Value loss from weevil-caused defects in eastern white pine lumber

Treesearch

Myron D. Ostrander; Carl H. Stoltenberg

1957-01-01

Owners of eastern white pine stands suffer financially in several ways from attacks by the white-pine weevil (Pissodes strobi). Crooks, forks, and other weevil-caused tree-bole deformities increase bucking, logging, and sawing costs, and they reduce recoverable volumes. The injuries also reduce the average value of the lumber recovered. It is only with this reduction...

Rinne revisited: steel versus aluminum tuning forks.

PubMed

MacKechnie, Cheryl A; Greenberg, Jesse J; Gerkin, Richard C; McCall, Andrew A; Hirsch, Barry E; Durrant, John D; Raz, Yael

2013-12-01

(1) Determine whether tuning fork material (aluminum vs stainless steel) affects Rinne testing in the clinical assessment of conductive hearing loss (CHL). (2) Determine the relative acoustic and mechanical outputs of 512-Hz tuning forks made of aluminum and stainless steel. Prospective, observational. Outpatient otology clinic. Fifty subjects presenting May 2011 to May 2012 with negative or equivocal Rinne in at least 1 ear and same-day audiometry. Rinne test results using aluminum and steel forks were compared and correlated with the audiometric air-bone gap. Bench top measurements using sound-level meter, microphone, and artificial mastoid. Patients with CHL were more likely to produce a negative Rinne test with a steel fork than with an aluminum fork. Logistic regression revealed that the probability of a negative Rinne reached 50% at a 19 dB air-bone gap for stainless steel versus 27 dB with aluminum. Bench top testing revealed that steel forks demonstrate, in effect, more comparable air and bone conduction efficiencies while aluminum forks have relatively lower bone conduction efficiency. We have found that steel tuning forks can detect a lesser air-bone gap compared to aluminum tuning forks. This is substantiated by observations of clear differences in the relative acoustic versus mechanical outputs of steel and aluminum forks, reflecting underlying inevitable differences in acoustic versus mechanical impedances of these devices, and thus efficiency of coupling sound/vibratory energy to the auditory system. These findings have clinical implications for using tuning forks to determine candidacy for stapes surgery.

Comparison of dynamic stall phenomena for pitching and vertical translation motions

NASA Technical Reports Server (NTRS)

Fukushima, T.; Dadone, L. U.

1977-01-01

Test data for vertical translation motions of the V0012 and V23010-1.58 airfoils were compared with force pitch and oscillation data to determine qualitative differences in dynamic stall behavior. Chordwise differential pressure variations were examined in detail for the test conditions displaying dynamic stall. The comparison revealed a number of differences both in the onset of stall and in the progression separation as a function of the type of motion. The evidence of secondary stall events following the recovery from initial stall were found to be dependent on the type of motion, but additional data will be needed to incorporate vertical translation effects into the empirical approximation of dynamic stall.

Flow Visualization of Dynamic Stall on an Oscillating Airfoil

DTIC Science & Technology

1989-09-01

Dynamic Stall; Dynamic lift, ’Unsteady lift; Helicopter retreating blade stall; Oscillating airfoil ; Flow visualization,’Schlieren method ;k ez.S-,’ .0...the degree of MASTER OF SCIENCE IN AERONAUTICAL ENGINEERING from the NAVAL POSTGRADUATE SCHOOL September 1989 Author...and moment behavior is quite different from the static stall associated with fixed-wing airfoils . Helicopter retreating blade stall is a dynamic

Stage effects on stalling and recovery of a high-speed 10-stage axial-flow compressor

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

Copenhaver, W.W.

1988-01-01

Results of a high-speed 10-stage axial-flow compressor test involving overall compressor and individual stage performance while stalling and operating in quasi-steady rotating stall are described. Test procedures and data-acquisition methods used to obtain the dynamic stalling and quasi-steady in-stall data are explained. Unstalled and in-stall time-averaged data obtained from the compressor operating at five different shaft speeds and one off-schedule variable vane condition are presented. Effects of compressor speed and variable geometry on overall compressor in-stall pressure rise and hysteresis extent are illustrated through the use of quasi-steady-stage temperature rise and pressure-rise characteristics. Results indicate that individual stage performance duringmore » overall compressor rotating stall operation varies considerably throughout the length of the compressor. The measured high-speed 10-stage test compressor individual stage pressure and temperature characteristics were input into a stage-by-stage dynamic compressor performance model. Comparison of the model results and measured pressures provided the additional validation necessary to demonstrate the model's ability to predict high-speed multistage compressor stalling and in-stall performance.« less

Preferences of dairy cows for three stall surface materials with small amounts of bedding.

PubMed

Norring, M; Manninen, E; de Passillé, A M; Rushen, J; Saloniemi, H

2010-01-01

Farmers' concerns about the economy, cost of labor, and hygiene have resulted in reduced use of organic bedding in stalls for dairy cows; however, the reduced use of organic bedding possibly impairs cow comfort. The effects of different stall surface materials were evaluated in an unheated building in which only a small amount of bedding was used. The lying time and preferences of 18 cows using 3 stall surface materials (concrete, soft rubber mat, and sand) were compared. All materials were lightly bedded with a small amount of straw, and the amount of straw added to each stall was measured. The cows only had access to stalls of one surface type while their lying time was observed. Lying times were longest on the rubber mats compared with other surfaces (rubber mat 768; concrete 727; sand 707+/-16 min/d). In a preference test, cows had access to 2 of the 3 types of stalls for 10 d and their stall preference was measured. Cows preferred stalls with rubber mats to stalls with a concrete floor (median 73 vs. 18 from a total of 160 observations per day; interquartile range was 27 and 12, respectively), but showed no preference for sand stalls compared with stalls with a concrete floor or with rubber mats. More straw was needed on sand stalls compared with concrete or mat (638+/-13 g/d on sand, 468+/-10 g/d on concrete, and 464+/-8 g/d on rubber mats). Lying times on bedded mats indicated that mats were comfortable for the cows. If availability or cost of bedding material requires limiting the amount of bedding used, rubber mats may help maintain cow comfort. Copyright 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

USAF Test Pilot School. Flying Qualities Textbook, Volume 2 Part 2

DTIC Science & Technology

1986-04-01

regime that precipitates entry into a PSG, spin, or deep stall condition (MIL-F-83691A, Reference 10.4, Paragraph 6.3.9). Notice two things about...motions may result after departure - the aircraft enters either a PSG, spin, or deep stall (of course, a PSG can progress into a spin or deep stall...gyration," "spin" and " deep stalls," used to define a departure. 10.3.1.3 Post-Stall Gyration. A post-stall gyration is an uncontrolled motion about one

Enhancement of DNA ligase I level by gemcitabine in human cancer cells.

PubMed

Sun, Daekyu; Urrabaz, Rheanna; Kelly, Susan; Nguyen, Myhanh; Weitman, Steve

2002-04-01

DNA ligase I is an essential enzyme for completing DNA replication and DNA repair by ligating Okazaki fragments and by joining single-strand breaks formed either directly by DNA-damaging agents or indirectly by DNA repair enzymes, respectively. In this study, we examined whether the DNA ligase I level could be modulated in human tumor cell lines by treatment with gemcitabine (2', 2'-difluoro-2'-deoxycytidine), which is a nucleoside analogue of cytidine with proven antitumor activity against a broad spectrum of human cancers in clinical studies. To determine the effect of gemcitabine on DNA ligase I expression, Western blot analysis was used to measure the DNA ligase I levels in MiaPaCa, NGP, and SK-N-BE cells treated with different concentrations of gemcitabine and harvested at different time intervals. Cell cycle analysis was also performed to determine the underlying mechanism of DNA ligase I level enhancement in response to gemcitabine. In addition, other agents that share the same mechanism of action with gemcitabine were used to elucidate further details. When different types of tumor cell lines, including MiaPaCa, NGP, and SK-N-BE, were treated with gemcitabine, the level of DNA ligase I increased severalfold despite significant cell growth inhibition. In contrast, other DNA ligases (III and IV) either remained unchanged or decreased with treatment. Cell cycle analysis showed that arrest in S-phase corresponded to an increase of DNA ligase I levels in gemcitabine treated cells. Other agents, such as 1-beta-D-arabinofuranosylcytosine and hydroxyurea, which partly share mechanisms of action with gemcitabine by targeting DNA polymerases and ribonucleotide reductase, respectively, also caused an increase of DNA ligase I levels. However, 5-fluorouracil, which predominantly targets thymidylate synthase, did not cause an increase of DNA ligase I level. Our results suggest that an arrest of DNA replication caused by gemcitabine treatment through incorporation of gemcitabine triphosphate into replicating DNA and inhibition of ribonucleotide reductase would trigger an increase in DNA ligase I levels in cancer cells. The elevated presence of DNA ligase I in S-phase-arrested cells leads us to speculate that DNA ligase I might have an important role in repairing DNA damage caused by stalled replication forks.

Differences in survival and growth in hatchery and stream environments, and in maturation of residuls in a stream, between progeny of hatchery and wild steelhead (Study sites: Brushy Fork Creek and Dworshak Hatchery; Stocks:Dworshak hatchery and Fish Creek wild; Year classes: 1992 and 1993): Chapter 1

USGS Publications Warehouse

Rubin, Stephen P.; Reisenbichler, Reginald R.; Wetzel, Lisa A.; Hensleigh, Jay E.; Leonetti,; Rubin, Stephen P.; Reisenbichler, Reginald R.; Wetzel, Lisa A.; Hayes, Michael C.

2012-01-01

Freshwater survival in hatchery and natural rearing environments was compared between progeny of hatchery (H) and wild (W) steelhead Oncorhynchus mykiss from the Clearwater River drainage in Idaho. Adults from Dworshak National Fish Hatchery and wild adults from Fish Creek fish were artificially spawned, and their progeny were genetically marked at the PEPA allozyme locus and released together as unfed fry in production facilities at the hatchery and in Brushy Fork Creek, also in the Clearwater River drainage, in a common garden design. Survival was higher for H than for W progeny at the hatchery but lower for H than for W progeny in Brushy Fork, indicating reduced fitness of the hatchery population for natural rearing and suggesting domestication as the cause. Survival at the hatchery was lower than is typical due to disease outbreaks. Survival of the first year-class of experimental fish to smolt release was only 18%. Survival of H fish was 3.8 times that of W fish under these poor survival conditions. All fish from the second year-class died halfway through the scheduled 10 month rearing period. Survival of H fish was 5.2 times that of W fish to when 1% of the initial fry were still alive indicating that W fish succumbed to the epizootic sooner than did H fish. Emigrants from the Brushy Fork study reach were sampled for three years and fish residing in the study reach were sampled for six years following fry release. Most emigrants were one or two years old and too small to be smolts (mean fork length at age-2 = 93 mm). Survival in Brushy Fork was lower for H than for W fish of the first year-class. Survival of the second year-class was higher for H than for W fish during the first two months in the stream but was lower for H than for W fish thereafter, and net survival from release to ages 3 and older was also lower for H than for W fish if our emigrant samples were representative (periods of inoperative emigrant traps prevented certainty about this). Differences between progeny groups were also found for growth (H>W) and condition (H>W) in the hatchery and downstream migration success of hatchery-reared fish after release (H>W), and for growth (HW for the other), condition (H>W), downstream dispersal (H>W for one year-class; H=W for the other), and maturation of residuals (ovaries weight was greater for H than for W females at ages 4 and 5; testes weight was less for H than for W age-3 males of one year-class) in Brushy Fork. A thunderstorm-induced power outage interrupted flow to the incubation trays at the hatchery and compromised a major tenet of the common garden design for the second year-class, possibly contributing to the inconsistency in relative survivals in Brushy Fork between year-classes. The storm caused the incubation environment to differ between the stocks as a result of reduced oxygen levels and substantially higher densities for H alevins. This difference was illustrated by a 55% loss for H fish during the event, about twice that for W fish.

Dimension Determination of Precursive Stall Events in a Single Stage High Speed Compressor

NASA Technical Reports Server (NTRS)

Bright, Michelle M.; Qammar, Helen K.; Hartley, Tom T.

1996-01-01

This paper presents a study of the dynamics for a single-stage, axial-flow, high speed compressor core, specifically, the NASA Lewis rotor stage 37. Due to the overall blading design for this advanced core compressor, each stage has considerable tip loading and higher speed than most compressor designs, thus, the compressor operates closer to the stall margin. The onset of rotating stall is explained as bifurcations in the dynamics of axial compressors. Data taken from the compressor during a rotating stall event is analyzed. Through the use of a box-assisted correlation dimension methodology, the attractor dimension is determined during the bifurcations leading to rotating stall. The intent of this study is to examine the behavior of precursive stall events so as to predict the entrance into rotating stall. This information may provide a better means to identify, avoid or control the undesirable event of rotating stall formation in high speed compressor cores.

Why do Cross-Flow Turbines Stall?

NASA Astrophysics Data System (ADS)

Cavagnaro, Robert; Strom, Benjamin; Polagye, Brian

2015-11-01

Hydrokinetic turbines are prone to instability and stall near their peak operating points under torque control. Understanding the physics of turbine stall may help to mitigate this undesirable occurrence and improve the robustness of torque controllers. A laboratory-scale two-bladed cross-flow turbine operating at a chord-based Reynolds number ~ 3 ×104 is shown to stall at a critical tip-speed ratio. Experiments are conducting bringing the turbine to this critical speed in a recirculating current flume by increasing resistive torque and allowing the rotor to rapidly decelerate while monitoring inflow velocity, torque, and drag. The turbine stalls probabilistically with a distribution generated from hundreds of such events. A machine learning algorithm identifies stall events and indicates the effectiveness of available measurements or combinations of measurements as predictors. Bubble flow visualization and PIV are utilized to observe fluid conditions during stall events including the formation, separation, and advection of leading-edge vortices involved in the stall process.

The fecundity of fork-tailed threadfin bream (Nemipterus furcosus) in Bangka, Bangka Belitung

NASA Astrophysics Data System (ADS)

Utami, E.; Safitriyani, E.; Gatra Persada, Leo

2018-04-01

Fork-tailed threadfin bream (Nemipterus furcosus) is one of important economic fishes in Bangka. The sustainability of fork-tailed threadfin bream is threatened by degradation of natural habitat. Information of reproductive is needed for further management. The objective of this study was to examine fecundity of fork-tailed threadfin bream. The mean values of temperature was 28.83 ± 0,37°C, respectively. Sex ratio during sampling showed that female fork-tailed threadfin bream greater than male population. Berried female fork-tailed threadfin bream found from March until November. The greatest number of berried female fork-tailed threadfin bream showed in July with berried female value of 25. Fork-tailed threadfin bream fecundity was 19951 and 66628, respectively. The fecundity data can be used to access the reproductive potential of fish stock and also as an assessment on stock size of their natural population.

Termination of DNA replication forks: "Breaking up is hard to do".

PubMed

Bailey, Rachael; Priego Moreno, Sara; Gambus, Agnieszka

2015-01-01

To ensure duplication of the entire genome, eukaryotic DNA replication initiates from thousands of replication origins. The replication forks move through the chromatin until they encounter forks from neighboring origins. During replication fork termination forks converge, the replisomes disassemble and topoisomerase II resolves the daughter DNA molecules. If not resolved efficiently, terminating forks result in genomic instability through the formation of pathogenic structures. Our recent findings shed light onto the mechanism of replisome disassembly upon replication fork termination. We have shown that termination-specific polyubiquitylation of the replicative helicase component - Mcm7, leads to dissolution of the active helicase in a process dependent on the p97/VCP/Cdc48 segregase. The inhibition of terminating helicase disassembly resulted in a replication termination defect. In this extended view we present hypothetical models of replication fork termination and discuss remaining and emerging questions in the DNA replication termination field.

Sortal Concepts and Pragmatic Inference in Children's Early Quantification of Objects

ERIC Educational Resources Information Center

Srinivasan, Mahesh; Chestnut, Eleanor; Li, Peggy; Barner, David

2013-01-01

It is typically assumed that count nouns like "fork" act as logical sortals, specifying whether objects are countable units of a kind (e.g., that a whole fork counts as "one fork") or not (e.g., that a piece of a fork does not count as "one fork"). In four experiments, we provide evidence from linguistic and conceptual development that nouns do…

11. Photocopy of photograph (original copy in Edison collection). Photographer ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

11. Photocopy of photograph (original copy in Edison collection). Photographer and date unknown, although photo taken prior to 1930 reconstruction of Project flumes. VIEW OF ORIGINAL SOUTH FORK OF THE TULE RIVER MIDDLE FORK "BOX" WOOD FLUME BRANCH SHOWING NORTH FORK OF TULE RIVER MIDDLE FORK CROSSING. VIEW TO NORTHWEST. - Tule River Hydroelectric Project, Water Conveyance System, Middle Fork Tule River, Springville, Tulare County, CA

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression.

PubMed

Franz, André; Pirson, Paul A; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

2016-02-04

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

PubMed Central

Franz, André; Pirson, Paul A.; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

2016-01-01

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. PMID:26842564

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

Day, I.J.; Breuer, T.; Escuret, J.

As part of a European collaborative project, four high-speed compressors were tested to investigate the generic features of stall inception in aero-engine type compressors. Tests were run over the full speed range to identify the design and operating parameters that influence the stalling process. A study of data analysis techniques was also conducted in the hope of establishing early warning of stall. The work presented here is intended to relate the physical happenings in the compressor to the signals that would be received by an active stall control system. The measurements show a surprising range of stall-related disturbances and suggestmore » that spike-type stall inception is a feature of low-speed operation while modal activity is clearest in the midspeed range. High-frequency disturbances were detected at both ends of the speed range and nonrotating stall, a new phenomenon, was detected in three out of the four compressors. The variety of the stalling patterns, and the ineffectiveness of the stall warning procedures, suggests that the ultimate goal of a flightworthy active control system remains some way off.« less

Grand Forks/East Grand Forks ITS strategy plan

DOT National Transportation Integrated Search

2001-01-15

The Grand Forks/East Grand Forks (GF/EGF) Area's Intelligent Transportation Systems (ITS) Strategy Plan is an effort by the GF/EGF Metropolitan Planning Organization (MPO) and its partners to develop a plan for deploying Intelligent Transportation Sy...

77 FR 39675 - Wallowa-Whitman National Forest, Baker County, OR; North Fork Burnt River Mining

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-05

...-Whitman National Forest, Baker County, OR; North Fork Burnt River Mining AGENCY: Forest Service, USDA... North Fork Burnt River Mining Record of Decision will replace and supercede the 2004 North Fork Burnt...

Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.

PubMed

Feng, Wenyi; Di Rienzi, Sara C; Raghuraman, M K; Brewer, Bonita J

2011-10-01

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or "collapsed" replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.

Close-loop Dynamic Stall Control on a Pitching Airfoil

NASA Astrophysics Data System (ADS)

Giles, Ian; Corke, Thomas

2017-11-01

A closed-loop control scheme utilizing a plasma actuator to control dynamic stall is presented. The plasma actuator is located at the leading-edge of a pitching airfoil. It initially pulses at an unsteady frequency that perturbs the boundary layer flow over the suction surface of the airfoil. As the airfoil approaches and enters stall, the amplification of the unsteady disturbance is detected by an onboard pressure sensor also located near the leading edge. Once detected, the actuator is switched to a higher voltage control state that in static airfoil experiments would reattach the flow. The threshold level of the detection is a parameter in the control scheme. Three stall regimes were examined: light, medium, and deep stall, that were defined by their stall penetration angles. The results showed that in general, the closed-loop control scheme was effective at controlling dynamic stall. The cycle-integrated lift improved in all cases, and increased by as much as 15% at the lowest stall penetration angle. As important, the cycle-integrated aerodynamic damping coefficient also increased in all cases, and was made to be positive at the light stall regime where it traditionally is negative. The latter is important in applications where negative damping can lead to stall flutter.

General perspective view of the North Fork Butter Creek Bridge, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

General perspective view of the North Fork Butter Creek Bridge, view looking southwest - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

Approach view of the North Fork Butter Creek Bridge, view ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Approach view of the North Fork Butter Creek Bridge, view looking south - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

General perspective view of the North Fork Butter Creek Bridge, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

General perspective view of the North Fork Butter Creek Bridge, view looking north - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

Elevation view of the North Fork Butter Creek Bridge, view ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Elevation view of the North Fork Butter Creek Bridge, view looking west - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

Approach view of the North Fork Butter Creek Bridge, view ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Approach view of the North Fork Butter Creek Bridge, view looking north - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

Detail perspective view of the North Fork Butter Creek Bridge, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Detail perspective view of the North Fork Butter Creek Bridge, view looking southwest - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

General perspective view of the North Fork Butter Creek Bridge, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

General perspective view of the North Fork Butter Creek Bridge, view looking south - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

The quest for stall-free dynamic lift

NASA Technical Reports Server (NTRS)

Tung, C.; Mcalister, K. W.; Carr, Lawrence W.; Duque, E.; Zinner, R.

1992-01-01

During the past decade, numerous major effects have addressed the question of how to control or alleviate dynamic stall effects on helicopter rotors, but little concrete evidence of any significant reduction of the adverse characteristics of the dynamic stall phenomenon has been demonstrated. Nevertheless, it is important to remember that the control of dynamic stall is an achievable goal. Experiments performed at the US Army Aeroflight-dynamics Directorate more than a decade ago demonstrated that dynamic stall is not an unavoidable penalty of high amplitude motion, and that airfoils can indeed operate dynamically at angles far above the static-stall angle without necessarily forming a stall vortex. These experiments, one of them featuring a slat that was designed from static airfoil considerations, showed that unsteadiness can be a very beneficial factor in the development of high-lift devices for helicopter rotors. The experience drawn from these early experiments is now being focused on a program for the alleviation of dynamic-stall effects on helicopter rotors. The purpose of this effort is to demonstrate that rotor stall can be controlled through an improved understanding of the unsteady effects on airfoil stall and to document the role of specific means that lead to stall alleviation in the three dimensional unsteady environment of helicopter rotors in forward flight. The first concept to be addressed in this program will be a slatted airfoil. A two dimensional unsteady Navier-Stokes code has been modified to compute the flow around a two-element airfoil.

77 FR 73279 - Airworthiness Directives; Saab AB, Saab Aerosystems Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-10

... AD was prompted by reports of stall events during icing conditions where the natural stall warning (buffet) was not identified. This AD requires replacing the stall warning computer (SWC) with a new SWC, which provides an artificial stall [[Page 73280

The "stall barrier" as a new preventive in general aviation accidents.

