Mouse embryonic stem cells have increased capacity for replication fork restart driven by the specific Filia-Floped protein complex.

PubMed

Zhao, Bo; Zhang, Weidao; Cun, Yixian; Li, Jingzheng; Liu, Yan; Gao, Jing; Zhu, Hongwen; Zhou, Hu; Zhang, Rugang; Zheng, Ping

2018-01-01

Pluripotent stem cells (PSCs) harbor constitutive DNA replication stress during their rapid proliferation and the consequent genome instability hampers their applications in regenerative medicine. It is therefore important to understand the regulatory mechanisms of replication stress response in PSCs. Here, we report that mouse embryonic stem cells (ESCs) are superior to differentiated cells in resolving replication stress. Specifically, ESCs utilize a unique Filia-Floped protein complex-dependent mechanism to efficiently promote the restart of stalled replication forks, therefore maintaining genomic stability. The ESC-specific Filia-Floped complex resides on replication forks under normal conditions. Replication stress stimulates their recruitment to stalling forks and the serine 151 residue of Filia is phosphorylated in an ATR-dependent manner. This modification enables the Filia-Floped complex to act as a functional scaffold, which then promotes the stalling fork restart through a dual mechanism: both enhancing recruitment of the replication fork restart protein, Blm, and stimulating ATR kinase activation. In the Blm pathway, the scaffolds recruit the E3 ubiquitin ligase, Trim25, to the stalled replication forks, and in turn Trim25 tethers and concentrates Blm at stalled replication forks through ubiquitination. In differentiated cells, the recruitment of the Trim25-Blm complex to replication forks and the activation of ATR signaling are much less robust