DOT National Transportation Integrated Search

1966-09-01

An elementary device, actuated by the conventional stall warning vane, is described which can be inexpensively installed in any aircraft. The new device, the Stall Barrier, prevents stalls through (1) warning the pilot through the 'touch sense' of th...

Assessment of hand-transmitted vibration exposure from motorized forks used for beach-cleaning operations.

PubMed

McDowell, Thomas W; Welcome, Daniel E; Warren, Christopher; Xu, Xueyan S; Dong, Ren G

2013-01-01

Motorized vibrating manure forks were used in beach-cleaning operations following the massive Deepwater Horizon oil spill in the Gulf of Mexico during the summer of 2010. The objectives of this study were to characterize the vibration emissions of these motorized forks and to provide a first approximation of hand-transmitted vibration exposures to workers using these forks for beach cleaning. Eight operators were recruited to operate the motorized forks during this laboratory study. Four fork configurations were used in the study; two motor speeds and two fork basket options were evaluated. Accelerations were measured near each hand as the operators completed the simulated beach-cleaning task. The dominant vibration frequency for these tools was identified to be around 20 Hz. Because acceleration was found to increase with motor speed, workers should consider operating these tools with just enough speed to get the job done. These forks exhibited considerable acceleration magnitudes when unloaded. The study results suggest that the motor should not be operated with the fork in the unloaded state. Anti-vibration gloves are not effective at attenuating the vibration frequencies produced by these forks, and they may even amplify the transmitted vibration and increase hand/arm fatigue. While regular work gloves are suitable, vibration-reducing gloves may not be appropriate for use with these tools. These considerations may also be generally applicable for the use of motorized forks in other workplace environments.

Note: Enhanced energy harvesting from low-frequency magnetic fields utilizing magneto-mechano-electric composite tuning-fork.

PubMed

Yang, Aichao; Li, Ping; Wen, Yumei; Yang, Chao; Wang, Decai; Zhang, Feng; Zhang, Jiajia

2015-06-01

A magnetic-field energy harvester using a low-frequency magneto-mechano-electric (MME) composite tuning-fork is proposed. This MME composite tuning-fork consists of a copper tuning fork with piezoelectric Pb(Zr(1-x)Ti(x))O3 (PZT) plates bonded near its fixed end and with NdFeB magnets attached at its free ends. Due to the resonance coupling between fork prongs, the MME composite tuning-fork owns strong vibration and high Q value. Experimental results show that the proposed magnetic-field energy harvester using the MME composite tuning-fork exhibits approximately 4 times larger maximum output voltage and 7.2 times higher maximum power than the conventional magnetic-field energy harvester using the MME composite cantilever.

Study of the Unsteady Flow Features on a Stalled Wing

NASA Technical Reports Server (NTRS)

Yon, Steven A.; Katz, Joseph

1997-01-01

The occurrence of large scale structures in the post stall flow over a rectangular wing at high angles of attack was investigated in a small-scale subsonic wind tunnel. Mean and time dependent measurements within the separated flow field suggest the existence of two distinct angle of attack regimes beyond wing stall. The shallow stall regime occurs over a narrow range of incidence angles (2-3 deg.) immediately following the inception of leading edge separation. In this regime, the principal mean flow structures, termed stall cells, are manifested as a distinct spanwise periodicity in the chordwise extent of the separated region on the model surface with possible lateral mobility not previously reported. Within the stall cells and on the wing surface, large amplitude pressure fluctuations occur with a frequency much lower than anticipated for bluff body shedding, and with minimum effect in the far wake. In the deep stall regime, stall cells are not observed and the separated region near the model is relatively free of large amplitude pressure disturbances.

Topographic view of the North Fork Butter Creek Bridge (located ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Topographic view of the North Fork Butter Creek Bridge (located center of frame), view looking west - North Fork Butter Creek Bridge, Spanning North Fork Butter Creek Bridge at Milepost 76.63 on Heppner Highway (Oregon Route 74), Pilot Rock, Umatilla County, OR

Effects of bedding quality on lying behavior of dairy cows.

PubMed

Fregonesi, J A; Veira, D M; von Keyserlingk, M A G; Weary, D M

2007-12-01

Cows prefer to spend more time lying down in free stalls with more bedding, but no research to date has addressed the effects of bedding quality. Bedding in stalls often becomes wet either from exposure to the elements or from feces and urine. The aim of this study was to test the effect of wet bedding on stall preference and use. Four groups of 6 nonlactating Holstein cows were housed in free stalls bedded daily with approximately 0.1 m of fresh sawdust. Following a 5-d adaptation period, each group of cows was tested sequentially with access to stalls with either dry or wet sawdust bedding (86.4 +/- 2.1 vs. 26.5 +/- 2.1% dry matter), each for 2 d. These no-choice phases were followed by a 2-d free-choice phase during which cows had simultaneous access to stalls containing either wet or dry bedding. Stall usage was assessed by using 24-h video recordings scanned at 10-min intervals, and responses were analyzed by using a mixed model, with group (n = 4) as the observational unit. The minimum and maximum environmental temperatures during the experiment were 3.4 +/- 2.2 and 6.8 +/- 2.5 degrees C, respectively. When cows had access only to stalls with wet bedding, they spent 8.8 +/- 0.8 h/d lying down, which increased to 13.8 +/- 0.8 h/d when stalls with dry bedding were provided. Cows spent more time standing with their front 2 hooves in the stall when provided with wet vs. dry bedding (92 +/- 10 vs. 32 +/- 10 min/d). During the free-choice phase, all cows spent more time lying down in the dry stalls, spending 12.5 +/- 0.3 h/d in the dry stalls vs. 0.9 +/- 0.3 h/ d in stalls with wet bedding. In conclusion, dairy cows show a clear preference for a dry lying surface, and they spend much more time standing outside the stall when only wet bedding is available.

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase.

PubMed

Takahashi, Shuntaro; Brazier, John A; Sugimoto, Naoki

2017-09-05

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase

PubMed Central

Takahashi, Shuntaro; Brazier, John A.; Sugimoto, Naoki

2017-01-01

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases. PMID:28827350

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

PubMed Central

Crampton, Neal; Bonass, William A.; Kirkham, Jennifer; Rivetti, Claudio; Thomson, Neil H.

2006-01-01

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned λpr promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after ‘collision’ has caused their arrest. PMID:17012275

A Comparative Study of Some Dynamic Stall Models

NASA Technical Reports Server (NTRS)

Reddy, T. S. R.; Kaza, K. R. V.

1987-01-01

Three semi-empirical aerodynamic stall models are compared with respect to their lift and moment hysteresis loop prediction, limit cycle behavior, easy implementation, and feasibility in developing the parameters required for stall flutter prediction of advanced turbines. For the comparison of aeroelastic response prediction including stall, a typical section model and a plate structural model are considered. The response analysis includes both plunging and pitching motions of the blades. In model A, a correction to the angle of attack is applied when the angle of attack exceeds the static stall angle. In model B, a synthesis procedure is used for angles of attack above static stall angles and the time history effects are accounted through the Wagner function. In both models the life and moment coefficients for angle of attack below stall are obtained from tabular data for a given Mach number and angle of attack. In model C, referred to an the ONERA model, the life and moment coefficients are given in the form of two differential equations, one for angles below stall, and the other for angles above stall. The parameters of those equations are nonlinear functions of the angle of attack.

Feasibility and safety of modified inverted T-shaped method using linear stapler with movable cartridge fork for esophagojejunostomy following laparoscopic total gastrectomy.

PubMed

Ohuchida, Kenoki; Nagai, Eishi; Moriyama, Taiki; Shindo, Koji; Manabe, Tatsuya; Ohtsuka, Takao; Shimizu, Shuji; Nakamura, Masafumi

2017-01-01

We previously reported the use of an inverted T-shaped method to obtain a suitable view for hand sewing to close the common entry hole when the linear stapler was fired for esophagojejunostomy after laparoscopic total gastrectomy (LTG). This conventional method involved insertion of the fixed cartridge fork to the Roux limb and the fine movable anvil fork to the esophagus to avoid perforation of the jejunum. However, insertion of the movable anvil fork to the esophagus during this procedure often requires us to strongly push down the main body of the stapler with the fixed cartridge fork to bring the direction of the anvil fork in line with the direction of the long axis of the esophagus while controlling the opening of the movable anvil fork. We therefore modified this complicated inverted T-shaped method using a linear stapler with a movable cartridge fork. This modified method involved insertion of the movable cartridge fork into the Roux limb followed by natural, easy insertion of the fixed anvil fork into the esophagus without controlling the opening of the movable cartridge fork. We performed LTG in a total of 155 consecutive patients with gastric cancer from November 2007 to December 2015 in Kyushu University Hospital. After LTG, we performed the conventional inverted T-shaped method using a linear stapler with a fixed cartridge fork in 61 patients from November 2007 to July 2011 (fixed cartridge group). From August 2011, we used a linear stapler with a movable cartridge fork and performed the modified inverted T-shaped method in 94 patients (movable cartridge group). We herein compare the short-term outcomes in 94 cases of LTG using the modified method (movable cartridge fork) with those in 61 cases using the conventional method (fixed cartridge fork). We found no significant differences in the perioperative or postoperative events between the movable and fixed cartridge groups. One case of anastomotic leakage occurred in the fixed cartridge group, but no anastomotic leakage occurred in the movable cartridge group. Although there were no remarkable differences in the short-term outcomes between the movable and fixed cartridge groups, we believe that the modified inverted T-shaped method is technically more feasible and reliable than the conventional method and will contribute to the improved safety of LTG.

Feasibility and safety of modified inverted T-shaped method using linear stapler with movable cartridge fork for esophagojejunostomy following laparoscopic total gastrectomy

PubMed Central

Ohuchida, Kenoki; Moriyama, Taiki; Shindo, Koji; Manabe, Tatsuya; Ohtsuka, Takao; Shimizu, Shuji; Nakamura, Masafumi

2017-01-01

Background We previously reported the use of an inverted T-shaped method to obtain a suitable view for hand sewing to close the common entry hole when the linear stapler was fired for esophagojejunostomy after laparoscopic total gastrectomy (LTG). This conventional method involved insertion of the fixed cartridge fork to the Roux limb and the fine movable anvil fork to the esophagus to avoid perforation of the jejunum. However, insertion of the movable anvil fork to the esophagus during this procedure often requires us to strongly push down the main body of the stapler with the fixed cartridge fork to bring the direction of the anvil fork in line with the direction of the long axis of the esophagus while controlling the opening of the movable anvil fork. We therefore modified this complicated inverted T-shaped method using a linear stapler with a movable cartridge fork. This modified method involved insertion of the movable cartridge fork into the Roux limb followed by natural, easy insertion of the fixed anvil fork into the esophagus without controlling the opening of the movable cartridge fork. Methods We performed LTG in a total of 155 consecutive patients with gastric cancer from November 2007 to December 2015 in Kyushu University Hospital. After LTG, we performed the conventional inverted T-shaped method using a linear stapler with a fixed cartridge fork in 61 patients from November 2007 to July 2011 (fixed cartridge group). From August 2011, we used a linear stapler with a movable cartridge fork and performed the modified inverted T-shaped method in 94 patients (movable cartridge group). We herein compare the short-term outcomes in 94 cases of LTG using the modified method (movable cartridge fork) with those in 61 cases using the conventional method (fixed cartridge fork). Results We found no significant differences in the perioperative or postoperative events between the movable and fixed cartridge groups. One case of anastomotic leakage occurred in the fixed cartridge group, but no anastomotic leakage occurred in the movable cartridge group. Conclusions Although there were no remarkable differences in the short-term outcomes between the movable and fixed cartridge groups, we believe that the modified inverted T-shaped method is technically more feasible and reliable than the conventional method and will contribute to the improved safety of LTG. PMID:28616606

[Effect of supply of fresh poultry products on reducing environment contamination of avian influenza virus in markets].

PubMed

Xie, Chaojun; Su, Wenzhe; Li, Kuibiao; Chen, Jiandong; Liu, Jianping; Feng, Jing; Liu, Yanhui; Ma, Xiaowei; Liu, Yufei; Yuan, Jun; Wang, Ming

2016-03-01

To evaluate the effect of supply of fresh poultry products on reducing environment contamination of avian influenza virus (AIV) in markets in Guangzhou. A total of 40 markets, including 20 selling alive poultry and 20 selling fresh poultry products, were selected randomly in Guangzhou to conduct environment surveillance in 80 poultry stalls every 4 months from July 2014 to April 2015. Four smear samples were collected from different sites of each poultry stall to detect nucleic acid of AIV. The positive samples were further detected for AIV subtype H5, H7 and H9 nucleic acids. Among 40 alive poultry stalls, 95.0% (38/40) kept alive poultry overnight, 25.0% (10/40) were disinfected daily, 95.0% (38/40) were cleaned up weekly, 95.0% (38/40) were closed for one day every month. Among 40 fresh poultry product stalls, 20.0% (8/40) were disinfected daily, 90.0% (36/40) were cleaned up weekly, and 96.0% (38/40) ever sold dressed poultry from alive poultry markets. The positive rate of AIV in alive poultry markets was 40.4% (252/623), higher than that in fresh poultry product markets (32.3%, 197/610), the difference was significant (χ(2)=8.85, P=0.003), and the positive rate of subtype H9 virus in alive poultry markets was 28.6% (178/623), higher than that in fresh poultry product markets (16.2%, 99/610), the difference was significant (χ(2)=26.95, P<0.001). In fresh poultry product markets, the positive rate of AIV in stalls selling dressed poultry was 37.3% (180/482), higher than that in stalls selling no dressed poultry (13.3%, 17/128), the difference was significant (χ(2)=26.78, P<0.001), and the positive rate of subtype H9 virus in stalls selling dressed poultry was 19.1% (92/482), higher than that in stalls selling no dressed poultry (5.5%, 7/128), the difference was significant (χ(2)=13.80, P<0.001). Both the positive rate of AIV and the positive rate of subtype H9 virus were highest in the second round surveillance (October 2014). The differences in AIV and its subtype H5, H7 and H9 virus positive rates of environmental samples from four different sites were not significant, respectively. In the same sample site, the positive rate of subtype H9 virus in alive poultry markets was higher than that in fresh poultry product markets the difference was significant (P<0.05). The supply of fresh poultry products could effectively reduce the level of environment contamination of AIV in markets. Dressed poultry supplement caused the risk of AIV spread in fresh poultry product markets.

Advance Ratio Effects on the Dynamic-stall Vortex of a Rotating Blade in Steady Forward Flight

DTIC Science & Technology

2014-08-06

dependence on advance ratio is used to relate the stability of the dynamic-stall vortex to Coriolis effects . Advance ratio effects on the dynamic-stall vortex...relate the stability of the dynamic-stall vortex to Coriolis effects . Keywords: Leading-edge vortex, Dynamic stall vortex, Vortex flows, Rotating wing...Reynolds number are not decoupled. 3. Radial flow field In the rotating environment the coupled effect of centripetal and Coriolis accelerations is ex

Active control system for high speed windmills

DOEpatents

Avery, D.E.

1988-01-12

A pump stroke is matched to the operating speed of a high speed windmill. The windmill drives a hydraulic pump for a control. Changes in speed of a wind driven shaft open supply and exhaust valves to opposite ends of a hydraulic actuator to lengthen and shorten an oscillating arm thereby lengthening and shortening the stroke of an output pump. Diminishing wind to a stall speed causes the valves to operate the hydraulic cylinder to shorten the oscillating arm to zero. A pressure accumulator in the hydraulic system provides the force necessary to supply the hydraulic fluid under pressure to drive the actuator into and out of the zero position in response to the windmill shaft speed approaching and exceeding windmill stall speed. 4 figs.

Active control system for high speed windmills

DOEpatents

Avery, Don E.

1988-01-01

A pump stroke is matched to the operating speed of a high speed windmill. The windmill drives a hydraulic pump for a control. Changes in speed of a wind driven shaft open supply and exhaust valves to opposite ends of a hydraulic actuator to lengthen and shorten an oscillating arm thereby lengthening and shortening the stroke of an output pump. Diminishing wind to a stall speed causes the valves to operate the hydraulic cylinder to shorten the oscillating arm to zero. A pressure accumulator in the hydraulic system provides the force necessary to supply the hydraulic fluid under pressure to drive the actuator into and out of the zero position in response to the windmill shaft speed approaching and exceeding windmill stall speed.

Subsychronous vibration of multistage centrifugal compressors forced by rotating stall

NASA Technical Reports Server (NTRS)

Fulton, J. W.

1987-01-01

A multistage centrifugal compressor, in natural gas re-injection service on an offshore petroleum production platform, experienced subsynchronous vibrations which caused excessive bearing wear. Field performance testing correlated the subsynchronous amplitude with the discharge flow coefficient, demonstrating the excitation to be aerodynamic. Adding two impellers allowed an increase in the diffuser flow angle (with respect to tangential) to meet the diffuser stability criteria based on factory and field tests correlated using the theory of Senoo (for rotating stall in a vaneless diffuser). This modification eliminated all significant subsynchronous vibrations in field service, thus confirming the correctness of the solution. Other possible sources of aerodynamically induced vibrations were considered, but the judgment that those are unlikely has been confirmed by subsequent experience with other similar compressors.

[History of the tuning fork. I: Invention of the tuning fork, its course in music and natural sciences. Pictures from the history of otorhinolaryngology, presented by instruments from the collection of the Ingolstadt German Medical History Museum].

PubMed

Feldmann, H

1997-02-01

G. Cardano, physician, mathematician, and astrologer in Pavia, Italy, in 1550 described how sound may be perceived through the skull. A few years later H. Capivacci, also a physician in Padua, realized that this phenomenon might be used as a diagnostic tool for differentiating between hearing disorders located either in the middle ear or in the acoustic nerve. The German physician G. C. Schelhammer in 1684 was the first to use a common cutlery fork in further developing the experiments initiated by Cardano and Capivacci. For a long time to come, however, there was no demand for this in practical otology. The tuning fork was invented in 1711 by John Shore, trumpeter and lutenist to H. Purcell and G.F. Händel in London. A picture of Händel's own tuning fork, probably the oldest tuning fork in existence, is presented here for the first time. There are a number of anecdotes connected with the inventor of the tuning fork, using plays on words involving the name Shore, and mixing up pitch-pipe and pitchfork. Some of these are related here. The tuning fork as a musical instrument soon became a success throughout Europe. The German physicist E. F. F. Chladni in Wittenberg around 1800 was the first to systematically investigate the mode of vibration of the tuning fork with its nodal points. Besides this, he and others tried to construct a complete musical instrument based on sets of tuning forks, which, however, were not widely accepted. J. H. Scheibler in Germany in 1834 presented a set of 54 tuning forks covering the range from 220 Hz to 440 Hz, at intervals of 4 Hz. J. Lissajous in Paris constructed a very elaborate tuning fork with a resonance box, which was intended to represent the international standard of the musical note A with 435 vibrations per second, but this remained controversial. K. R. Koenig, a German physicist living in Paris, invented a tuning fork which was kept in continuous vibration by a clockwork. H. Helmholtz, physiologist in Heidelberg, in 1863 used sets of electromagnetically powered tuning forks for his famous experiments on the sensations of tone. Until the invention of the electronic valve, tuning forks remained indispensible instruments for producing defined sinusoidal vibrations. The history of this development is presented in detail. The diagnostic use of the tuning fork in otology will be described in a separate article.

Chemical and biological sensing using tuning forks

DOEpatents

Tao, Nongjian; Boussaad, Salah

2012-07-10

A device for sensing a chemical analyte is disclosed. The device is comprised of a vibrating structure having first and second surfaces and having an associated resonant frequency and a wire coupled between the first and second surfaces of the vibrating structure, wherein the analyte interacts with the wire and causes a change in the resonant frequency of the vibrating structure. The vibrating structure can include a tuning fork. The vibrating structure can be comprised of quartz. The wire can be comprised of polymer. A plurality of vibrating structures are arranged in an array to increase confidence by promoting a redundancy of measurement or to detect a plurality of chemical analytes. A method of making a device for sensing a chemical analyte is also disclosed.

Short revolving wings enable hovering animals to avoid stall and reduce drag

NASA Astrophysics Data System (ADS)

Lentink, David; Kruyt, Jan W.; Heijst, Gertjan F.; Altshuler, Douglas L.

2014-11-01

Long and slender wings reduce the drag of airplanes, helicopters, and gliding animals, which operate at low angle of attack (incidence). Remarkably, there is no evidence for such influence of wing aspect ratio on the energetics of hovering animals that operate their wings at much higher incidence. High incidence causes aircraft wings to stall, hovering animals avoid stall by generating an attached vortex along the leading edge of their wings that elevates lift. Hypotheses that explain this capability include the necessity for a short radial distance between the shoulder joint and wing tip, measured in chord lengths, instead of the long tip-to-tip distance that elevates aircraft performance. This stems from how hovering animals revolve their wings around a joint, a condition for which the precise effect of aspect ratio on stall performance is unknown. Here we show that the attachment of the leading edge vortex is determined by wing aspect ratio with respect to the center of rotation-for a suite of aspect ratios that represent both animal and aircraft wings. The vortex remains attached when the local radius is shorter than 4 chord lengths, and separates outboard on more slender wings. Like most other hovering animals, hummingbirds have wing aspect ratios between 3 and 4, much stubbier than helicopters. Our results show this makes their wings robust against flow separation, which reduces drag below values obtained with more slender wings. This revises our understanding of how aspect ratio improves performance at low Reynolds numbers.

Accessory replicative helicases and the replication of protein-bound DNA.

PubMed

Brüning, Jan-Gert; Howard, Jamieson L; McGlynn, Peter

2014-12-12

Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes. Removal of these barriers demands energy input not only to separate the DNA strands but also to disrupt multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein-DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability. Copyright © 2014. Published by Elsevier Ltd.

Simulator Studies of the Deep Stall

NASA Technical Reports Server (NTRS)

White, Maurice D.; Cooper, George E.

1965-01-01

Simulator studies of the deep-stall problem encountered with modern airplanes are discussed. The results indicate that the basic deep-stall tendencies produced by aerodynamic characteristics are augmented by operational considerations. Because of control difficulties to be anticipated in the deep stall, it is desirable that adequate safeguards be provided against inadvertent penetrations.

75 FR 80735 - Special Conditions: Gulfstream Model GVI Airplane; High Incidence Protection

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-23

..., Aircraft Certification Service, 1601 Lind Avenue, SW., Renton, Washington, 98057-3356; telephone (425) 227... from stalling, limits the angle of attack at which the airplane can be flown during normal low speed... limit impacts the stall speed determination, the stall characteristics, the stall warning demonstration...

21 CFR 882.1525 - Tuning fork.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Tuning fork. 882.1525 Section 882.1525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Diagnostic Devices § 882.1525 Tuning fork. (a) Identification. A tuning fork...

21 CFR 882.1525 - Tuning fork.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tuning fork. 882.1525 Section 882.1525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Diagnostic Devices § 882.1525 Tuning fork. (a) Identification. A tuning fork...

36 CFR 292.62 - Valid existing rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... RECREATION AREAS Smith River National Recreation Area § 292.62 Valid existing rights. (a) Definition. For the... “wild” segments of the Wild and Scenic Smith River, Middle Fork Smith River, North Fork Smith River, Siskiyou Fork Smith River, South Fork Smith River, and their designated tributaries, except Peridotite...

A flight investigation of the ultra-deep-stall descent and spin recovery characteristics of a 1/6 scale radiocontrolled model of the Piper PA38 Tomahawk

NASA Technical Reports Server (NTRS)

Blanchard, W. S., Jr.

1981-01-01

Ultradeep stall descent and spin recovery characteristics of a 1/6 scale radio controlled model of the Piper PA38 Tomahawk aircraft was investigated. It was shown that the full scale PA38 is a suitable aircraft for conducting ultradeep stall research. Spin recovery was accomplished satisfactorily by entry to the ultradeep stall mode, followed by the exit from the ultradeep stall mode. It is concluded that since the PA38 has excellent spin recovery characteristics using normal recovery techniques (opposite rudder and forward control colum pressure), recovery using ultradeep stall would be beneficial only if the pilot suffered from disorientation.

Stall flutter analysis of propfans

NASA Technical Reports Server (NTRS)

Reddy, T. S. R.

1988-01-01

Three semi-empirical aerodynamic stall models are compared with respect to their lift and moment hysteresis loop prediction, limit cycle behavior, easy implementation, and feasibility in developing the parameters required for stall flutter prediction of advanced turbines. For the comparison of aeroelastic response prediction including stall, a typical section model and a plate structural model are considered. The response analysis includes both plunging and pitching motions of the blades. In model A, a correction of the angle of attack is applied when the angle of attack exceeds the static stall angle. In model B, a synthesis procedure is used for angles of attack above static stall angles, and the time history effects are accounted for through the Wagner function.

Aerodynamic study of a stall regulated horizontal-axis wind turbine

NASA Astrophysics Data System (ADS)

Constantinescu, S. G.; Crunteanu, D. E.; Niculescu, M. L.

2013-10-01

The wind energy is deemed as one of the most durable energetic variants of the future because the wind resources are immense. Furthermore, one predicts that the small wind turbines will play a vital role in the urban environment. Unfortunately, the complexity and the price of pitch regulated small horizontal-axis wind turbines represent ones of the main obstacles to widespread the use in populated zones. Moreover, the energetic efficiency of small stall regulated wind turbines has to be high even at low and medium wind velocities because, usually the cities are not windy places. During the running stall regulated wind turbines, due to the extremely broad range of the wind velocity, the angle of attack can reach high values and some regions of the blade will show stall and post-stall behavior. This paper deals with stall and post-stall regimes because they can induce significant vibrations, fatigue and even the wind turbine failure.

76 FR 74649 - Harmonization of Various Airworthiness Standards for Transport Category Airplanes-Flight Rules

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-01

...-directional stability, speed increase and recovery characteristics, and the stall warning margin for the... which the onset of natural or artificial stall warning occurs. VSR reference stall speed. VSR1 reference.... Lastly, this rule adds a requirement that the non-icing stall warning requirements prescribing the speed...

Environmental Assessment: Construct POV Parking Lot Main Gate-Separated at Grand Forks AFB, North Dakota

DTIC Science & Technology

2004-09-01

water quality could be degraded, both in the short-term, during actual construction, and over the long-term due to reduced storm water quality caused by...due to reduced storm water quality caused by the increase of 37 paved area. The short-term effects come from possible erosion contributing to...construction, and over the long-term due to reduced storm water quality caused by the increase of paved area. The short-term effects come from

Intelligent Prediction of Fan Rotation Stall in Power Plants Based on Pressure Sensor Data Measured In-Situ

PubMed Central

Xu, Xiaogang; Wang, Songling; Liu, Jinlian; Liu, Xinyu

2014-01-01

Blower and exhaust fans consume over 30% of electricity in a thermal power plant, and faults of these fans due to rotation stalls are one of the most frequent reasons for power plant outage failures. To accurately predict the occurrence of fan rotation stalls, we propose a support vector regression machine (SVRM) model that predicts the fan internal pressures during operation, leaving ample time for rotation stall detection. We train the SVRM model using experimental data samples, and perform pressure data prediction using the trained SVRM model. To prove the feasibility of using the SVRM model for rotation stall prediction, we further process the predicted pressure data via wavelet-transform-based stall detection. By comparison of the detection results from the predicted and measured pressure data, we demonstrate that the SVRM model can accurately predict the fan pressure and guarantee reliable stall detection with a time advance of up to 0.0625 s. This superior pressure data prediction capability leaves significant time for effective control and prevention of fan rotation stall faults. This model has great potential for use in intelligent fan systems with stall prevention capability, which will ensure safe operation and improve the energy efficiency of power plants. PMID:24854057

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

PubMed Central

Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

PubMed

Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Monitoring indices of cow comfort in free-stall-housed dairy herds.

PubMed

Cook, N B; Bennett, T B; Nordlund, K V

2005-11-01

Indices of cow comfort are used widely by consultants in the dairy industry, with a general understanding that they are representative of lying behavior. This study examines the influence of stall base type (sand or a geotextile mattress filled with rubber crumbs) and time of measurement on 4 indices of comfort collected at hourly intervals in 12 herds, aligned by morning and afternoon milking. Stall base type significantly influenced all indices of comfort. For example, the least squares mean (SE) cow comfort index (proportion of cows touching a stall that are lying down) was 0.76 (0.015) in herds with mattresses compared with 0.86 (0.015) in herds with sand stalls. Significant hourly variation was also identified suggesting that timing of measurement is important. None of the indices of cow comfort derived from the high-yielding group pen was associated with the mean 24-h lying time of 10 sentinel cows whose time budgets were known in each herd. However, the cow comfort index was associated with the herd mean 24-h stall standing time, with the strongest relationships occurring 2 h before the morning and afternoon milking, when stall base type did not significantly influence the association. When measured at these times, we recommend use of the stall standing index (proportion of cows touching a stall that are standing), with values greater than 0.20 being associated with abnormally long herd mean stall standing times greater than 2 h/d.

Contamination of the environment by strongylid (Nematoda: Strongylidae) infective larvae at horse farms of various types in Ukraine.

PubMed

Kuzmina, Tetiana A

2012-05-01

Analysis of the influence of horse-keeping conditions by contamination of the environment (pastures, paddocks, and stalls) by the strongylid infective larvae (L(3)) was carried out at various types of horse farms, hippodromes, and riding clubs in Ukraine. A total of 1,237 horses from three types of horse-keeping conditions were examined. Epidemiological studies of stall and grazing area (pasture and paddocks) contamination by L(3) were performed at hippodrome (stalled horse-keeping) and horse farms with stall/paddock-keeping and stall/pasture-keeping conditions. Grass and stall litter samples were examined by the Baermann procedure. It was found that horses of stall-keeping conditions had the lowest level of strongylid infection (prevalence 46.4-77.8%, average infection 25.6-92.9 eggs per gram of feces (EPG)) and lowest proportion of large strongyle L(3) in coprocultures (1.6-11.3%). Horses of stall/pasture-keeping conditions were the most infected (prevalence 95.1-100%, average infection 198.2-453.7 EPG), and the proportion of large strongyle L(3) was 17.3-24.7%. Strongyle L(3) were found in litter of all parts of individual stalls; areas at the stall center, "toilet", and entrance were the most contaminated. The highest L(3) number in stall litter was registered in summer. Contamination of permanent pasture grass by L(3) was notably lower than grass in paddocks (86.3-161.4 L(3)/kg compared with 305.9-409.1 L(3)/kg). The highest level of pasture grass contamination was observed in the middle of summer (July)--up 970.7 L(3)/kg. The results obtained confirmed importance of environmental contamination in epidemiology of horse strongylidosis at various types of horse-keeping conditions.

Effects of sand and straw bedding on the lying behavior, cleanliness, and hoof and hock injuries of dairy cows.

PubMed

Norring, M; Manninen, E; de Passillé, A M; Rushen, J; Munksgaard, L; Saloniemi, H

2008-02-01

This experiment compared the effects of sand and straw bedding in free stalls on resting time, cleanliness, hock injuries, and hoof health of dairy cows and tested whether cow preferences for a bedding material depended on the familiarity with the material. A total of 52 dairy cows were kept either on straw bedded concrete stalls or sand stalls for at least 21 wk. The lying behavior was observed, and hock lesions, hoof health, and cleanliness of the cows and stalls were measured. A 5-d preference test between sand and straw stalls was conducted at the end of the experiment. The total daily duration of lying was longer for cows on straw bedding than on sand bedding (straw 749 +/- 16 vs. sand 678 +/- 19 min). During the preference test, cows that had been kept on straw bedding preferred lying in straw stalls [straw 218.7 (133.4 to 239.7) vs. sand 9.0 min (2.8 to 44.8)]; however, cows that had been kept on sand showed no preference [straw 101.3 (51.7 to 205.9) vs. sand 94.3 min (54.1 to 156.1, median and interquartile range)]. Although there were no differences in the dirtiness of stalls, the cows using straw stalls were dirtier than cows using sand stalls [straw 6.04 (5.39 to 6.28) vs. sand 4.19 (3.62 to 5.16)]. At the end of experiment the severity of hock lesions was lower for cows on sand than for cows on straw [sand 0.5 (0.0 to 1.0) vs. straw 1.0 (1.0 to 2.0)]. The improvement in overall hoof health over the observation period was greater for cows kept on sand compared with cows kept on straw [sand -2.00 (-3.75 to -0.25) vs. straw 0.00 (-2.00 to 2.00)]. Straw bedding increased the time that cows spend lying, and cows preferred straw stalls to sand stalls. However, previous experience with sand reduces avoidance of sand stalls. Sand stalls were advantageous for cow cleanliness and health; hock lesions and claw diseases healed more quickly for cows using sand stalls compared with straw.

Runoff generation from neighboring headwater basins with differing glacier coverage using the distributed hydrological model WaSiM, Eklutna, Alaska

NASA Astrophysics Data System (ADS)

Ostman, J. S.; Loso, M.; Liljedahl, A. K.; Gaedeke, A.; Geck, J. E.

2017-12-01

Many Alaska glaciers are thinning and retreating, and glacier wastage is projected to affect runoff processes from glacierized basins. Accordingly, effective resource management in glacierized watersheds requires quantification of a glacier's role on streamflow generation. The Eklutna catchment (311 km2) supplies water and electricity for Anchorage, Alaska (pop. 300,000) via Eklutna Lake. The Eklutna headwaters include the West Fork (64 km2, 46% glacier), and the East Fork (101 km2, 12% glacier). Total average annual discharge (2009-2015) is similar from the West (42,100 m3) and East (42,200 m3) forks, while specific annual runoff from the West Fork (2940 mm) exceeds that of the East Fork (1500 mm). To better understand what controls runoff, we are simulating the Eklutna annual water budget using a distributed watershed-level hydrological model. We force the Water Flow and Balance Simulation Model (WaSiM) using continuous air temperature, precipitation, wind speed, shortwave incoming radiation, and relative humidity primarily measured in the West Fork basin. We use Eklutna Glacier snow accumulation and ablation to calibrate the snowmelt and glacier sub-modules. Melt season discharge from the West and East forks is used for runoff comparison. Preliminary results show 2013-2015 simulated glacier point balances (accumulation and melt) are within 15% of glacier stake observations. Runoff was effectively modeled in the West Fork (NSE=0.80), while being over-predicted in the East Fork , which we attribute to a lack of forcing data in the less-glacierized basin. The simulations suggest that 78% of West Fork total runoff is from glacier melt, compared with <40% in the East Fork where glacier runoff contribution is higher during low-snow years.

Wider stall space affects behavior, lesion scores, and productivity of gestating sows.

PubMed

Salak-Johnson, J L; DeDecker, A E; Levitin, H A; McGarry, B M

2015-10-01

Limited space allowance within the standard gestation stall is an important welfare concern because it restricts the ability of the sow to make postural adjustments and hinders her ability to perform natural behaviors. Therefore, we evaluated the impacts of increasing stall space and/or providing sows the freedom to access a small pen area on sow well-being using multiple welfare metrics. A total of 96 primi- and multiparous crossbred sows were randomly assigned in groups of 4 sows/treatment across 8 replicates to 1 of 3 stall treatments (TRT): standard stall (CTL; dimensions: 61 by 216 cm), width-adjustable stall (flex stall [FLX]; dimensions: adjustable width of 56 to 79 cm by 216 cm), or an individual walk-in/lock-in stall with access to a small communal open-pen area at the rear of the stall (free-access stall [FAS]; dimensions: 69 by 226 cm). Lesion scores, behavior, and immune and productivity traits were measured at various gestational days throughout the study. Total lesion scores were greatest for sows in FAS and least for sows in FLX ( < 0.001). Higher-parity sows in FAS had the most severe lesion scores (TRT × parity, < 0.0001) and scores were greatest at all gestational days (TRT × day, < 0.05). Regardless of parity, sows in FLX had the least severe scores ( < 0.0001). As pregnancy progressed, lesion scores increased among sows in CTL ( < 0.05). Sow BW and backfat (BF) were greater for sows in FLX and FAS ( < 0.05), and BCS and BF were greater for parity 1 and 2 sows in FAS than the same parity sows in CTL (TRT × parity, < 0.05). Duration and frequency of some postural behaviors and sham chew behavior were affected by TRT ( < 0.05) and time of day (TRT × day, < 0.05). These data indicate that adequate stall space, especially late in gestation, may improve the well-being of higher-parity and heavier-bodied gestating sows as assessed by changes in postural behaviors, lesion severity scores, and other sow traits. Moreover, compromised welfare measures found among sows in various stall environments may be partly attributed to the specific constraints of each stall system such as restricted stall space in CTL, insufficient floor space in the open-pen area of the FAS system, and gate design of the FLX (e.g., direction of bars and feeder space). These results also indicate that parity and gestational day are additional factors that may exacerbate the effects of restricted stall space or insufficient pen space, further compromising sow well-being.

A numerical strategy for modelling rotating stall in core compressors

NASA Astrophysics Data System (ADS)

Vahdati, M.

2007-03-01

The paper will focus on one specific core-compressor instability, rotating stall, because of the pressing industrial need to improve current design methods. The determination of the blade response during rotating stall is a difficult problem for which there is no reliable procedure. During rotating stall, the blades encounter the stall cells and the excitation depends on the number, size, exact shape and rotational speed of these cells. The long-term aim is to minimize the forced response due to rotating stall excitation by avoiding potential matches between the vibration modes and the rotating stall pattern characteristics. Accurate numerical simulations of core-compressor rotating stall phenomena require the modelling of a large number of bladerows using grids containing several tens of millions of points. The time-accurate unsteady-flow computations may need to be run for several engine revolutions for rotating stall to get initiated and many more before it is fully developed. The difficulty in rotating stall initiation arises from a lack of representation of the triggering disturbances which are inherently present in aeroengines. Since the numerical model represents a symmetric assembly, the only random mechanism for rotating stall initiation is provided by numerical round-off errors. In this work, rotating stall is initiated by introducing a small amount of geometric mistuning to the rotor blades. Another major obstacle in modelling flows near stall is the specification of appropriate upstream and downstream boundary conditions. Obtaining reliable boundary conditions for such flows can be very difficult. In the present study, the low-pressure compression (LPC) domain is placed upstream of the core compressor. With such an approach, only far field atmospheric boundary conditions are specified which are obtained from aircraft speed and altitude. A chocked variable-area nozzle, placed after the last compressor bladerow in the model, is used to impose boundary conditions downstream. Such an approach is representative of modelling an engine.Using a 3D viscous time-accurate flow representation, the front bladerows of a core compressor were modelled in a whole-annulus fashion whereas the rest of bladerows are modelled in a single-passage fashion. The rotating stall behaviour at two different compressor operating points was studied by considering two different variable-vane scheduling conditions for which experimental data were available. Using a model with nine whole-assembly models, the unsteady-flow calculations were conducted on 32-CPUs of a parallel cluster, typical run times being around 3-4 weeks for a grid with about 60 million points. The simulations were conducted over several engine rotations. As observed on the actual development engine, there was no rotating stall for the first scheduling condition while mal-scheduling of the stator vanes created a 12-band rotating stall which excited the 1st flap mode.

Movement of bull trout in the upper Jarbidge River watershed, Idaho and Nevada, 2008-09--A supplement to Open-File Report 2010-1033

USGS Publications Warehouse

Munz, Carrie S.; Allen, M. Brady; Connolly, Patrick J.

2011-01-01

We monitored bull trout (Salvelinus confluentus) in 2008 and 2009 as a continuation of our work in 2006 and 2007, which involved the tagging of 1,536 bull trout with passive integrated transponder (PIT) tags in the East Fork Jarbidge River and West Fork Jarbidge River and their tributaries in northeastern Nevada and southern Idaho. We installed PIT tag interrogation systems (PTISs) at established locations soon after ice-out, and maintained the PTISs in order to collect information on bull trout movements through December of each year. We observed a marked increase of movement in 2008 and 2009. Bull trout tagged in the uppermost portions of the East Fork Jarbidge River at altitudes greater than 2,100 meters moved to the confluence of the East Fork Jarbidge River and West Fork Jarbidge River in summer and autumn. Ten bull trout tagged upstream of the confluence of Pine Creek and the West Fork Jarbidge River moved downstream and then upstream in the East Fork Jarbidge River, and then past the PTIS at Murphy Hot Springs (river kilometer [rkm] 4.1). Two of these fish ascended Dave Creek, a tributary of the East Fork Jarbidge River, past the PTIS at rkm 0.4. One bull trout that was tagged at rkm 11 in Dave Creek on June 28, 2007 moved downstream to the confluence of the East Fork Jarbidge River and West Fork Jarbidge River (rkm 0) on July 28, 2007, and it was then detected in the West Fork Jarbidge River moving past our PTIS at rkm 15 on May 4, 2008. Combined, the extent and types of bull trout movements observed indicated that the primarily age-1 and age-2 bull trout that we tagged in 2006 and 2007 showed increased movement with age and evidence of a substantial amount of fluvial life history. The movements suggest strong connectivity between spawning areas and downstream mainstem areas, as well as between the East Fork Jarbidge River and West Fork Jarbidge River.

Salmon Supplementation Studies in Idaho Rivers, 1999-2000 Progress Report.

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

Kohler, Andy; Taki, Doug; Teton, Angelo

2001-11-01

As part of the Idaho Supplementation Studies, fisheries crews from the Shoshone-Bannock Tribes have been snorkeling tributaries of the Salmon River to estimate chinook salmon (Oncorhynchus tshawytscha) parr abundance; conducting surveys of spawning adult chinook salmon to determine the number of redds constructed and collect carcass information; operating a rotary screw trap on the East Fork Salmon River and West Fork Yankee Fork Salmon River to enumerate and PIT-tag emigrating juvenile chinook salmon; and collecting and PIT-tagging juvenile chinook salmon on tributaries of the Salmon River. The Tribes work in the following six tributaries of the Salmon River: Bear Valleymore » Creek, East Fork Salmon River, Herd Creek, South Fork Salmon River, Valley Creek, and West Fork Yankee Fork Salmon River. Snorkeling was used to obtain parr population estimates for ISS streams from 1992 to 1997. However, using the relatively vigorous methods described in the ISS experimental design to estimate summer chinook parr populations, results on a project-wide basis showed extraordinarily large confidence intervals and coefficients of variation. ISS cooperators modified their sampling design over a few years to reduce the variation around parr population estimates without success. Consequently, in 1998 snorkeling to obtain parr population estimates was discontinued and only General Parr Monitoring (GPM) sites are snorkeled. The number of redds observed in SBT-ISS streams has continued to decline as determined by five year cycles. Relatively weak strongholds continue to occur in the South Fork Salmon River and Bear Valley Creek. A rotary screw trap was operated on the West Fork Yankee Fork during the spring and fall of 1999 and the spring of 2000 to monitor juvenile chinook migration. A screw trap was also operated on the East Fork of the Salmon River during the spring and fall from 1993 to 1997 and 1999 (fall only) to 2000. Significant supplementation treatments have occurred in the South Fork Salmon River (IDFG). The East Fork Salmon River received supplementation treatments yearly through 1995. There have been no treatments since 1995, and no significant future treatments from local broodstock are conceivable due to extremely poor escapement. The West Fork Yankee Fork received a single presmolt treatment in 1994. Similarly, no significant future treatments are planned for the WFYF due to extremely poor escapement. However, small scale experimental captive rearing and broodstock techniques are currently being tested with populations from the EFSR and WFYF. Captive rearing/broodstock techniques could potentially provide feedback for evaluation of supplementation. The other three SBT-ISS streams are control streams and do not receive hatchery treatments.« less

Evaluation of fish kills during November 1986 and July 1987 in upper East Fork Poplar Creek near the Y-12 Plant

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

Ryon, M.G.; Loar, J.M.; Southworth, G.R.

1990-09-01

The Environmental Sciences Division (ESD) investigated two fish kills that occurred on November 21, 1986, and July 9, 1987, in upper East Fork Poplar Creek at the outfall of New Hope Pond (NHP) below the Oak Ridge Y-12 Plant. Investigative procedures included sampling of water at the inlet and outfall of NHP for water quality, examination of operating procedures at the Y-12 Plant and in the biomonitoring program that may have adversely affected the fish populations, review of results of concurrent ambient toxicity tests of the inlet and outfall water of NHP, autopsy investigations of the cause of death ofmore » the stonerollers, and laboratory experimentation to evaluate potential causes. The investigations revealed that the cause of death was bacterial hemorrhagic septicemia caused by Aeromonas hydrophila, which is a stress-mediated disease. The specific stressor responsible for the outbreak of the disease was not identified. Several possible stresses were indicated, including elevated concentrations of mercury and chlorine, excessive electroshocking activity, and elevated levels of the pathogen. Cumulative stress due to the combination of several factors was also suggested. Elevated temperatures and overcrowding may have enhanced the spread of the epizootic but were not the primary causes. The impact on the stoneroller population below NHP was not ecologically significant. 23 refs., 3 figs., 12 tabs.« less

16 CFR 1505.50 - Stalled motor testing.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Stalled motor testing. 1505.50 Section 1505... USE BY CHILDREN Policies and Interpretations § 1505.50 Stalled motor testing. (a) § 1505.6(e)(4)(ii) requires that a motor-operated toy be tested with the motor stalled if the construction of the toy is such...

16 CFR 1505.50 - Stalled motor testing.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Stalled motor testing. 1505.50 Section 1505... USE BY CHILDREN Policies and Interpretations § 1505.50 Stalled motor testing. (a) § 1505.6(e)(4)(ii) requires that a motor-operated toy be tested with the motor stalled if the construction of the toy is such...

Investigation of Rotating Stall Phenomena in Axial Flow Compressors. Volume I. Basic Studies of Rotating Stall

DTIC Science & Technology

1976-06-01

rotating stall control system which was tested both on a low speed rig and a J-85-S engine. The second objective was to perform fundamental studies of the...Stator Stage 89 6 Annular Cascade Configuration Used for Rotating Stall Studies on Rotoi-Stator Stage ..... .............. ... 90 7 Static Pressure Rise...ground tests on a J-8S-S turbojet engine. The work i3 reported in three separate volumes. Volume I entitled, "Basic Studies of Rotating Stall", covers

Stalling of Helicopter Blades

NASA Technical Reports Server (NTRS)

Gustafson, F B; Myers, G C , Jr

1946-01-01

Theoretical studies have predicted that operation of helicopter rotor beyond certain combinations of thrust, forward speed, and rotational speed might be prevented by rapidly increasing stalling of the retreating blade. The same studies also indicate that the efficiency of the rotor will increase until these limits are reached or closely approached, so that it is desirable to design helicopter rotors for operation close to the limits imposed by blade stalling. Inasmuch as the theoretical predictions of blade stalling involve numerous approximations and assumptions, an experimental investigation was needed to determine whether, in actual practice, the stall did occur and spread as predicted and to establish the amount of stalling that could be present without severe vibration or control difficulties being introduced. This report presents the results of such an investigation.

27 CFR 9.113 - North Fork of Long Island.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries of...

27 CFR 9.113 - North Fork of Long Island.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries of...

27 CFR 9.113 - North Fork of Long Island.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false North Fork of Long Island... North Fork of Long Island. (a) Name. The name of the viticultural area described in this section is “North Fork of Long Island.” (b) Approved maps. The appropriate maps for determining the boundaries of...

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

PubMed Central

Bastia, Deepak; Srivastava, Pankaj; Zaman, Shamsu; Choudhury, Malay; Mohanty, Bidyut K.; Bacal, Julien; Langston, Lance D.; Pasero, Philippe; O’Donnell, Michael E.

2016-01-01

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex. PMID:27298353

Initial Geomorphic Responses to Removal of Milltown Dam, Clark Fork River, Montana, USA

NASA Astrophysics Data System (ADS)

Wilcox, A. C.; Brinkerhoff, D.; Woelfle-Erskine, C.

2008-12-01

The removal of Milltown Dam on the Clark Fork River, Montana, USA, is creating a field-scale experiment on upstream and downstream responses to dam removal and on how gravel-bed rivers respond to sediment pulses. Milltown Dam was removed in 2008, reconnecting the Clark Fork River to its upstream basin in terms of sediment transport and fish passage. This dam removal is especially notable because (1) it is the largest dam removal to date in the United States in terms of the volume of reservoir sediment potentially available for downstream transport (over 3 million m3; 1.7 million m3 are being mechanically removed); and (2) the dam is the downstream end of the largest Superfund site in the United States, the Clark Fork Complex, and reservoir sediments are composed largely of contaminated mine tailings. Data collection on pre- and post-dam removal channel morphology, bed sediment characteristics, and sediment loads are being used to investigate spatial and temporal patterns of sediment transport and deposition associated with this dam removal. In the first several months following breaching of the dam, snowmelt runoff with a 3-year recurrence interval peak caused substantial erosion and downstream transport of metals-laden sediments from Milltown reservoir. Reservoir sediments in the Clark Fork arm of Milltown reservoir eroded at levels far exceeding modeling predictions as a result of both incision to the new base level created by dam removal and bank retreat of over 200 m in reaches upstream of a constructed bypass reach and remediation area. Copper and other metals in these eroded reservoir sediments provide a tracer for identifying whether sediment deposits observed downstream of the dam originated from Milltown reservoir or uncontaminated tributaries and indicate that Milltown sediments have reached over 200 km downstream. Downstream deposition has been greatest along channel margins and in side-channel areas, whereas the transport capacity of the active channel has limited channel changes there.

Multiscale thermal refugia and stream habitat associations of chinook salmon in northwestern Oregon

USGS Publications Warehouse

Torgersen, Christian E.; Price, David M.; Li, Hiram W.; McIntosh, B.A.

1999-01-01

We quantified distribution and behavior of adult spring chinook salmon (Oncorhynchus tshawytscha) related to patterns of stream temperature and physical habitat at channel-unit, reach-, and section-level spatial scales in a wilderness stream and a disturbed stream in the John Day River basin in northeastern Oregon. We investigated the effectiveness of thermal remote sensing for analyzing spatial patterns of stream temperature and assessed habitat selection by spring chinook salmon, evaluating whether thermal refugia might be responsible for the persistence of these stocks in rivers where water temperatures frequently exceed their upper tolerance levels (25A?C) during spawning migration. By presenting stream temperature and the ecology of chinook salmon in a historical context, we could evaluate how changes in riverine habitat and thermal spatial structure, which can be caused by land-use practices, may influence distributional patterns of chinook salmon. Thermal remote sensing provided spatially continuous maps of stream temperature for reaches used by chinook salmon in the upper subbasins of the Middle Fork and North Fork John Day River. Electivity analysis and logistic regression were used to test for associations between the longitudinal distribution of salmon and cool-water areas and stream habitat characteristics. Chinook salmon were distributed nonuniformly in reaches throughout each stream. Salmon distribution and cool water temperature patterns were most strongly related at reach-level spatial scales in the warm stream, the Middle Fork (maximum likelihood ratio: P 0.30). Pools were preferred by adult chinook salmon in both subbasins (Bonferroni confidence interval: P a?? 0.05); however, riffles were used proportionately more frequently in the North Fork than in the Middle Fork. Our observations of thermal refugia and their use by chinook salmon at multiple spatial scales reveal that, although heterogeneity in the longitudinal stream temperature profile may be viewed as an ecological warning sign, thermal patchiness in streams also should be recognized for its biological potential to provide habitat for species existing at the margin of their environmental tolerances.

Numerical study of delta wing leading edge blowing

NASA Technical Reports Server (NTRS)

Yeh, David; Tavella, Domingo; Roberts, Leonard

1988-01-01

Spanwise and tangential leading edge blowing as a means of controlling the position and strength of the leading edge vortices are studied by numerical solution of the three-dimensional Navier-Stokes equations. The leading edge jet is simulated by defining a permeable boundary, corresponding to the jet slot, where suitable boundary conditions are implemented. Numerical results are shown to compare favorably with experimental measurements. It is found that the use of spanwise leading edge blowing at moderate angle of attack magnifies the size and strength of the leading edge vortices, and moves the vortex cores outboard and upward. The increase in lift primarily comes from the greater nonlinear vortex lift. However, spanwise blowing causes earlier vortex breakdown, thus decreasing the stall angle. The effects of tangential blowing at low to moderate angles of attack tend to reduce the pressure peaks associated with leading edge vortices and to increase the suction peak around the leading edge, so that the integrated value of the surface pressure remains about the same. Tangential leading edge blowing in post-stall conditions is shown to re-establish vortical flow and delay vortex bursting, thus increasing C sub L sub max and stall angle.

Experimental investigation on the effects of non-cyclical frequency and amplitude variation on dynamic stall

NASA Astrophysics Data System (ADS)

Heintz, Kyle C.

An experimental study of a cambered airfoil undergoing non-cyclical, transient pitch trajectories and the resulting effects on the dynamic stall phenomenon is presented. Surface pressure measurements and airfoil incidence angle are acquired simultaneously to resolve instantaneous aerodynamic load coefficients at Mach numbers ranging from 0.2 to 0.4. Derived from these coefficients are various formulations of the aerodynamic damping factor, referred to copiously throughout. Using a two-motor mechanism, each providing independent frequency and amplitude input to the airfoil, unique pitch motions can be implemented by actively controlling the phase between inputs. This work primarily focuses on three pitch motion schemas, the first of which is a "chirp" style trajectory featuring concurrent exponential frequency growth and amplitude decay. Second, these parameters are tested separately to determine their individual contributions. Lastly, a novel dual harmonic pitch motion is devised which rapidly traverses dynamic stall regimes on an inter-cycle basis by modulating the static-stall penetration angle. Throughout all results presented, there is evidence that for consecutive pitch-cycles, the process of dynamic stall is affected when prior oscillations prior have undergone deeper stall-penetration angles. In other words when stall-penetration is descending, retreating from a regime of light or deep stall, statistics of load coefficients, such as damping coefficient, maximum lift, minimum quarter-chord moment, and their phase relationships, do not match the values seen when stall-penetration was growing. The outcomes herein suggest that the airfoil retains some memory of previous flow separation which has the potential to change the influence of the dynamic stall vortex.

Effects of Transposable Elements on the Expression of the Forked Gene of Drosophila Melanogaster

PubMed Central

Hoover, K. K.; Chien, A. J.; Corces, V. G.

1993-01-01

The products of the forked gene are involved in the formation and/or maintenance of a temporary fibrillar structure within the developing bristle rudiment of Drosophila melanogaster. Mutations in the forked locus alter this structure and result in aberrant development of macrochaetae, microchaetae and trichomes. The locus has been characterized at the molecular level by walking, mutant characterization and transcript analysis. Expression of the six forked transcripts is temporally restricted to midlate pupal development. At this time, RNAs of 6.4, 5.6, 5.4, 2.5, 1.9 and 1.1 kilobases (kb) are detected by Northern analysis. The coding region of these RNAs has been found to be within a 21-kb stretch of genomic DNA. The amino terminus of the proteins encoded by the 5.4- and 5.6-kb forked transcripts contain tandem copies of ankyrin-like repeats that may play an important role in the function of forked-encoded products. The profile of forked RNA expression is altered in seven spontaneous mutations characterized during this study. Three forked mutations induced by the insertion of the gypsy retrotransposon contain a copy of this element inserted into an intron of the gene. In these mutants, the 5.6-, 5.4- and 2.5-kb forked mRNAs are truncated via recognition of the polyadenylation site in the 5' long terminal repeat of the gypsy retrotransposon. These results help explain the role of the forked gene in fly development and further our understanding of the role of transposable elements in mutagenesis. PMID:8244011

Development of Tuning Fork Based Probes for Atomic Force Microscopy

NASA Astrophysics Data System (ADS)

Jalilian, Romaneh; Yazdanpanah, Mehdi M.; Torrez, Neil; Alizadeh, Amirali; Askari, Davood

2014-03-01

This article reports on the development of tuning fork-based AFM/STM probes in NaugaNeedles LLC for use in atomic force microscopy. These probes can be mounted on different carriers per customers' request. (e.g., RHK carrier, Omicron carrier, and tuning fork on a Sapphire disk). We are able to design and engineer tuning forks on any type of carrier used in the market. We can attach three types of tips on the edge of a tuning fork prong (i.e., growing Ag2Ga nanoneedles at any arbitrary angle, cantilever of AFM tip, and tungsten wire) with lengths from 100-500 μm. The nanoneedle is located vertical to the fork. Using a suitable insulation and metallic coating, we can make QPlus sensors that can detect tunneling current during the AFM scan. To make Qplus sensors, the entire quartz fork will be coated with an insulating material, before attaching the nanoneedle. Then, the top edge of one prong is coated with a thin layer of conductive metal and the nanoneedle is attached to the fork end of the metal coated prong. The metal coating provides electrical connection to the tip for tunneling current readout and to the electrodes and used to read the QPlus current. Since the amount of mass added to the fork is minimal, the resonance frequency spectrum does not change and still remains around 32.6 KHz and the Q factor is around 1,200 in ambient condition. These probes can enhance the performance of tuning fork based atomic microscopy.

In Situ Distribution Guided Analysis and Visualization of Transonic Jet Engine Simulations.

PubMed

Dutta, Soumya; Chen, Chun-Ming; Heinlein, Gregory; Shen, Han-Wei; Chen, Jen-Ping

2017-01-01

Study of flow instability in turbine engine compressors is crucial to understand the inception and evolution of engine stall. Aerodynamics experts have been working on detecting the early signs of stall in order to devise novel stall suppression technologies. A state-of-the-art Navier-Stokes based, time-accurate computational fluid dynamics simulator, TURBO, has been developed in NASA to enhance the understanding of flow phenomena undergoing rotating stall. Despite the proven high modeling accuracy of TURBO, the excessive simulation data prohibits post-hoc analysis in both storage and I/O time. To address these issues and allow the expert to perform scalable stall analysis, we have designed an in situ distribution guided stall analysis technique. Our method summarizes statistics of important properties of the simulation data in situ using a probabilistic data modeling scheme. This data summarization enables statistical anomaly detection for flow instability in post analysis, which reveals the spatiotemporal trends of rotating stall for the expert to conceive new hypotheses. Furthermore, the verification of the hypotheses and exploratory visualization using the summarized data are realized using probabilistic visualization techniques such as uncertain isocontouring. Positive feedback from the domain scientist has indicated the efficacy of our system in exploratory stall analysis.

3-D Stall Cell Inducement Using Static Trips on a NACA0015 Airfoil

NASA Astrophysics Data System (ADS)

Dell'Orso, Haley; Amitay, Michael

2015-11-01

Stall cells typically occur at high angles of attack and moderate to high Reynolds numbers (105 to 106) , which are applicable to High Altitude Long Endurance (HALE) vehicles. Under certain conditions stall cells can form abruptly and have a severe and detrimental impact on flight. In order to better understand this phenomenon, stall cell formation is studied using oil flow visualization and SPIV on a NACA0015 airfoil with AR = 2.67. It was shown that there is a critical Reynolds number above which stall cells begin to form, and that Recrit varies with angle of attack. Zig-zag tape and balsa wood trips were used to induce stall cells at lower Reynolds numbers than they would otherwise be present. This will aid in understanding the formation mechanism of these cells. It was also demonstrated that, in the case of full span trips, stall cells are induced by the 3-D nature of zig-zag trips and did not appear when balsa wood trips were used. This suggests that the formation of the stall cell might be due to 3-D disturbances that are naturally present in a flow field. AFOSR Grant Number FA9550-13-1-0059.

High-Speed Experiments on Combustion-Powered Actuation for Dynamic Stall Suppression

NASA Technical Reports Server (NTRS)

Matalanis, Claude; Bowles, Patrick; Lorber, Peter; Crittenden, Thomas; Glezer, Ari; Schaeffler, Norman; Min, Byung-Young; Jee, Solkeun; Kuczek, Andrzej; Wake, Brian

2016-01-01

This work documents high-speed wind tunnel experiments conducted on a pitching airfoil equipped with an array of combustion-powered actuators (COMPACT). The main objective of these experiments was to demonstrate the stall-suppression capability of COMPACT on a high-lift rotorcraft airfoil, the VR-12, at relevant Mach numbers. Through dynamic pressure measurements at the airfoil surface it was shown that COMPACT can positively affect the stall behavior of the VR-12 at Mach numbers up to 0.4. Static airfoil results demonstrated 25% and 50% increases in post-stall lift at Mach numbers of 0.4 and 0.3, respectively. Deep dynamic stall results showed cycle-averaged lift coefficient increases up to 11% at Mach 0.4. Furthermore, it was shown that these benefits could be achieved with relatively few pulses during down-stroke and with no need to pre-anticipate the stall event. The flow mechanisms responsible for stall suppression were investigated using particle image velocimetry.

Water quality of some logged and unlogged California streams

Treesearch

Fredric R. Kopperdahl; James W. Burns; Gary E. Smith

1971-01-01

Water quality was monitored in 1968 and 1969 in six coastal streams in northern California, four of which were subjected to logging and/or road building (Bummer Lake Creek, South Fork Yager Creek, Little North Fork Noyo River, and South Fork Caspar Creek), while the others remained undisturbed (Godwood Creek and North Fork Caspar Creek). The purposes of this study were...

Eukaryotic DNA Replication Fork.

PubMed

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Compressor Stall Recovery Through Tip Injection Assessed

NASA Technical Reports Server (NTRS)

Suder, Ken L.

2001-01-01

Aerodynamic stability is a fundamental limit in the compressor design process. The development of robust techniques for increasing stability has several benefits: enabling higher loading and fewer blades, increasing safety throughout a mission, increasing tolerance to stage mismatch during part-speed operation and speed transients, and providing an opportunity to match stages at the compressor maximum efficiency point, thus reducing fuel burn. Mass injection upstream of the tip of a high-speed axial compressor rotor is a stability enhancement approach known to be effective in suppressing stall in tip-critical rotors if the injection is activated before stall occurs. This approach to stall suppression requires that a reliable stall warning system be available. Tests have recently been performed to assess whether steady injection can also be used to recover from fully developed stall. If mass injection is effective in recovering from stall quickly enough to avoid structural damage or loss of engine power, then a stall warning system may not be required. The stall recovery tests were performed on a transonic compressor rotor at its design tip speed of 1475 ft/sec using four injectors evenly spaced around the compressor case upstream of the rotor. The injectors were connected to an external air source. In an actual engine application, the injected air would be supplied with compressor bleed air. The injectors were isolated from the air source by a fast-acting butterfly valve. With the injectors turned off, the compressor was throttled into stall. Air injection was then activated with no change in throttle setting by opening the butterfly valve. The compressor recovered from stall at a fixed throttle setting with the aid of tip injection. The unsteady operating characteristic of the rotor was measured during these tests using high-response pressure sensors located upstream and downstream of the rotor. The figure shows the results, where the unsteady pressure and mass flow are superimposed on the steady operating characteristic. The total injected mass flow was equal to 1.3 percent of the compressor flow. The solid line with no solid squares on it denotes the operating point during the beginning of throttle closure and the initial drop into stall. The gray traces denote the operating point during an additional throttle closure that occurred over the next 1200 rotor revolutions (4 sec). The dashed line denotes the recovery from stall that occurred during 90 rotor revolutions (0.3 sec) after the injectors were activated with no change in throttle setting. Tip injection not only recovers the compressor from stall, but also restores the compressor to its pre-stall level of pressure rise. In contrast, standard stall recovery schemes such as compressor bleed, stator vane actuation, or engine throttle modulation result in a loss of pressure rise across the compressor, which results in a loss of engine power.

Determinants of G quadruplex-induced epigenetic instability in REV1-deficient cells

PubMed Central

Schiavone, Davide; Guilbaud, Guillaume; Murat, Pierre; Papadopoulou, Charikleia; Sarkies, Peter; Prioleau, Marie-Noëlle; Balasubramanian, Shankar; Sale, Julian E

2014-01-01

REV1-deficient chicken DT40 cells are compromised in replicating G quadruplex (G4)-forming DNA. This results in localised, stochastic loss of parental chromatin marks and changes in gene expression. We previously proposed that this epigenetic instability arises from G4-induced replication fork stalls disrupting the accurate propagation of chromatin structure through replication. Here, we test this model by showing that a single G4 motif is responsible for the epigenetic instability of the BU-1 locus in REV1-deficient cells, despite its location 3.5 kb from the transcription start site (TSS). The effect of the G4 is dependent on it residing on the leading strand template, but is independent of its in vitro thermal stability. Moving the motif to more than 4 kb from the TSS stabilises expression of the gene. However, loss of histone modifications (H3K4me3 and H3K9/14ac) around the transcription start site correlates with the position of the G4 motif, expression being lost only when the promoter is affected. This supports the idea that processive replication is required to maintain the histone modification pattern and full transcription of this model locus. PMID:25190518

Timing matters: error-prone gap filling and translesion synthesis in immunoglobulin gene hypermutation

PubMed Central

Sale, Julian E.; Batters, Christopher; Edmunds, Charlotte E.; Phillips, Lara G.; Simpson, Laura J.; Szüts, Dávid

2008-01-01

By temporarily deferring the repair of DNA lesions encountered during replication, the bypass of DNA damage is critical to the ability of cells to withstand genomic insults. Damage bypass can be achieved either by recombinational mechanisms that are generally accurate or by a process called translesion synthesis. Translesion synthesis involves replacing the stalled replicative polymerase with one of a number of specialized DNA polymerases whose active sites are able to tolerate a distorted or damaged DNA template. While this property allows the translesion polymerases to synthesize across damaged bases, it does so with the trade-off of an increased mutation rate. The deployment of these enzymes must therefore be carefully regulated. In addition to their important role in general DNA damage tolerance and mutagenesis, the translesion polymerases play a crucial role in converting the products of activation induced deaminase-catalysed cytidine deamination to mutations during immunoglobulin gene somatic hypermutation. In this paper, we specifically consider the control of translesion synthesis in the context of the timing of lesion bypass relative to replication fork progression and arrest at sites of DNA damage. We then examine how recent observations concerning the control of translesion synthesis might help refine our view of the mechanisms of immunoglobulin gene somatic hypermutation. PMID:19008194

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

PubMed Central

Sasatani, Megumi; Xu, Yanbin; Kawai, Hidehiko; Cao, Lili; Tateishi, Satoshi; Shimura, Tsutomu; Li, Jianxiang; Iizuka, Daisuke; Noda, Asao; Hamasaki, Kanya; Kusunoki, Yoichiro; Kamiya, Kenji

2015-01-01

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure. PMID:25675240

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance

PubMed Central

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E.; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O’Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O’Driscoll, Mark; Semple, Robert

2014-01-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress. PMID:25105364

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance.

PubMed

Payne, Felicity; Colnaghi, Rita; Rocha, Nuno; Seth, Asha; Harris, Julie; Carpenter, Gillian; Bottomley, William E; Wheeler, Eleanor; Wong, Stephen; Saudek, Vladimir; Savage, David; O'Rahilly, Stephen; Carel, Jean-Claude; Barroso, Inês; O'Driscoll, Mark; Semple, Robert

2014-09-01

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

Topological Behavior of Plasmid DNA

PubMed Central

Higgins, N. Patrick; Vologodskii, Alexander V.

2015-01-01

The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells. PMID:26104708

Resolving complex chromosome structures during meiosis: versatile deployment of Smc5/6.

PubMed

Verver, Dideke E; Hwang, Grace H; Jordan, Philip W; Hamer, Geert

2016-03-01

The Smc5/6 complex, along with cohesin and condensin, is a member of the structural maintenance of chromosome (SMC) family, large ring-like protein complexes that are essential for chromatin structure and function. Thanks to numerous studies of the mitotic cell cycle, Smc5/6 has been implicated to have roles in homologous recombination, restart of stalled replication forks, maintenance of ribosomal DNA (rDNA) and heterochromatin, telomerase-independent telomere elongation, and regulation of chromosome topology. The nature of these functions implies that the Smc5/6 complex also contributes to the profound chromatin changes, including meiotic recombination, that characterize meiosis. Only recently, studies in diverse model organisms have focused on the potential meiotic roles of the Smc5/6 complex. Indeed, Smc5/6 appears to be essential for meiotic recombination. However, due to both the complexity of the process of meiosis and the versatility of the Smc5/6 complex, many additional meiotic functions have been described. In this review, we provide a clear overview of the multiple functions found so far for the Smc5/6 complex in meiosis. Additionally, we compare these meiotic functions with the known mitotic functions in an attempt to find a common denominator and thereby create clarity in the field of Smc5/6 research.

The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[OPEN

PubMed Central

Le Ret, Monique; Bergdoll, Marc; Bichara, Marc; Dietrich, André

2015-01-01

The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift. PMID:26462909

Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication.

PubMed

Bj Rås, Karine Ø; Sousa, Mirta M L; Sharma, Animesh; Fonseca, Davi M; S Gaard, Caroline K; Bj Rås, Magnar; Otterlei, Marit

2017-08-21

Base lesions in DNA can stall the replication machinery or induce mutations if bypassed. Consequently, lesions must be repaired before replication or in a post-replicative process to maintain genomic stability. Base excision repair (BER) is the main pathway for repair of base lesions and is known to be associated with DNA replication, but how BER is organized during replication is unclear. Here we coupled the iPOND (isolation of proteins on nascent DNA) technique with targeted mass-spectrometry analysis, which enabled us to detect all proteins required for BER on nascent DNA and to monitor their spatiotemporal orchestration at replication forks. We demonstrate that XRCC1 and other BER/single-strand break repair (SSBR) proteins are enriched in replisomes in unstressed cells, supporting a cellular capacity of post-replicative BER/SSBR. Importantly, we identify for the first time the DNA glycosylases MYH, UNG2, MPG, NTH1, NEIL1, 2 and 3 on nascent DNA. Our findings suggest that a broad spectrum of DNA base lesions are recognized and repaired by BER in a post-replicative process. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

PubMed Central

2015-01-01

The kinetic mechanisms by which DNA polymerases catalyze DNA replication and repair have long been areas of active research. Recently discovered Y-family DNA polymerases catalyze the bypass of damaged DNA bases that would otherwise block replicative DNA polymerases and stall replication forks. Unlike DNA polymerases from the five other families, the Y-family DNA polymerases have flexible, solvent-accessible active sites that are able to tolerate various types of damaged template bases and allow for efficient lesion bypass. Their promiscuous active sites, however, also lead to fidelities that are much lower than those observed for other DNA polymerases and give rise to interesting mechanistic properties. Additionally, the Y-family DNA polymerases have several other unique structural features and undergo a set of conformational changes during substrate binding and catalysis different from those observed for replicative DNA polymerases. In recent years, pre-steady-state kinetic methods have been extensively employed to reveal a wealth of information about the catalytic properties of these fascinating noncanonical DNA polymerases. Here, we review many of the recent findings on the kinetic mechanisms of DNA polymerization with undamaged and damaged DNA substrates by the Y-family DNA polymerases, and the conformational dynamics employed by these error-prone enzymes during catalysis. PMID:24716482

Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

PubMed Central

Ciesielski, Grzegorz L; Nadalutti, Cristina A; Oliveira, Marcos T; Griffith, Jack D; Kaguni, Laurie S

2018-01-01

Abstract Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of the mitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization. PMID:29432582

Salmon Supplementation Studies in Idaho Rivers, 1996-1998 Progress Report.

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

Reighn, Christopher A.; Lewis, Bert; Taki, Doug

1999-06-01

Information contained in this report summarizes the work that has been done by the Shoshone-Bannock Tribes Fisheries Department under BPA Project No. 89-098-3, Contract Number 92-BI-49450. Relevant data generated by the Shoshone-Bannock Tribe will be collated with other ISS cooperator data collected from the Salmon and Clearwater rivers and tributary streams. A summary of data presented in this report and an initial project-wide level supplementation evaluation will be available in the ISS 5 year report that is currently in progress. The Shoshone-Bannock Tribal Fisheries Department is responsible for monitoring a variety of chinook salmon (Oncorhynchus tshawytscha) production parameters as partmore » of the Idaho Supplementation Studies (BPA Project No. 89-098-3, Contract Number 92-BI-49450). Parameters include parr abundance in tributaries to the upper Salmon River; adult chinook salmon spawner abundance, redd counts, and carcass collection. A rotary screw trap is operated on the East Fork Salmon River and West Fork Yankee Fork Salmon River to enumerate and PIT-tag chinook smolts. These traps are also used to monitor parr movement, and collect individuals for the State and Tribal chinook salmon captive rearing program. The SBT monitors fisheries parameters in the following six tributaries of the Salmon River: Bear Valley Creek, East Fork Salmon River, Herd Creek, South Fork Salmon River, Valley Creek, and West Fork Yankee Fork. Chinook populations in all SBT-ISS monitored streams continue to decline. The South Fork Salmon River and Bear Valley Creek have the strongest remaining populations. Snorkel survey methodology was used to obtain parr population estimates for ISS streams from 1992 to 1997. Confidence intervals for the parr population estimates were large, especially when the populations were low. In 1998, based on ISS cooperator agreement, snorkeling to obtain parr population estimates was ceased due to the large confidence intervals. A rotary screw trap was operated on the West Fork Yankee Fork during the spring, summer, and fall of 1998 to monitor juvenile chinook migration. A screw trap was also operated on the East Fork of the Salmon River during the spring and fall from 1993 to 1997. Supplementation treatments have occurred on the South Fork Salmon River (IDFG), the East Fork Salmon River (EFSR), and the West Fork Yankee Fork of the Salmon River (WFYF). The EFSR received supplementation treatments yearly through 1995. There have been no treatments since 1995, and no significant future treatments from local broodstock are planned due to extremely poor escapement. The WFYF received a single presmolt treatment in 1994. There was an egg and adult release treatment in 1998 from the captive rearing program, not part of the original ISS study. Similarly, no significant future treatments are planned for the West Fork Yankee Fork due to extremely poor escapement. However, small scale experimental captive rearing and broodstock techniques are currently being tested with populations from the EFSR and WFYF. Captive rearing/broodstock techniques could potentially provide feedback for evaluation of supplementation. The other three SBT-ISS streams are control streams and do not receive supplementation treatments.« less

An experimental investigation of compressor stall using an on-line distortion indicator and signal conditioner

NASA Technical Reports Server (NTRS)

Costakis, W. G.; Wenzel, L. M.

1975-01-01

The relation of the steady-state and dynamic distortions and the stall margin of a J85-13 turbojet engine was investigated. A distortion indicator capable of computing two distortion indices was used. A special purpose signal conditioner was also used as an interface between transducer signals and distortion indicator. A good correlation of steady-state distortion and stall margin was established. The prediction of stall by using the indices as instantaneous distortion indicators was not successful. A sensitivity factor that related the loss of stall margin to the turbulence level was found.

Near Stall Flow Analysis in the Transonic Fan of the RTA Propulsion System

NASA Technical Reports Server (NTRS)

Hah, Chunill

2010-01-01

Turbine-based propulsion systems for access to space have been investigated at NASA Glenn Research center. A ground demonstrator engine for validation testing has been developed as a part of the program. The demonstrator, the Revolutionary Turbine Accelerator (RTA-1), is a variable cycle turbofan ramjet designed to transition from an augmented turbofan to a ramjet that produces the thrust required to accelerate the vehicle to Mach 4. The RTA-1 is designed to accommodate a large variation in bypass ratio from sea level static to Mach 4 flight condition. A key component of this engine is a new fan stage that accommodates these large variations in bypass ratio and flow ranges. In the present study, unsteady flow behavior in the fan of the RTA-1 is studied in detail with large eddy simulation (LES) and the numerical results are compared with measured data. During the experimental study of the fan stage, humming sound was detected at 100 % speed near stall operation. The main purpose of the study is to investigate details of the unsteady flow behavior at near stall operation and to identify a possible cause of the hum. The large eddy simulation of the current flow field reproduces main features of the measured flow very well. The LES simulation indicates that non-synchronous flow instability develops as the fan operates toward the stall limit. The FFT analysis of the calculated wall pressure shows that the rotating flow instability has the characteristic frequency that is about 50% of the blade passing frequency.

The dynamics underlying the regeneration and stalling of Hurricane Harvey

NASA Astrophysics Data System (ADS)

Liang, X. S.

2017-12-01

The explosive regeneration and stalling make the hurricane Harvey go from a little-noticed storm to an extremely destructive behemoth in late August 2017 that incurred an estimated economic loss at 70-200 billion USD. In this study, we use a recently developed analysis tool, namely, multiscale window transform (MWT), and the MWT-based theory of canonical transfer, to investigate the dynamics underlying this regeneration and stalling. The atmospheric fields are reconstructed onto three scale ranges or windows, namely, large-scale, tropical cyclone-scale, and cumulus convection-scale windows. The intertwined cyclone-scale nonlinear energy process is uniquely separated into a transport of energy within the cyclone window and an interscale transfer through reconstructing the "atomic" energy fluxes on the multiple scale windows. The resulting transfer bears a Lie bracket form, reminiscent of the Poisson bracket in Hamiltonian mechanics, and is hence referred to as canonical. It is found that within the Gulf of Mexico, Harvey gains much energy from the cumulus convection window through an inverse energy cascade, leading to its explosive growth. In the mean time, there is a barotropic instability (positive canonical transfer) center of the mean circulation in the lower and mid troposphere which lies quasi-steadily over Houston during August 22 through early September. The northwestward propagating Harvey meets that center and then stalls for two days near the coastline, dropping torrential and unprecedented amounts of rainfall and causing catastrophic flooding. It moves out of the instability center by the end of August, and then dissipates quickly in the following days.

Incidence rate of clinical mastitis on Canadian dairy farms.

PubMed

Olde Riekerink, R G M; Barkema, H W; Kelton, D F; Scholl, D T

2008-04-01

No nationwide studies of the incidence rate of clinical mastitis (IRCM) have been conducted in Canada. Because the IRCM and distribution of mastitis-causing bacteria may show substantial geographic variation, the primary objective of this study was to determine regional pathogen-specific IRCM on Canadian dairy farms. Additionally, the association of pathogen-specific IRCM with bulk milk somatic cell count (BMSCC) and barn type were determined. In total, 106 dairy farms in 10 provinces of Canada participated in the study for a period of 1 yr. Participating producers recorded 3,149 cases of clinical mastitis. The most frequently isolated mastitis pathogens were Staphylococcus aureus, Escherichia coli, Streptococcus uberis, and coagulase-negative staphylococci. Overall mean and median IRCM were 23.0 and 16.7 cases per 100 cow-years in the selected herds, respectively, with a range from 0.7 to 97.4 per herd. No association between BMSCC and overall IRCM was found, but E. coli and culture-negative IRCM were highest and Staph. aureus IRCM was lowest in low and medium BMSCC herds. Staphylococcus aureus, Strep. uberis, and Streptococcus dysgalactiae IRCM were lowest in the Western provinces. Staphylococcus aureus and Strep. dysgalactiae IRCM were highest in Québec. Cows in tie-stalls had higher incidences of Staph. aureus, Strep. uberis, coagulase-negative staphylococci, and other streptococcal IRCM compared with those in free-stalls, whereas cows in free stalls had higher Klebsiella spp. and E. coli IRCM than those in tie-stall barns. The focus of mastitis prevention and control programs should differ between regions and should be tailored to farms based on housing type and BMSCC.

Hydraulic geometry and sediment data for the South Fork Salmon River, Idaho, 1985-86

USGS Publications Warehouse

Williams, Rhea P.; O'Dell, Ivalou; Megahan, Walter F.

1989-01-01

Hydraulic geometry, suspended-sediment, and bedload samples were collected at three sites in the upper reach of the South Fork Salmon River drainage basin from April 1985 to June 1986. Sites selected were South Fork Salmon River near Krassel Ranger Station, Buckhorn Creek, and North Fork Lick Creek. Results of the data collection are presented in this report.

Effects of three types of free-stall surfaces on preferences and stall usage by dairy cows.

PubMed

Tucker, C B; Weary, D M; Fraser, D

2003-02-01

One important criterion in choosing appropriate housing systems for dairy cattle is that the freestall provides a comfortable surface for the cow. This paper describes two experiments testing the effects of commonly used lying surfaces on stall preference and stall usage by Holstein cows. In both experiments, 12 cows were housed individually in separate pens. Each pen contained three free stalls with a different surface: deep-bedded sawdust, deep-bedded sand, and a geotextile mattress covered with 2 to 3 cm of sawdust. The animals were restricted to each surface in turn, in a random order for either 2 (Experiment 1) or 3 d (Experiment 2). Both before and after this restriction phase, the animals were allowed access to all three surfaces, and preference was determined, based on lying times. Of the 12 cows used in Experiment 1, 10 preferred sawdust before and nine after the restriction phase. During the restriction phase, average lying times and number of lying events during the restriction phase were significantly lower for the sand-bedded stalls (P < or = 0.05), and standing times were higher on mattresses (P < or = 0.05), compared with sawdust. Although these cows had some experience with all three surfaces during the experiment, they had been housed in sawdust-bedded stalls during their previous lactation. Cows used in Experiment 2 had spent their previous lactation in sand bedded stalls. In this experiment, about half the cows preferred sand and half sawdust, after the restriction phase. During the restriction phase of experiment, lying times and number of lying events were lower, and standing times were higher when the animals were restricted to the mattresses compared to either sand or sawdust (P < or = 0.05). These results indicate that (1) free stall surface can affect both stall preferences and stall usage, and (2) mattresses are less preferred.

A comparison of free-stall barns used by modernized Wisconsin dairies.

PubMed

Bewley, J; Palmer, R W; Jackson-Smith, D B

2001-02-01

A primary objective of the Wisconsin Dairy Modernization Survey was to compare features of free-stall barns available to dairy producers. This study used data from a large random sample of expanding dairy farms to determine whether the theoretical benefits of particular free-stall configurations bear out under on-farm conditions. Comparisons were made among herds using free-stall barns as their primary housing for new versus remodeled facilities, barn design, bedding used, feed-delivery design, manure removal strategies, animal restraint, maternity areas, overcrowding, and cooling methods. Producers who made the transition from tie-stall housing to free-stall housing were satisfied with this decision. New free-stall barns provided a more desirable environment for the herds than remodeled free-stall barns, although initial investments were higher. When new free-stall barns were compared, herds with four-row barns had higher production, lower somatic cell count, and higher stocking rates than herds with six-row barns. Respondents were more satisfied with four- and six-row barns than with two- and three-row barns. Respondents felt sand provided some advantages for cow comfort, while satisfaction with bedding cost and manure handling was higher with mattresses. Dairy Herd Improvement data showed no difference in milk production or somatic cell count for producers who chose sand or mattress-based free stalls. Respondents were more satisfied with the use of drive-through feeding than other feed-delivery designs. Most producers chose to use tractor scrapers to remove manure; however, producers who used automated systems were more satisfied with manure management. Few differences were observed when comparing self-locking head gates to palpation rails. Overcrowding did not have any adverse affect on production or user satisfaction with feed intake or cow comfort. Using supplemental cooling appeared to facilitate higher production.

Short communication: Bacterial counts in recycled manure solids bedding replaced daily or deep packed in freestalls.

PubMed

Sorter, D E; Kester, H J; Hogan, J S

2014-05-01

An experiment was conducted to compare bacterial counts of mastitis pathogens in deep-packed manure solids bedding with those in manure solids bedding replaced daily from mattresses. Eighteen Holstein cows were housed in 1 pen with 18 stalls. One row of 9 stalls was equipped with mattresses topped with bedding. The back one-third of these stalls toward the alleyway was covered in 25 mm of recycled manure solids, which was removed daily for the next 6 d and replaced with bedding from the brisket board and lunge space areas of stalls. The second row of 9 stalls was bedded for 3 wk with 100 to 150 mm of deep-pack recycled manure bedding from which only fecal matter was removed daily. After 3 wk, bedding treatments were changed between rows in a switchback design. Mean total gram-negative bacterial counts did not differ between treatments throughout the experiment. Coliform and Klebsiella spp. bacterial counts were lower in daily replaced bedding compared with deep pack across the experiment and on each of d 0, 1, 2, and 6. Streptococcal counts were reduced in daily replacement stalls compared with deep-pack stalls on d 0 and greater in daily replacement stalls compared with deep-pack stalls on d 1, 2, and 6. Daily replacement of recycled manure bedding from the back one-third of the stalls appeared to be an effective approach to reducing exposure to coliforms, specifically Klebsiella, but not streptococci. However, bacterial counts in bedding from both treatments were elevated throughout the trial and resulted in considerable risk for exposure to teats and development of intramammary infections. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Investigation of a bio-inspired lift-enhancing effector on a 2D airfoil.

PubMed

Johnston, Joe; Gopalarathnam, Ashok

2012-09-01

A flap mounted on the upper surface of an airfoil, called a 'lift-enhancing effector', has been shown in wind tunnel tests to have a similar function to a bird's covert feathers, which rise off the wing's surface in response to separated flows. The effector, fabricated from a thin Mylar sheet, is allowed to rotate freely about its leading edge. The tests were performed in the NCSU subsonic wind tunnel at a chord Reynolds number of 4 × 10(5). The maximum lift coefficient with the effector was the same as that for the clean airfoil, but was maintained over an angle-of-attack range from 12° to almost 20°, resulting in a very gentle stall behavior. To better understand the aerodynamics and to estimate the deployment angle of the free-moving effector, fixed-angle effectors fabricated out of stiff wood were also tested. A progressive increase in the stall angle of attack with increasing effector angle was observed, with diminishing returns beyond the effector angle of 60°. Drag tests on both the free-moving and fixed effectors showed a marked improvement in drag at high angles of attack. Oil flow visualization on the airfoil with and without the fixed-angle effectors proved that the effector causes the separation point to move aft on the airfoil, as compared to the clean airfoil. This is thought to be the main mechanism by which an effector improves both lift and drag. A comparison of the fixed-effector results with those from the free-effector tests shows that the free effector's deployment angle is between 30° and 45°. When operating at and beyond the clean airfoil's stall angle, the free effector automatically deploys to progressively higher angles with increasing angles of attack. This slows down the rapid upstream movement of the separation point and avoids the severe reduction in the lift coefficient and an increase in the drag coefficient that are seen on the clean airfoil at the onset of stall. Thus, the effector postpones the stall by 4-8° and makes the stall behavior more gentle. The benefits of using the effector could include care-free operations at high angles of attack during perching and maneuvering flight, especially in gusty conditions.

1. Roaring Fork Motor Nature Trail, entrance sign. Great ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. Roaring Fork Motor Nature Trail, entrance sign. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

Roaring Fork Motor Nature Trail, Title Sheet Great Smoky ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Roaring Fork Motor Nature Trail, Title Sheet - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

9. Roaring Fork Motor Nature Trail, Reagan House. Great ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

9. Roaring Fork Motor Nature Trail, Reagan House. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

Regulation of Replication Fork Advance and Stability by Nucleosome Assembly

PubMed Central

Prado, Felix; Maya, Douglas

2017-01-01

The advance of replication forks to duplicate chromosomes in dividing cells requires the disassembly of nucleosomes ahead of the fork and the rapid assembly of parental and de novo histones at the newly synthesized strands behind the fork. Replication-coupled chromatin assembly provides a unique opportunity to regulate fork advance and stability. Through post-translational histone modifications and tightly regulated physical and genetic interactions between chromatin assembly factors and replisome components, chromatin assembly: (1) controls the rate of DNA synthesis and adjusts it to histone availability; (2) provides a mechanism to protect the integrity of the advancing fork; and (3) regulates the mechanisms of DNA damage tolerance in response to replication-blocking lesions. Uncoupling DNA synthesis from nucleosome assembly has deleterious effects on genome integrity and cell cycle progression and is linked to genetic diseases, cancer, and aging. PMID:28125036

Flight Measurements of the Flying Qualities of a Lockheed P-80A Airplane (Army No. 44-85099) - Stalling Characteristics

NASA Technical Reports Server (NTRS)

Anderson, Seth B.; Cooper, George E.

1947-01-01

This report contains the flight-test results of the stalling characteristics measured during the flying-qualities investigation of the Lockheed P-8OA airplane (Army No. 44-85099). The tests were conducted in straight and turning flight with and without wing-tip tanks. These tests showed satisfactory stalling characteristics and adequate stall warning for all configurations and conditions tested.

Mercury Contributions from Flint Creek and other Tributaries to the Upper Clark Fork River in Northwestern Montana

NASA Astrophysics Data System (ADS)

Langner, H.; Young, M.; Staats, M. F.

2013-12-01

Methylmercury contamination in biota is a major factor diminishing the environmental quality of the Upper Clark Fork River (CFR), e.g. by triggering human consumption limits of fish. The CFR is subject to one of the largest Superfund cleanup projects in the US, but remediation and restoration is currently focused exclusively on other mining-related contaminants (As, Cu, Zn, Pb, Cd), which may be counterproductive with respect to the bio-availability of mercury, for example by creation of wetlands along mercury-contaminated reaches of the river. The identification and elimination of Hg sources is an essential step toward reducing the methylmercury exposure in the biota of the CFR watershed because a strong correlation exists between total mercury levels in river sediment and methylmercury levels in aquatic life. We analyzed duplicate samples from the top sediment layer of the main stem and significant tributaries to the Clark Fork River along a 240 km reach between Butte, MT and downstream of the Missoula Valley. Mercury concentrations were 1.3 × 1.6 (mean × SD, n = 35) in the main stem. Concentrations in tributaries varied widely (0.02 to 85 mg/kg) and seemed only loosely related to the number of historic precious metal mines in the watershed. In the upper reach of the CFR, elevated Hg levels are likely caused by residual contaminated sediments in the flood plain. Levels tend to decrease downstream until Drummond, MT, where Flint Creek contributes a significant amount of mercury, causing Hg levels in the main stem CFR to increase from 0.7 to 4 mg/kg. Levels continue to decrease downstream. Flint Creek is the single largest contributor of Hg to the CFR. Detailed sampling of the main stem Flint Creek and tributaries (26 sites) showed extremely high levels in two tributaries (22 to 85 mg/kg) where historic milling operations were located. Elimination of these point sources may be accomplished comparatively economically and may significantly reduce mercury levels in Flint Creek and the Clark Fork River basin.

Channelization and floodplain forests: impacts of accelerated sedimentation and valley plug formation on floodplain forests of the Middle Fork Forked Deer River, Tennessee, USA

Treesearch

Sonja N. Oswalt; Sammy L. King

2005-01-01

We evaluated the severe degradation of floodplain habitats resulting from channelization and concomitant excessive coarse sedimentation on the Middle Fork Forked Deer River in west Tennessee from 2000 to 2003. Land use practices have resulted in excessive sediment in the tributaries and river system eventually resulting in sand deposition on the floodplain, increased...

76 FR 53964 - Dale J. Bingham, P.A.; Revocation of Registration

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-30

... DEPARTMENT OF JUSTICE Drug Enforcement Administration Dale J. Bingham, P.A.; Revocation of... Enforcement Administration, issued an Order to Show Cause to Dale J. Bingham, P.A. (Registrant), of Ash Fork... 28 CFR 0.100(b), I order that DEA Certificate of Registration MB1048746, issued to Dale J. Bingham, P...

At the centre of the tuning fork

NASA Image and Video Library

2015-11-02

This galaxy is known as Mrk 820 and is classified as a lenticular galaxy — type S0 on the Hubble Tuning Fork. The Hubble Tuning Fork is used to classify galaxies according to their morphology. Elliptical galaxies look like smooth blobs in the sky and lie on the handle of the fork. They are arranged along the handle based on how elliptical they are, with the more spherical galaxies furthest from the tines of the fork, and the more egg-shaped ones closest to the end of the handle where it divides. The two prongs of the tuning fork represent types of unbarred and barred spiral galaxies. Lenticular galaxies like Mrk 820 are in the transition zone between ellipticals and spirals and lie right where the fork divides. A closer look at the appearance of Mrk 820 reveals hints of a spiral structure embedded in a circular halo of stars. Surrounding Mrk 820 in this image is good sampling of other galaxy types, covering almost every type found on the Hubble Tuning Fork, both elliptical and spiral. Most of the smears and specks are distant galaxies, but the prominent bright object at the bottom is a foreground star called TYC 4386-787-1. A version of this image was entered into the Hubble's Hidden Treasures image processing competition by contestant Judy Schmidt.

Co-localization of polar replication fork barriers and rRNA transcription terminators in mouse rDNA.

PubMed

López-estraño, C; Schvartzman, J B; Krimer, D B; Hernández, P

1998-03-27

We investigated the replication of the region where transcription terminates in mouse rDNA. It contains a replication fork barrier (RFB) that behaves in a polar manner, arresting only replication forks moving in the direction opposite to transcription. This RFB consists of several closely spaced fork arrest sites that co-localize with the transcription terminator elements, known as Sal boxes. Sal boxes are the target for mTTF-I (murine transcription termination factor I). These results suggest that both termination of rRNA transcription and replication fork arrest may share cis-acting as well as trans-acting factors. Copyright 1998 Academic Press Limited.

On the sound field radiated by a tuning fork

NASA Astrophysics Data System (ADS)

Russell, Daniel A.

2000-12-01

When a sounding tuning fork is brought close to the ear, and rotated about its long axis, four distinct maxima and minima are heard. However, when the same tuning fork is rotated while being held at arm's length from the ear only two maxima and minima are heard. Misconceptions concerning this phenomenon are addressed and the fundamental mode of the fork is described in terms of a linear quadrupole source. Measured directivity patterns in the near field and far field of several forks agree very well with theoretical predictions for a linear quadrupole. Other modes of vibration are shown to radiate as dipole and lateral quadrupole sources.

Two-stage fan. 2: Data and performance with redesigned second stage rotor uniform and distorted inlet flows

NASA Technical Reports Server (NTRS)

Messenger, H. E.; Keenan, M. J.

1974-01-01

A two-stage fan with a first rotor tip speed of 1450 ft/sec (441.96 m/sec) and no inlet guide vanes was tested with uniform and distorted inlet flows, with a redesigned second rotor having a part span shroud to prevent flutter, with variable-stagger stators set in nominal positions, and without rotor casing treatment. The fan achieved a pressure ratio 2.8 at a corrected flow of 185.4 lbm/sec (84.0 kg/sec), an adiabatic efficiency of 85.0 percent, and a stall margin of 12 percent. The redesigned second rotor did not flutter. Tip radial distortion reduced the stall margin at intermediate speed, but had little effect on stall margin at high or low speeds. Hub radial distortion reduced the stall margin at design speed but increased stall margin at low speed. Circumferential distortion reduced stall pressure ratio and flow to give approximately the same stall lines with uniform inlet flow. Distortions were attenuated by the fan. For Vol. 1, see N74-11421.

8. Roaring Fork Motor Nature Trail, handbuilt rock pile. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

8. Roaring Fork Motor Nature Trail, hand-built rock pile. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress

PubMed Central

Rodriguez, Jairo; Tsukiyama, Toshio

2013-01-01

Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks. PMID:23307868

Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities.

PubMed

Ward, T R; Hoang, M L; Prusty, R; Lau, C K; Keil, R L; Fangman, W L; Brewer, B J

2000-07-01

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).

Rotating Stall Suppression Using Oscillatory Blowing Actuation on Blades

DTIC Science & Technology

2010-06-30

severe mechanical vibrations. Certainly, violent surge cannot be tolerated in an aircraft jet engine because of the danger of mechanical failure or...isolated airfoils increases the stall angle. Therefore, herein it was hypothesized that when a stall cell reaches a blade with jet actuation, the stall...Detailed view of the jet slot. Figure 2.30: Wing fences mounted on test blade (with the neighboring airfoils re- moved). (a) Attachment and pipe (b

A theory of post-stall transients in axial compression systems. II - Application

NASA Technical Reports Server (NTRS)

Greitzer, E. M.; Moore, F. K.

1985-01-01

Using the theory developed in Part I, calculations have been carried out to show the evolution of the mass flow, pressure rise, and rotating-stall cell amplitude during compression system post-stall transients. In particular, it is shown that the unsteady growth or decay of the stall cell can have a significant effect on the instantaneous compressor pumping characteristic and hence on the overall system behavior. A limited parametric study is carried out to illustrate the impact of different system features on transient behavior. It is shown, for example, that the ultimate mode of system response, surge or stable rotating stall, depends not only on the B parameter, but also on the compressor length-to-radius ratio. Small values of this latter quantity tend to favor the occurrence of surge, as do large values of B. Based on the analytical and numerical results, several specific topics are suggested for future research on post-stall transients.

Comparison of Milk Yield and Animal Health in Turkish Farms with Differing Stall Types and Resting Surfaces

PubMed Central

Kara, Nurcan Karslioglu; Galic, Askin; Koyuncu, Mehmet

2015-01-01

The current study was carried out to determine the influence of different resting surfaces and stall types on milk yield and animal health. Study was carried out in Bursa that is one of the most important cities of Turkey in terms of dairy production. Effects of resting surfaces and stall types on milk yield were found to be important. Also influence of different resting surfaces and stall types on lactation length was examined and found that rubber mats were different from the two other options. Relationships between different resting surfaces or stall types and health problems were examined and connection between stall type and repeat breeding (RB), dystocia, retained placenta and a connection between resting surface types and RB and clinical mastitis were found to be important. Considering their economic reflections, it can be said that results are quite important to the Turkish dairy industry. PMID:25557824

Vertical Motion Simulator Experiment on Stall Recovery Guidance

NASA Technical Reports Server (NTRS)

Schuet, Stefan; Lombaerts, Thomas; Stepanyan, Vahram; Kaneshige, John; Shish, Kimberlee; Robinson, Peter; Hardy, Gordon H.

2017-01-01

A stall recovery guidance system was designed to help pilots improve their stall recovery performance when the current aircraft state may be unrecognized under various complicating operational factors. Candidate guidance algorithms were connected to the split-cue pitch and roll flight directors that are standard on large transport commercial aircraft. A new thrust guidance algorithm and cue was also developed to help pilots prevent the combination of excessive thrust and nose-up stabilizer trim. The overall system was designed to reinforce the current FAA recommended stall recovery procedure. A general transport aircraft model, similar to a Boeing 757, with an extended aerodynamic database for improved stall dynamics simulation fidelity was integrated into the Vertical Motion Simulator at NASA Ames Research Center. A detailed study of the guidance system was then conducted across four stall scenarios with 30 commercial and 10 research test pilots, and the results are reported.

Preliminary analysis of dynamic stall effects on a 91-meter wind turbine rotor

NASA Technical Reports Server (NTRS)

Wilson, Robert E.

1995-01-01

Analytical investigation of dynamic stall on HAWT (horizontal-axis wind turbines) rotor loads was conducted. Dynamic stall was modeled using the Gormont approach on the MOD-2 rotor, treating the blade as a rigid body teetering about a fixed axis. Blade flapwise bending moments at station 370 were determined with and without dynamic stall for spatial variations in local wind speed due to wind shear and yaw. The predicted mean flapwise bending moments were found to be in good agreement with test results. Results obtained with and without dynamic stall showed no significant difference for the mean flapwise bending moment. The cyclic bending moments calculated with and without dynamic stall effects were substantially the same. None of the calculated cyclic loads reached the level of the cyclic loads measured on the MOD-2 using the Boeing five-minute-average technique.

Effect of stall design on dairy calf transition to voluntary feeding on an automatic milk feeder after introduction to group housing.

PubMed

Wilson, Tanya R; LeBlanc, Stephen J; DeVries, Trevor J; Haley, Derek B

2018-06-01

Automatic milk feeders (AMF) for young dairy calves are widely used in the dairy industry. These feeders are thought to have benefits for calf health and welfare and may reduce labor required for feeding; however, little is known about how calves adapt to feeding with AMF. The objective of this study was to observe the effects of feeding stall design on calves learning to use the AMF. The hypothesis was that solid side stalls, compared with steel bar stalls, would result in a longer latency to approach and feed from the AMF without assistance. A total of 147 Holstein calves (80 male and 67 female) were enrolled at 4 d of age, introduced to a group pen, and, at the same time, trained on an AMF. For training, calves were allowed to suck on the trainer's fingers and guided to the teat. Calves were allocated to 1 of 2 stall designs at the pen level, depending on which treatment cohort they were born into, either with steel bar stall walls (n = 46 male, 34 female calves) or with solid side stall walls (n = 34 male, 33 female calves). For 72 h after introductory training on the AMF, data from the feeders were collected and calf behavior was monitored by video. Outcomes measured included latency to first voluntary visit to the feeder and to first feeding, time spent in the feeder, amount of milk consumed over 72 h, number of retraining sessions required (retrained if <2 L was consumed every 12 h), and exploratory behavior, such as sniffing and licking of the feeder. Data were analyzed using mixed effects linear regression models or a Poisson model for the outcome of retraining. For certain outcomes the effects of stall design interacted with difficulty of training (willingness to enter feeder and drink); for the 38% of calves that were scored as moderately difficult to train on a scale of easy, moderate, or difficult, treatment (stall design) differences were detected. These calves took 2× longer to lick or bite toward the nipple, 2× longer to first voluntarily feeding, and consumed less milk over 72 h following training when trained on the steel bar stall design. These results suggest simple features of a stall may influence how quickly calves learn to use an AMF, but that the influence of stall wall design was affected by how easy calves were to train on the feeder upon initial introduction, which may depend in part on certain aspects of calf temperament. For many calves, solid side stalls at an AMF resulted faster in adaption than the steel bar stalls. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

LOOKING EASTSOUTHEAST. Showing downstream side of completed bridge, from confluence ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

LOOKING EAST-SOUTHEAST. Showing downstream side of completed bridge, from confluence of Trinity and South Fork Trinity Rivers - South Fork Trinity River Bridge, State Highway 299 spanning South Fork Trinity River, Salyer, Trinity County, CA

7. Roaring Fork Motor Nature Trail, rocks along edge of ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

7. Roaring Fork Motor Nature Trail, rocks along edge of road. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

5. Roaring Fork Motor Nature Trail, vista at stop three. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. Roaring Fork Motor Nature Trail, vista at stop three. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

15. INSIDE VIEW OF FLUME, LOOKING DOWNSTREAM, LEFT FORK TO ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

15. INSIDE VIEW OF FLUME, LOOKING DOWNSTREAM, LEFT FORK TO SETTLING BASIN, SHOWING RIGHT FORK WITH GATE IN PLACE AND A FEW NEEDLES IN PLACE - Electron Hydroelectric Project, Along Puyallup River, Electron, Pierce County, WA

2. Roaring Fork Motor Nature Trail, road view before first ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. Roaring Fork Motor Nature Trail, road view before first stop. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

6. Roaring Fork Motor Nature Trail, road view after stop ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

6. Roaring Fork Motor Nature Trail, road view after stop four. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

5. VIEW EAST ACROSS BRIDGE SHOWING RAILING DETAILS AND WATERFALLS ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. VIEW EAST ACROSS BRIDGE SHOWING RAILING DETAILS AND WATERFALLS OF THE SOUTH FORK OF THE TUOLUMNE. - South Fork Tuolumne River Bridge, Spanning South Fork Tuolumne River on Tioga Road, Mather, Tuolumne County, CA

Evaluating the impacts of logging activities on erosion and suspended sediment transport in the Caspar Creek watersheds

Treesearch

Jack Lewis

1998-01-01

Suspended sediment has been sampled at both the North and South Fork weirs of Caspar Creek in northwestern California since 1963, and at 13 tributary locations in the North Fork since 1986. The North Fork gaging station (NFC) was used as a control to evaluate the effects of logging in the South Fork, in the 1970's, on annual sediment loads. In the most...

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

PubMed

Tokarsky, E John; Gadkari, Varun V; Zahurancik, Walter J; Malik, Chanchal K; Basu, Ashis K; Suo, Zucai

2016-10-01

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG C8- N -ABA ), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dG C8- N -ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dG C8- N -ABA in vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dG C8- N- ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dG C8- N -ABA . In contrast, hPolι and hRev1 were severely stalled by dG C8- N -ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dG C8- N- ABA . Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dG C8- N -ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation. Copyright © 2016 Elsevier B.V. All rights reserved.

Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

PubMed

Henderson, Morgan L; Kreuzer, Kenneth N

2015-01-01

Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

PubMed

Duggin, Iain G; Matthews, Jacqueline M; Dixon, Nicholas E; Wake, R Gerry; Mackay, Joel P

2005-04-01

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

Progress has Stalled in U.S. Stroke Death Rates after Decades of Decline

MedlinePlus

... Library (PHIL) Progress has stalled in US stroke death rates after decades of decline More timely stroke ... cdc.gov/vitalsigns/stroke/infographic.html#graphic) Stroke death declines have stalled in 3 out of every ...

Aerodynamic analysis of the Darrieus wind turbines including dynamic-stall effects

NASA Astrophysics Data System (ADS)

Paraschivoiu, Ion; Allet, Azeddine

Experimental data for a 17-m wind turbine are compared with aerodynamic performance predictions obtained with two dynamic stall methods which are based on numerical correlations of the dynamic stall delay with the pitch rate parameter. Unlike the Gormont (1973) model, the MIT model predicts that dynamic stall does not occur in the downwind part of the turbine, although it does exist in the upwind zone. The Gormont model is shown to overestimate the aerodynamic coefficients relative to the MIT model. The MIT model is found to accurately predict the dynamic-stall regime, which is characterized by a plateau oscillating near values of the experimental data for the rotor power vs wind speed at the equator.

South Fork Latrine, east elevation showing structure in context, view ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Latrine, east elevation showing structure in context, view west - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

3. Roaring Fork Motor Nature Trail, view between second and ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. Roaring Fork Motor Nature Trail, view between second and third stops - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

11. Roaring Fork Motor Nature Trail, boulders along road after ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

11. Roaring Fork Motor Nature Trail, boulders along road after stop 13. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

2. VIEW OF NORTH FACE SHOWING SUBSTRUCTURE AND ABUTMENTS OF ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. VIEW OF NORTH FACE SHOWING SUBSTRUCTURE AND ABUTMENTS OF BRIDGE CROSSING THE SOUTH FORK OF THE TUOLUMNE RIVER. - South Fork Tuolumne River Bridge, Spanning South Fork Tuolumne River on Tioga Road, Mather, Tuolumne County, CA

Arabidopsis homologues of the histone chaperone ASF1 are crucial for chromatin replication and cell proliferation in plant development.

PubMed

Zhu, Yan; Weng, Minjie; Yang, Yue; Zhang, Chi; Li, Ziyu; Shen, Wen-Hui; Dong, Aiwu

2011-05-01

Anti-silencing function1 (ASF1) is an evolutionarily conserved histone chaperone. Studies in yeast and animals indicate that ASF1 proteins play important roles in various chromatin-based processes, including gene transcription, DNA replication and repair. While two genes encoding ASF1 homologues, AtASF1A and AtASF1B, are found in the Arabidopsis genome, their function has not been studied. Here we report that both AtASF1A and AtASF1B proteins bind histone H3, and are localized in the cytoplasm and the nucleus. Loss-of-function of either AtASF1A or AtASF1B did not show obvious defects, whereas simultaneous knockdown of both genes in the double mutant Atasf1ab drastically inhibited plant growth and caused abnormal vegetative and reproductive organ development. The Atasf1ab mutant plants exhibit cell number reduction, S-phase delay/arrest, and reduced polyploidy levels. Selective up-regulation of expression of a subset of genes, including those involved in S-phase checkpoints and the CYCB1;1 gene at the G₂-to-M transition, was observed in Atasf1ab. Furthermore, the Atasf1ab-triggered replication fork stalling constitutively activates the DNA damage checkpoint and repair genes, including ATM, ATR, PARP1 and PARP2 as well as several genes of the homologous recombination (HR) pathway but not genes of the non-homologous end joining (NHEJ) pathway. In spite of the activation of repair genes, an increased level of DNA damage was detected in Atasf1ab, suggesting that defects in the mutant largely exceed the available capacity of the repair machinery. Taken together, our study establishes crucial roles for the AtASF1A and AtASF1B genes in chromatin replication, maintenance of genome integrity and cell proliferation during plant development. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η.

PubMed

Ucisik, Melek N; Hammes-Schiffer, Sharon

2017-04-20

Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine-thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation-π, and π-π interactions of the side chains with the dATP and the TTD or thymine-thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment.

Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η

PubMed Central

2016-01-01

Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine–thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation−π, and π–π interactions of the side chains with the dATP and the TTD or thymine–thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment. PMID:28423907

Distribution and movement of bull trout in the upper Jarbidge River watershed, Nevada

USGS Publications Warehouse

Allen, M. Brady; Connolly, Patrick J.; Mesa, Matthew G.; Charrier, Jodi; Dixon, Chris

2010-01-01

In 2006 and 2007, we surveyed the occurrence of bull trout (Salvelinus confluentus), the relative distributions of bull trout and redband trout (Oncorhynchus mykiss), and stream habitat conditions in the East and West Forks of the Jarbidge River in northeastern Nevada and southern Idaho. We installed passive integrated transponder (PIT) tag interrogation systems at strategic locations within the watershed, and PIT-tagged bull trout were monitored to evaluate individual fish growth, movement, and the connectivity of bull trout between streams. Robust bull trout populations were found in the upper portions of the East Fork Jarbidge River, the West Fork Jarbidge River, and in the Pine, Jack, Dave, and Fall Creeks. Small numbers of bull trout also were found in Slide and Cougar Creeks. Bull trout were numerically dominant in the upper portions of the East Fork Jarbidge River, and in Fall, Dave, Jack, and Pine Creeks, whereas redband trout were numerically dominant throughout the rest of the watershed. The relative abundance of bull trout was notably higher at altitudes above 2,100 m. This study was successful in documenting bull trout population connectivity within the West Fork Jarbidge River, particularly between West Fork Jarbidge River and Pine Creek. Downstream movement of bull trout to the confluence of the East Fork and West Fork Jarbidge River both from Jack Creek (rkm 16.6) in the West Fork Jarbidge River and from Dave Creek (rkm 7.5) in the East Fork Jarbidge River was detected. Although bull trout exhibited some downstream movement during the spring and summer, much of their emigration occurred in the autumn, concurrent with decreasing water temperatures and slightly increasing flows. The bull trout that emigrated were mostly age-2 or older, but some age-1 fish also emigrated. Upstream movement by bull trout was detected less than downstream movement. The overall mean annual growth rate of bull trout in the East Fork and West Fork Jarbidge River was 36 mm. This growth rate is within the range reported in other river systems and is indicative of good habitat conditions. Mark-recapture methods were used to estimate a population of 147 age-1 or older bull trout in the reach of Jack Creek upstream of Jenny Creek.

Task I: A Computational Model for Short Wavelength Stall Inception and Development In Multi-Stage Compressors

NASA Technical Reports Server (NTRS)

Suder, Kenneth (Technical Monitor); Tan, Choon-Sooi

2003-01-01

A computational model is presented for simulating axial compressor stall inception and development via disturbances with length scales on the order of several (typically about three) blade pitches. The model was designed for multi-stage compressors in which stall is initiated by these short wavelength disturbances, also referred to as spikes. The inception process described is fundamentally nonlinear, in contrast to the essentially linear behavior seen in so-called modal stall inception . The model was able to capture the following experimentally observed phenomena: (1) development of rotating stall via short wavelength disturbances, (2) formation and evolution of localized short wavelength stall cells in the first stage of a mismatched compressor, (3) the switch from long to short wavelength stall inception resulting from the re-staggering of the inlet guide vane, (4) the occurrence of rotating stall inception on the negatively sloped portion of the compressor characteristic. Parametric investigations indicated that (1) short wavelength disturbances were supported by the rotor blade row, (2) the disturbance strength was attenuated within the stators, and (3) the reduction of inter-blade row gaps can suppress the growth of short wavelength disturbances. It is argued that each local component group (rotor plus neighboring stators) has its own instability point (i.e. conditions at which disturbances are sustained) for short wavelength disturbances, with the instability point for the compressor set by the most unstable component group.

Use of impact testing to predict softness, cow preference, and hardening over time of stall bases.

PubMed

Fulwider, W K; Palmer, R W

2004-09-01

The objective of this study was to assess the softness and durability of commercially available free-stall bases, and to determine the relationship of stall base softness to cow preference. Clegg impact values were recorded at the University of Wisconsin-Madison Arlington Agricultural Research Station on June 19, 2002, and again on July 24, 2003. The Clegg Impact Soil Tester (model 95051, Lafayette Instruments, Lafayette, IN) with a 20-kg hammer was used in this study. The impact of the hammer on the free-stall base results in a digital display based on peak deceleration of the hammer's impact with the free-stall base in tens of gravities (CIV/H). The CIV/H value, as measured by the Clegg Impact hammer, is based on peak deceleration of the 20-kg hammer's impact with the surface, from a height of 30 cm. Clegg impact measures were highly correlated with cow preference measurements. This relationship suggests that Clegg impact measures of compressibility were good indicators for predicting stall-base acceptance. A cork mattress, 4 foam mattresses, 4 rubber mattresses, 4 rubber mats, and a waterbed were evaluated in this study. Foam-based mattresses lost cushioning ability faster than rubber mattresses or rubber mats. Clegg impact values increased over the 13-mo time period for most stall base types, which indicated a tendency of stall bases to harden.

A Cultural Resource Reconnaissance of Two Proposed Bank Unloading Areas, East Grand Forks, Polk County, Minnesota,

DTIC Science & Technology

1984-12-01

architectural or archeotogical evidence was identified. The southern unloading area includes the former sites of a brewery and a sawmill, both of... brewery and a sawmill, both of which were associated with 0 significant historic events and themes in the late nineteenth and early twentieth century...Forks Brewery (1888) . . . . . . . 16 Figure 4. The Grand Forks Lumber Company mill is located at A. -. Building at B is probably the East Grand Forks

Archaeological Investigations on the East Fork of the Salmon River, Custer County, Idaho.

DTIC Science & Technology

1984-01-01

coniferous environment in addition to pine marten (Martes americana), red squirrel (Tamiasciurus hudsonicus), porcupine (Erithizon dorsatum), mountain vole...can be seen in small herds throughout the East Fork valley from the Salmon River to Big Boulder Creek. Two bands of Rocky Mountain bighorn sheep...utilize the Challis Planning Unit, one on the East Fork and the other in the Birch Creek area. The East Fork herd is comprised of approximately 50-70

Dissolved-Solids Load in Henrys Fork Upstream from the Confluence with Antelope Wash, Wyoming, Water Years 1970-2009

USGS Publications Warehouse

Foster, Katharine; Kenney, Terry A.

2010-01-01

Annual dissolved-solids load at the mouth of Henrys Fork was estimated by using data from U.S. Geological Survey streamflow-gaging station 09229500, Henrys Fork near Manila, Utah. The annual dissolved-solids load for water years 1970-2009 ranged from 18,300 tons in 1977 to 123,300 tons in 1983. Annual streamflows for this period ranged from 14,100 acre-feet in 1977 to 197,500 acre-feet in 1983. The 25-percent trimmed mean dissolved-solids load for water years 1970-2009 was 44,300 tons per year at Henrys Fork near Manila, Utah. Previous simulations using a SPAtially Referenced Regression On Watershed attributes (SPARROW) model for dissolved solids specific to water year 1991 conditions in the Upper Colorado River Basin predicted an annual dissolved-solids load of 25,000 tons for the Henrys Fork Basin upstream from Antelope Wash. On the basis of computed dissolved-solids load data from Henrys Fork near Manila, Utah, together with estimated annual dissolved-solids load from Antelope Wash and Peoples Canal, this prediction was adjusted to 37,200 tons. As determined by simulations with the Upper Colorado River Basin SPARROW model, approximately 56 percent (14,000 tons per year) of the dissolved-solids load at Henrys Fork upstream from Antelope Wash is associated with the 21,500 acres of irrigated agricultural lands in the upper Henrys Fork Basin.

South Fork Latrine, oblique view showing south and east sides; ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Latrine, oblique view showing south and east sides; view northwest - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

South Fork Latrine showing north and west sides, general view ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Latrine showing north and west sides, general view to southeast - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

COMPLETED STRUCTURE. View is eastsoutheast of downstream side of bridge, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

COMPLETED STRUCTURE. View is east-southeast of downstream side of bridge, from beyond confluence of Trinity and South Fork Trinity Rivers - South Fork Trinity River Bridge, State Highway 299 spanning South Fork Trinity River, Salyer, Trinity County, CA

7. View to southeast. View of downstream side of bridge ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

7. View to southeast. View of downstream side of bridge from confluence of Trinity and South Fork Trinity Rivers. (90mm Lens) - South Fork Trinity River Bridge, State Highway 299 spanning South Fork Trinity River, Salyer, Trinity County, CA

50 CFR 226.211 - Critical habitat for Seven Evolutionarily Significant Units (ESUs) of Salmon (Oncorhynchus spp...

Code of Federal Regulations, 2014 CFR

2014-10-01

... form and maintain physical habitat conditions and support juvenile growth and mobility; (ii) Water...); Fossil Creek (39.9447, -123.0403); Middle Fork Eel River (40.0780, -123.0442); North Fork Middle Fork Eel...

50 CFR 226.211 - Critical habitat for Seven Evolutionarily Significant Units (ESUs) of Salmon (Oncorhynchus spp...

Code of Federal Regulations, 2011 CFR

2011-10-01

... form and maintain physical habitat conditions and support juvenile growth and mobility; (ii) Water...); Fossil Creek (39.9447, -123.0403); Middle Fork Eel River (40.0780, -123.0442); North Fork Middle Fork Eel...

50 CFR 226.211 - Critical habitat for Seven Evolutionarily Significant Units (ESUs) of Salmon (Oncorhynchus spp...

Code of Federal Regulations, 2013 CFR

2013-10-01

... form and maintain physical habitat conditions and support juvenile growth and mobility; (ii) Water...); Fossil Creek (39.9447, -123.0403); Middle Fork Eel River (40.0780, -123.0442); North Fork Middle Fork Eel...

50 CFR 226.211 - Critical habitat for Seven Evolutionarily Significant Units (ESUs) of Salmon (Oncorhynchus spp...

Code of Federal Regulations, 2012 CFR

2012-10-01

... form and maintain physical habitat conditions and support juvenile growth and mobility; (ii) Water...); Fossil Creek (39.9447, -123.0403); Middle Fork Eel River (40.0780, -123.0442); North Fork Middle Fork Eel...

50 CFR 226.211 - Critical habitat for Seven Evolutionarily Significant Units (ESUs) of Salmon (Oncorhynchus spp...

Code of Federal Regulations, 2010 CFR

2010-10-01

... form and maintain physical habitat conditions and support juvenile growth and mobility; (ii) Water...); Fossil Creek (39.9447, -123.0403); Middle Fork Eel River (40.0780, -123.0442); North Fork Middle Fork Eel...

LAKE FORK

EPA Science Inventory

The Lake Fork of the Arkansas River Watershed has been adversely affected through mining, water diversion and storage projects, grazing, logging, and other human influences over the past 120 years. It is the goals of the LFWWG to improve the health of Lake fork by addressing th...

12. Roaring Fork Motor Nature Trail, place of a thousand ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. Roaring Fork Motor Nature Trail, place of a thousand drips, view from road. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

Measurements in an axisymmetric turbulent wake with rotation downstream of a model wind turbine

NASA Astrophysics Data System (ADS)

Dufresne, Nathaniel; Wosnik, Martin

2012-11-01

Energy production data from several of the existing offshore wind farms indicate that turbine arrays may enter a stall condition which can cause an overall energy production shortfall (which can exceed 10%). This deep array stall is (presumably) due to the wakes generated by turbines upstream interacting with turbine rotors downstream. It is hypothesized that there is a critical array spacing at which this stall occurs, but that this spacing is dependent on rotor thrust cT (which is determined by tip-speed ratio λ and power coefficient cP of the rotor), Reynolds number, upstream conditions, and possibly wall roughness. An experimental investigation of the axial and azimuthal velocity field measurements in the wake of a single 3-bladed wind turbine with rotor diameter of 0.91m was conducted. The turbine was positioned in the free stream, near the entrance of the 6m × 2.5m test section of the UNH FPF, which can achieve test section velocities of up to 15 m/s and Reynolds numbers δ+ = δuτ / ν ~ 30 , 000 . Hot-wire anemometry was used to obtain velocity field measurements. The data obtained will be used to examine similarity scaling functions for velocity, wake growth, and turbulence derived from an equilibrium similarity analysis of the far wake.

Active flow control of the laminar separation bubble on a plunging airfoil near stall

NASA Astrophysics Data System (ADS)

Pande, Arth; Agate, Mark; Little, Jesse; Fasel, Hermann

2017-11-01

The effects of small amplitude (A/c = 0.048) high frequency (πfc/U∞ = 0.70) plunging motion on the X-56A airfoil are examined experimentally at Re = 200,000 for 12° angle of attack (CL,MAX = 12.25°) . The purpose of this research is to study the aerodynamic influence of structural motion when the wing is vibrating close to its eigenfrequency near static stall. Specific focus is placed on the laminar separation bubble (LSB) near the leading edge and its control via plasma actuation. In the baseline case, the leading edge bubble bursts during the oscillation cycle causing moment stall. A collaborative computational effort has shown that small amplitude forcing at a frequency that is most amplified by the primary instability of the LSB (FLSB+= 1, Fc+= 52) generates coherent spanwise vortices that entrain freestream momentum, thus reducing separation all while maintaining a laminar flow state. Results (PIV and surface pressure) indicate that a similar control mechanism is effective in the experiments. This is significant given the existence of freestream turbulence in the wind tunnel which has been shown to limit the efficacy of this active flow control technique in a model problem using Direct Numerical Simulation. The implications of these results are discussed.

Protistan Predation Affects Trichloroethene Biodegradation in a Bedrock Aquifer▿

PubMed Central

Cunningham, Joseph J.; Kinner, Nancy E.; Lewis, Maureen

2009-01-01

Despite extensive research on the bottom-up force of resource availability (e.g., electron donors and acceptors), slow biodegradation rates and stalling at cis-dichloroethene (cDCE) and vinyl chloride continue to be observed in aquifers contaminated with trichloroethene (TCE). The objective of this research was to gauge the impact of the top-down force of protistan predation on TCE biodegradation in laboratory microcosms. When indigenous bacteria from an electron donor-limited TCE-contaminated bedrock aquifer were present, the indigenous protists inhibited reductive dechlorination altogether. The presence of protists during organic carbon-amended conditions caused the bacteria to elongate (length:width, ≥10:1), but reductive dechlorination was still inhibited. When a commercially available dechlorinating bacterial culture and an organic carbon amendment were added in he presence of protists, the elongated bacteria predominated and reductive dechlorination stalled at cDCE. When protists were removed under organic carbon-amended conditions, reductive dechlorination stalled at cDCE, whereas in the presence organic carbon and bacterial amendments, the total chlorinated ethene concentration decreased, indicating TCE was converted to ethene and/or CO2. The data suggested that indigenous protists grazed dechlorinators to extremely low levels, inhibiting dechlorination altogether. Hence, in situ bioremediation/bioaugmentation may not be successful in mineralizing TCE unless the top-down force of protistan predation is inhibited. PMID:19820148

Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress

PubMed Central

Basile, Giorgia; Leuzzi, Giuseppe; Pichierri, Pietro; Franchitto, Annapaola

2014-01-01

Werner syndrome (WS) is a human chromosomal instability disorder associated with cancer predisposition and caused by mutations in the WRN gene. WRN helicase activity is crucial in limiting breakage at common fragile sites (CFS), which are the preferential targets of genome instability in precancerous lesions. However, the precise function of WRN in response to mild replication stress, like that commonly used to induce breaks at CFS, is still missing. Here, we establish that WRN plays a role in mediating CHK1 activation under moderate replication stress. We provide evidence that phosphorylation of CHK1 relies on the ATR-mediated phosphorylation of WRN, but not on WRN helicase activity. Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress. Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels. Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility. PMID:25352544

Dairy producer attitudes to pain in cattle in relation to disbudding calves.

PubMed

Wikman, I; Hokkanen, A-H; Pastell, M; Kauppinen, T; Valros, A; Hänninen, L

2013-01-01

Pain is an important indicator of poor welfare of livestock. Despite this, pain has largely gone unrecognized in farm animals due to attitudes of producers and veterinarians, although they play a key role in monitoring and managing the perception of animal pain. Producer attitudes toward animal welfare influence livestock management and production. The aim was to quantify dairy producer attitudes to the painfulness of various cattle diseases and disbudding, a painful routine procedure performed on farm to ensure safer handling of cattle. A questionnaire on disbudding-related opinions and practices was sent to 1,000 Finnish dairy producers (response rate: 45%). Attitudes toward disbudding were gauged using a 5-point Likert scale and attitudes to cattle pain scored on an 11-point numerical rating scale. Principal components analysis was used to assess the loadings, which were further tested for differences between producer gender and housing systems with Mann-Whitney U-tests, and between herd milk yield, herd size, and age and work experience of producers with a Kruskal-Wallis test. Four main factors were identified: factor I ("taking disbudding pain seriously"), factor II ("sensitivity to pain caused by cattle diseases"), factor III ("ready to medicate calves myself"), and factor IV ("pro horns"). Female producers took disbudding pain more seriously, were more sensitive to pain caused to cattle by diseases, and were more ready to medicate disbudded calves than male producers. Producers with tie-stalls favored horns over producers with freestalls. Male producers with tie-stalls were sensitive to cattle pain and preferred horns over male producers with freestalls. Female producers with freestalls were more ready to medicate calves, but did not prefer horns more than female producers with tie-stalls. Taking disbudding seriously correlated with sensitivity to pain caused by cattle diseases. Producers with low-milk-yielding herds were less willing to medicate calves and more willing to keep cattle with horns than producers with higher-yielding herds. Older producers were more sensitive to cattle pain than middle-aged and younger producers. No effect was established for taking disbudding pain seriously: the pro-horn factor was associated with work experience, age, and herd size. Women rated pain higher and were more positive toward pain medication for animals than men. Maintaining horns are more important for producers with tie-stalls than for those with freestalls. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Pre-Stall Behavior of a Transonic Axial Compressor Stage via Time-Accurate Numerical Simulation

NASA Technical Reports Server (NTRS)

Chen, Jen-Ping; Hathaway, Michael D.; Herrick, Gregory P.

2008-01-01

CFD calculations using high-performance parallel computing were conducted to simulate the pre-stall flow of a transonic compressor stage, NASA compressor Stage 35. The simulations were run with a full-annulus grid that models the 3D, viscous, unsteady blade row interaction without the need for an artificial inlet distortion to induce stall. The simulation demonstrates the development of the rotating stall from the growth of instabilities. Pressure-rise performance and pressure traces are compared with published experimental data before the study of flow evolution prior to the rotating stall. Spatial FFT analysis of the flow indicates a rotating long-length disturbance of one rotor circumference, which is followed by a spike-type breakdown. The analysis also links the long-length wave disturbance with the initiation of the spike inception. The spike instabilities occur when the trajectory of the tip clearance flow becomes perpendicular to the axial direction. When approaching stall, the passage shock changes from a single oblique shock to a dual-shock, which distorts the perpendicular trajectory of the tip clearance vortex but shows no evidence of flow separation that may contribute to stall.

Dynamic stall characterization using modal analysis of phase-averaged pressure distributions

NASA Astrophysics Data System (ADS)

Harms, Tanner; Nikoueeyan, Pourya; Naughton, Jonathan

2017-11-01

Dynamic stall characterization by means of surface pressure measurements can simplify the time and cost associated with experimental investigation of unsteady airfoil aerodynamics. A unique test capability has been developed at University of Wyoming over the past few years that allows for time and cost efficient measurement of dynamic stall. A variety of rotorcraft and wind turbine airfoils have been tested under a variety of pitch oscillation conditions resulting in a range of dynamic stall behavior. Formation, development and separation of different flow structures are responsible for the complex aerodynamic loading behavior experienced during dynamic stall. These structures have unique signatures on the pressure distribution over the airfoil. This work investigates the statistical behavior of phase-averaged pressure distribution for different types of dynamic stall by means of modal analysis. The use of different modes to identify specific flow structures is being investigated. The use of these modes for different types of dynamic stall can provide a new approach for understanding and categorizing these flows. This work uses airfoil data acquired under Army contract W911W60160C-0021, DOE Grant DE-SC0001261, and a gift from BP Alternative Energy North America, Inc.

Evaluation of the Life History of Native Salmonids in the Malheur River Basin; Cooperative Bull Trout/Redband Trout Research Project, 1999-2000 Annual Report.

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

Schwabe, Lawrence; Tiley, Mark; Perkins, Raymond R.

2000-11-01

The purpose of this study is to document the seasonal distribution of adult/sub-adult bull trout (Salvelinus confluentus) in the Malheur River basin. Due to the decline of bull trout in the Columbia Basin, the United States Fish and Wildlife Service listed bull trout as a threatened species in June 1998. Past land management activities; construction of dams; and fish eradication projects in the North Fork and Middle Fork Malheur River by poisoning have worked in concert to cumulatively impact native species in the Malheur Basin (Bowers et. al. 1993). Survival of the remaining bull trout populations is severely threatened (Buchananmore » 1997). 1999 Research Objects are: (1) Document the migratory patterns of adult/sub-adult bull trout in the North Fork Malheur River; (2) Determine the seasonal bull trout use of Beulah Reservoir and bull trout entrainment; and (3) Timing and location of bull trout spawning in the North Fork Malheur River basin. The study area includes the Malheur basin from the mouth of the Malheur River located near Ontario, Oregon to the headwaters of the North Fork Malheur River (Map 1). All fish collected and most of the telemetry effort was done on the North Fork Malheur River subbasin (Map 2). Fish collection was conducted on the North Fork Malheur River at the tailwaters of Beulah Reservoir (RK 29), Beulah Reservoir (RK 29-RK 33), and in the North Fork Malheur River at Crane Crossing (RK 69) to the headwaters of the North Fork Malheur. Radio telemetry was done from the mouth of the Malheur River in Ontario, Oregon to the headwaters of the North Fork Malheur. This report will reflect all migration data collected from 3/1/99 to 12/31/99.« less

An approach for aerodynamic optimization of transonic fan blades

NASA Astrophysics Data System (ADS)

Khelghatibana, Maryam

Aerodynamic design optimization of transonic fan blades is a highly challenging problem due to the complexity of flow field inside the fan, the conflicting design requirements and the high-dimensional design space. In order to address all these challenges, an aerodynamic design optimization method is developed in this study. This method automates the design process by integrating a geometrical parameterization method, a CFD solver and numerical optimization methods that can be applied to both single and multi-point optimization design problems. A multi-level blade parameterization is employed to modify the blade geometry. Numerical analyses are performed by solving 3D RANS equations combined with SST turbulence model. Genetic algorithms and hybrid optimization methods are applied to solve the optimization problem. In order to verify the effectiveness and feasibility of the optimization method, a singlepoint optimization problem aiming to maximize design efficiency is formulated and applied to redesign a test case. However, transonic fan blade design is inherently a multi-faceted problem that deals with several objectives such as efficiency, stall margin, and choke margin. The proposed multi-point optimization method in the current study is formulated as a bi-objective problem to maximize design and near-stall efficiencies while maintaining the required design pressure ratio. Enhancing these objectives significantly deteriorate the choke margin, specifically at high rotational speeds. Therefore, another constraint is embedded in the optimization problem in order to prevent the reduction of choke margin at high speeds. Since capturing stall inception is numerically very expensive, stall margin has not been considered as an objective in the problem statement. However, improving near-stall efficiency results in a better performance at stall condition, which could enhance the stall margin. An investigation is therefore performed on the Pareto-optimal solutions to demonstrate the relation between near-stall efficiency and stall margin. The proposed method is applied to redesign NASA rotor 67 for single and multiple operating conditions. The single-point design optimization showed +0.28 points improvement of isentropic efficiency at design point, while the design pressure ratio and mass flow are, respectively, within 0.12% and 0.11% of the reference blade. Two cases of multi-point optimization are performed: First, the proposed multi-point optimization problem is relaxed by removing the choke margin constraint in order to demonstrate the relation between near-stall efficiency and stall margin. An investigation on the Pareto-optimal solutions of this optimization shows that the stall margin has been increased with improving near-stall efficiency. The second multi-point optimization case is performed with considering all the objectives and constraints. One selected optimized design on the Pareto front presents +0.41, +0.56 and +0.9 points improvement in near-peak efficiency, near-stall efficiency and stall margin, respectively. The design pressure ratio and mass flow are, respectively, within 0.3% and 0.26% of the reference blade. Moreover the optimized design maintains the required choking margin. Detailed aerodynamic analyses are performed to investigate the effect of shape optimization on shock occurrence, secondary flows, tip leakage and shock/tip-leakage interactions in both single and multi-point optimizations.

Force regulated dynamics of RPA on a DNA fork

PubMed Central

Kemmerich, Felix E.; Daldrop, Peter; Pinto, Cosimo; Levikova, Maryna; Cejka, Petr; Seidel, Ralf

2016-01-01

Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg2+ concentrations, such that human RPA can melt DNA in absence of force. PMID:27016742

Demographics and density estimates of two three-toed box turtle (Terrapene carolina triunguis) populations within forest and restored prairie sites in central Missouri.

PubMed

O'Connor, Kelly M; Rittenhouse, Chadwick D; Millspaugh, Joshua J; Rittenhouse, Tracy A G

2015-01-01

Box turtles (Terrapene carolina) are widely distributed but vulnerable to population decline across their range. Using distance sampling, morphometric data, and an index of carapace damage, we surveyed three-toed box turtles (Terrapene carolina triunguis) at 2 sites in central Missouri, and compared differences in detection probabilities when transects were walked by one or two observers. Our estimated turtle densities within forested cover was less at the Thomas S. Baskett Wildlife Research and Education Center, a site dominated by eastern hardwood forest (d = 1.85 turtles/ha, 95% CI [1.13, 3.03]) than at the Prairie Fork Conservation Area, a site containing a mix of open field and hardwood forest (d = 4.14 turtles/ha, 95% CI [1.99, 8.62]). Turtles at Baskett were significantly older and larger than turtles at Prairie Fork. Damage to the carapace did not differ significantly between the 2 populations despite the more prevalent habitat management including mowing and prescribed fire at Prairie Fork. We achieved improved estimates of density using two rather than one observer at Prairie Fork, but negligible differences in density estimates between the two methods at Baskett. Error associated with probability of detection decreased at both sites with the addition of a second observer. We provide demographic data on three-toed box turtles that suggest the use of a range of habitat conditions by three-toed box turtles. This case study suggests that habitat management practices and their impacts on habitat composition may be a cause of the differences observed in our focal populations of turtles.

Geomorphic Change Induced by 100 years of Flow Alteration on the Diamond Fork River, Central Utah

NASA Astrophysics Data System (ADS)

Jones, J.; Belmont, P.; Wilcock, P. R.

2017-12-01

Changes in hydrology and sediment supply affect the form of rivers. The rate of change of fluvial form is controlled by a variety of factors, including valley confinement, sediment size, and antecedent condition. The Diamond Fork River in central Utah has been altered by trans-basin flows delivered from the Colorado River system for over a century. Beginning in 1915, water used for irrigation was delivered through a tributary, Sixth Water Creek, with daily summer flows regularly exceeding the 50 - 100 year flood. Elevated flows caused drastic geomorphic change - resulting in incision and widening of the channel, and the destruction of riparian vegetation. Beginning in 1997, the outlet for the trans-basin diversion was moved downstream on Sixth Water, bypassing a large landslide, and flows were drastically reduced in 2004 through management actions. We delineated eight distinct process domains for the Sixth Water-Diamond Fork system and examined the response of each process domain to the altered flow and sediment regimes through the analysis of aerial photographs and repeat cross-sections. We measured a variety of channel metrics, including channel width, areal extent of bars and islands, and sinuosity in ArcGIS. Results indicate that unconfined reaches that were wide and braided during the period of elevated flows have narrowed to become single threaded and meandering in response to the reduced flows. Confined reaches have experienced minor changes since the reduction in flows, suggesting that confinement is a primary control on the degree of channel response. These findings and complimentary studies will provide managers of Sixth Water and Diamond Fork with a greater understanding of the physical response of the streams, and the resulting effects on ecological communities.

South Fork Telephone Switchboard Building, oblique view of east side; ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Telephone Switchboard Building, oblique view of east side; view northwest - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

South Fork Telephone Switchboard Building, interior west room showing hardwood ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Telephone Switchboard Building, interior west room showing hardwood floor; view south - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

South Fork Latrine, interior showing head with steel tank mounted ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Latrine, interior showing head with steel tank mounted to wall; view south - Fort McKinley, South Fork Latrine, West side of East Side Drive, approximately 225 feet south of Weymouth Way, Great Diamond Island, Portland, Cumberland County, ME

14. Roaring Fork Motor Nature Trail, Place of a thousand ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

14. Roaring Fork Motor Nature Trail, Place of a thousand drips, view with three culvert pipes. - Great Smoky Mountains National Park Roads & Bridges, Roaring Fork Motor Nature Trail, Between Cherokee Orchard Road & U.S. Route 321, Gatlinburg, Sevier County, TN

Caffeine toxicity is inversely related to DNA repair in simian virus 40-transformed xeroderma pigmentosum cells irradiated with ultraviolet light

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

Cleaver, J.E.

1989-01-01

Human cells transformed by simian virus 40 (SV40) are more sensitive to killing by ultraviolet light when grown in caffeine after irradiation. The degree of sensitization at 2 mM caffeine (expressed as the ratio of the 37% survival dose for control cells divided by the 37% survival dose for cells grown in caffeine, i.e., the dose modification factor) was approximately 1.9 in transformed normal cells and 3.8-5.8 in excision-defective xeroderma pigmentosum (XP) groups A, C, and D cells. A large dose modification factor of 12 was observed in a transformed XP variant cell line. Chinese hamster ovary cells were notmore » significantly different from transformed normal human cells, with a maximum dose modification factor of 1.5. Two radioresistant XP revertants that do not excise cyclobutane dimers gave different responses; one resembled its group A parent in being sensitized by caffeine, and one did not. These results can be interpreted on the basis of a single hypothesis that cells are killed as a result of attempts to replicate damaged DNA. Increased replication rates caused by transformation, increased numbers of replication forks in DNA caused by caffeine, and increased numbers of damaged sites ahead of replication forks in excision-defective cells are all processes that will consequently increase killing according to this hypothesis. A corollary is that the XP variant may be highly sensitized to caffeine because of excision defects at the DNA replication forks, an idea that may be important in designing cloning strategies for the XP variant gene.« less

Preliminary assessment of aggradation potential in the North Fork Stillaguamish River downstream of the State Route 530 landslide near Oso, Washington

USGS Publications Warehouse

Magirl, Christopher S.; Keith, Mackenzie K.; Anderson, Scott W.; O'Connor, Jim; Robert Aldrich,; Mastin, Mark C.

2015-12-28

On March 22, 2014, the State Route 530 Landslide near Oso, Washington, traveled almost 2 kilometers (km), destroyed more than 40 structures, and impounded the North Fork Stillaguamish River to a depth of 8 meters (m) and volume of 3.3×106 cubic meters (m3). The landslide killed 43 people. After overtopping and establishing a new channel through the landslide, the river incised into the landslide deposit over the course of 10 weeks draining the impoundment lake and mobilizing an estimated 280,000±56,000 m3 of predominantly sand-sized and finer sediment. During the first 4 weeks after the landslide, this eroded sediment caused downstream riverbed aggradation of 1–2 m within 1 km of the landslide and 0.4 m aggradation at Whitman Road Bridge, 3.5 km downstream. Winter high flows in 2014–15 were anticipated to mobilize an additional 220,000±44,000 m3 of sediment, potentially causing additional aggradation and exacerbating flood risk downstream of the landslide. Analysis of unit stream power and bed-material transport capacity along 35 km of the river corridor indicated that most fine-grained sediment will transport out of the North Fork Stillaguamish River, although some localized additional aggradation was possible. This new aggradation was not likely to exceed 0.1 m except in reaches within a few kilometers downstream of the landslide, where additional aggradation of up to 0.5 m is possible. Alternative river response scenarios, including continued mass wasting from the landslide scarp, major channel migration or avulsion, or the formation of large downstream wood jams, although unlikely, could result in reaches of significant local aggradation or channel change.

Cow comfort in tie-stalls: increased depth of shavings or straw bedding increases lying time.

PubMed

Tucker, C B; Weary, D M; von Keyserlingk, M A G; Beauchemin, K A

2009-06-01

Over half of US dairy operations use tie-stalls, but these farming systems have received relatively little research attention in terms of stall design and management. The current study tested the effects of the amount of 2 bedding materials, straw and shavings, on dairy cattle lying behavior. The effects of 4 levels of shavings, 3, 9, 15, and 24 kg/stall (experiment 1, n = 12), and high and low levels of straw in 2 separate experiments: 1, 3, 5, and 7 kg/stall (experiment 2, n = 12) and 0.5, 1, 2, and 3 kg/stall (experiment 3, n = 12) were assessed. Treatments were compared using a crossover design with lactating cows housed in tie-stalls fitted with mattresses. Treatments were applied for 1 wk. Total lying time, number of lying bouts, and the length of each lying bout was recorded with data loggers. In experiment 1, cows spent 3 min more lying down for each additional kilogram of shavings (11.0, 11.7, 11.6, and 12.1 +/- 0.24 h/d for 3, 9, 15, and 24 kg/stall shavings, respectively). In experiment 2, cows increased lying time by 12 min for every additional kilogram of straw (11.2, 12.0, 11.8, and 12.4 +/- 0.24 h/d for 1, 3, 5, and 7 kg/stall of straw, respectively). There were no differences in lying behavior among the lower levels of straw tested in experiment 3 (11.7 +/- 0.32 h/d). These results indicated that additional bedding above a scant amount improves cow comfort, as measured by lying time, likely because a well-bedded surface is more compressible.

Stalling Tropical Cyclones over the Atlantic Basin

NASA Astrophysics Data System (ADS)

Nielsen-Gammon, J. W.; Emanuel, K.

2017-12-01

Hurricane Harvey produced massive amounts of rain over southeast Texas and southwest Louisiana. Average storm total rainfall amounts over a 10,000 square mile (26,000 square km) area exceeded 30 inches (750 mm). An important aspect of the storm that contributed to the large rainfall totals was its unusual motion. The storm stalled shortly after making landfall, then moved back offshore before once again making landfall five days later. This storm motion permitted heavy rainfall to occur in the same general area for an extended period of time. The unusual nature of this event motivates an investigation into the characteristics and potential climate change influences on stalled tropical cyclones in the Atlantic basin using the HURDAT 2 storm track database for 1866-2016 and downscaled tropical cyclones driven by simulations of present and future climate. The motion of cyclones is quantified as the size of a circle circumscribing all storm locations during a given length of time. For a three-day period, Harvey remained inside a circle with a radius of 123 km. This ranks within the top 0.6% of slowest-moving historical storm instances. Among the 2% of slowest-moving storm instances prior to Harvey, only 13 involved storms that stalled near the continental United States coast, where they may have produced substantial rainfall onshore while tapping into marine moisture. Only two such storms stalled in the month of September, in contrast to 20 September stalls out of the 36 storms that stalled over the nearby open Atlantic. Just four of the stalled coastal storms were hurricanes, implying a return frequency for such storms of much less than once per decade. The synoptic setting of these storms is examined for common features, and historical and projected trends in occurrences of stalled storms near the coast and farther offshore are investigated.

A theory of post-stall transients in axial compression systems. I - Development of equations

NASA Technical Reports Server (NTRS)

Moore, F. K.; Greitzer, E. M.

1985-01-01

An approximate theory is presented for post-stall transients in multistage axial compression systems. The theory leads to a set of three simultaneous nonlinear third-order partial differential equations for pressure rise, and average and disturbed values of flow coefficient, as functions of time and angle around the compressor. By a Galerkin procedure, angular dependence is averaged, and the equations become first order in time. These final equations are capable of describing the growth and possible decay of a rotating-stall cell during a compressor mass-flow transient. It is shown how rotating-stall-like and surgelike motions are coupled through these equations, and also how the instantaneous compressor pumping characteristic changes during the transient stall process.

14 CFR 23.691 - Artificial stall barrier system.

Code of Federal Regulations, 2012 CFR

2012-01-01

... AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Design and Construction Control Systems § 23.691 Artificial stall barrier system. If the function of an artificial stall... downward pitching control will be provided must be established. (b) Considering the plus and minus airspeed...

14 CFR 23.691 - Artificial stall barrier system.

Code of Federal Regulations, 2011 CFR

2011-01-01

... AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Design and Construction Control Systems § 23.691 Artificial stall barrier system. If the function of an artificial stall... downward pitching control will be provided must be established. (b) Considering the plus and minus airspeed...

14 CFR 23.691 - Artificial stall barrier system.

Code of Federal Regulations, 2013 CFR

2013-01-01

... AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Design and Construction Control Systems § 23.691 Artificial stall barrier system. If the function of an artificial stall... downward pitching control will be provided must be established. (b) Considering the plus and minus airspeed...

14 CFR 23.691 - Artificial stall barrier system.

Code of Federal Regulations, 2010 CFR

2010-01-01

... AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Design and Construction Control Systems § 23.691 Artificial stall barrier system. If the function of an artificial stall... downward pitching control will be provided must be established. (b) Considering the plus and minus airspeed...

14 CFR 23.691 - Artificial stall barrier system.

Code of Federal Regulations, 2014 CFR

2014-01-01

... AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Design and Construction Control Systems § 23.691 Artificial stall barrier system. If the function of an artificial stall... downward pitching control will be provided must be established. (b) Considering the plus and minus airspeed...

South Fork Telephone Switchboard Building, general view in setting showing ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Telephone Switchboard Building, general view in setting showing (N) side; view (S) - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

South Fork Telephone Switchboard Building, oblique view of (W) and ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

South Fork Telephone Switchboard Building, oblique view of (W) and (S) sides, view to northeast - Fort McKinley, South Fork Telephone Switchboard Building, South side of Weymouth Way, approximately 100 feet west of East Side Drive, Great Diamond Island, Portland, Cumberland County, ME

STEEL ERECTION. View of downstream of bridge, looking southeast from ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

STEEL ERECTION. View of downstream of bridge, looking southeast from confluence of Trinity and South Fork Trinity rivers. The old suspension bridge is in background - South Fork Trinity River Bridge, State Highway 299 spanning South Fork Trinity River, Salyer, Trinity County, CA

Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe.

PubMed

Margalef, Pol; Kotsantis, Panagiotis; Borel, Valerie; Bellelli, Roberto; Panier, Stephanie; Boulton, Simon J

2018-01-25

Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1 -/- cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Base flow (1966-2009) and streamflow gain and loss (2010) of the Brazos River from the New Mexico-Texas State line to Waco, Texas

USGS Publications Warehouse

Baldys, Stanley; Schalla, Frank E.

2012-01-01

Streamflow was measured at 66 sites from June 6–9, 2010, and at 68 sites from October 16–19, 2010, to identify reaches in the upper Brazos River Basin that were gaining or losing streamflow. Gaining reaches were identified in each of the five subbasins. The gaining reach in the Salt Fork Brazos River Basin began at USGS streamflow-gaging station 08080940 Salt Fork Brazos River at State Highway 208 near Clairemont, Tex. (site SF–6), upstream from where Duck Creek flows into the Salt Fork Brazos River and continued downstream past USGS streamflow-gaging station 08082000 Salt Fork Brazos River near Aspermont, Tex. (site SF–9), to the outlet of the basin. In the Double Mountain Fork Brazos River Basin, a gaining reach from near Post, Tex., downstream to the outlet of the basin was identified. Two gaining reaches were identified in the Clear Fork Brazos River Basin—one from near Roby, Tex., downstream to near Noodle, Tex., and second from Hawley, Tex., downstream to Nugent, Tex. Most of the North Bosque River was characterized as gaining streamflow. Streamflow gains were identified in the main stem of the Brazos River from where the Brazos River main stem forms at the confluence of the Salt Fork Brazos River and Double Mountain Fork Brazos River near Knox City, Tex., downstream to near Seymour, Tex.

The effectiveness of front fork systems at damping accelerations during isolated aspects specific to cross-country mountain biking.

PubMed

Macdermid, Paul W; Miller, Matthew C; Fink, Philip W; Stannard, Stephen R

2017-11-01

Cross-country mountain bike suspension reportedly enhances comfort and performance through reduced vibration and impact exposure. This study analysed the effectiveness of three different front fork systems at damping accelerations during the crossing of three isolated obstacles (stairs, drop, and root). One participant completed three trials on six separate occasions in a randomised order using rigid, air-sprung, and carbon leaf-sprung forks. Performance was determined by time to cross obstacles, while triaxial accelerometers quantified impact exposure and damping response. Results identified significant main effect of fork type for performance time (p < 0.05). The air-sprung and leaf-sprung forks were significantly slower than the rigid forks for the stairs (p < 0.05), while air-sprung suspension was slower than the rigid for the root protocol (p < 0.05). There were no differences for the drop protocol (p < 0.05). Rigid forks reduced overall exposure (p < 0.05), specifically at the handlebars for the stairs and drop trials. More detailed analysis presented smaller vertical accelerations at the handlebar for air-sprung and leaf-sprung forks on the stairs (p < 0.05), and drop (p < 0.05) but not the root. As such, it appears that the suspension systems tested were ineffective at reducing overall impact exposure at the handlebar during isolated aspects of cross-country terrain features which may be influenced to a larger extent by rider technique.

High Fidelity Aeroelasticity Simulations of Aircraft and Turbomachinery with Fully-Coupled Fluid-Structure Interaction

NASA Astrophysics Data System (ADS)

Gan, Jiaye

The purpose of this research is to develop high fidelity numerical methods to investigate the complex aeroelasticity fluid-structural problems of aircraft and aircraft engine turbomachinery. Unsteady 3D compressible Navier-Stokes equations in generalized coordinates are solved to simulate the complex fluid dynamic problems in aeroelasticity. An efficient and low diffusion E-CUSP (LDE) scheme designed to minimize numerical dissipation is used as a Riemann solver to capture shock waves in transonic and supersonic flows. An improved hybrid turbulence modeling, delayed detached eddy simulation (DDES), is implemented to simulate shock induced separation and rotating stall flows. High order accuracy (3rd and 5th order) weighted essentially non-oscillatory (WENO) schemes for inviscid flux and a conservative 2nd and 4th order viscous flux differencing are employed. To resolve the nonlinear interaction between flow and vibrating blade structures, a fully coupled fluid-structure interaction (FSI) procedure that solves the structural modal equations and time accurate Navier-Stokes equations simultaneously is adopted. A rotor/stator sliding interpolation technique is developed to accurately capture the blade rows interaction at the interface with general grid distribution. Phase lag boundary conditions (BC) based on the time shift (direct store) method and the Fourier series phase lag BC are applied to consider the effect of phase difference for a sector of annulus simulation. Extensive validations are conducted to demonstrate high accuracy and robustness of the high fidelity FSI methodology. The accuracy and robustness of RANS, URANS and DDES turbulence models with high order schemes for predicting the lift and drag of the DLR-F6 configuration are verified. The DDES predicts the drag very well whereas the URANS model significantly over predicts the drag. DDES of a finned projectile base flows is conducted to further validate the high fidelity methods with vortical flow. The DDES is demonstrated to be superior to the URANS for the projectile flow prediction. DDES of a 3D transonic wing flutter is validated with AGARD Wing 445.6 aeroelasticity experiment at free stream Mach number varied from subsonic to supersonic. The predicted flutter boundary at different free stream Mach number including the sonic dip achieves very good agreement with the experiment. In particular, the predicted flutter boundaries at the supersonic conditions match the experiment accurately. The mechanism of sonic dip is investigated. Simulation of supersonic fluid-structural interaction of a flat panel is performed by using DDES with high order shock capturing scheme. The panel vibration induced by the shock boundary layer interaction is well resolved by the high fidelity method. The dominant panel response agrees well with the experiment in terms of the mean panel displacement and frequency. The DDES methodology is used to investigate the stall inception of NASA Stage 35 compressor. The process of rotating stall is compared between the results using both URANS and DDES with full annulus. The stall process begins with spike inception and develops to full stall. The numbers of stall cell, and the size and propagating speed of the stall cells are well captured by both URANS and DDES. Two stall cells with 42% rotor rotating speed are resolved by DDES and one stall cell with 90% rotor rotating speed by URANS. It is not conclusive which method is more accurate since there is no experimental data, but the DDES does show more realistic vortical turbulence with more small scale structures. The non-synchronous vibration (NSV) of a high speed 1-1/2 stage axial compressor is investigated by using rigid blade and vibrating blade with fluid-structural interaction. An interpolation sliding boundary condition is used for the rotor-stator interaction. The URANS simulation with rigid blades shows that the leading edge(LE) circumferentially traveling vortices, roughly above 80% rotor span, travel backwards relative to the rotor rotation and cause an excitation with the frequency agreeing with the measured NSV frequency. The predicted excitation frequency of the traveling vortices in the rigid blade simulation is a non-engine order frequency of 2603 Hz, which agrees very well with the rig measured frequency of 2600 Hz. For the FSI simulation, the results show that there exist two dominant frequencies in the spectrum of the blade vibration. The lower dominant frequency is close to the first bending mode. The higher dominant frequency close to the first torsional mode agrees very well with the measured NSV frequency. To investigate whether the NSV is caused by flow excitation or by flow-structure locked-in phenomenon, the rotating speed is varied within a small RPM range, in which the rig test detected the NSV. The unsteady flows with rigid blades are simulated first at several RPMs. A dominant excitation NSV frequency caused by the circumferentially traveling tip vortices are captured. The simulation then switches to fluid structure interaction that allows the blades to vibrate freely. (Abstract shortened by ProQuest.).

Academic Learning Time in the District of Columbia Public Schools.

ERIC Educational Resources Information Center

District of Columbia Public Schools, Washington, DC. Research Information Center.

Papers generated for a symposium entitled "Effectiveness of Stallings' Use of Time Training for Teachers in Washington, D.C." are presented. The intitial presentation, "Academic Learning Time: The Current Status of the Stallings Training" (Geraldine Williams Bethune), reviews the Stallings research and describes the Academic…

14 CFR 23.49 - Stalling period.

Code of Federal Regulations, 2011 CFR

2011-01-01

... on the stalling speed, with engine(s) idling and throttle(s) closed; (3) The propeller(s) in the... which the airplane is controllable with— (1) For reciprocating engine-powered airplanes, the engine(s... more than 110 percent of the stalling speed; (2) For turbine engine-powered airplanes, the propulsive...

An annotated bibliography of the hydrology and fishery studies of the South Fork Salmon River

Treesearch

Kathleen A. Seyedbagheri; Michael L. McHenry; William S. Platts

1987-01-01

A brief summary of the land management history of the South Fork Salmon River (Idaho) watershed includes citations and annotations of published and unpublished reports of fishery and hydrology studies conducted in the South Fork drainage for 1960 to 1986.

OLD SUSPENSION BRIDGE. This view appears to be looking southeast, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

OLD SUSPENSION BRIDGE. This view appears to be looking southeast, at the downstream side of the suspension bridge, which was replaced by the South Fork Trinity River Bridge - South Fork Trinity River Bridge, State Highway 299 spanning South Fork Trinity River, Salyer, Trinity County, CA

Impact. The southwest end is down, as the buckled middle ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Impact. The southwest end is down, as the buckled middle section splashes into the river. View is to southeast from confluence of Trinity and South Fork Trinity Rivers - South Fork Trinity River Bridge, State Highway 299 spanning South Fork Trinity River, Salyer, Trinity County, CA

76 FR 17022 - Special Conditions: Gulfstream Model GVI Airplane; High Incidence Protection

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-28

... Directorate, Aircraft Certification Service, 1601 Lind Avenue, SW., Renton, Washington 98057-3356; telephone..., limits the angle of attack at which the airplane can be flown during normal low speed operation, and... the stall speed determination, the stall characteristics, the stall warning demonstration, and the...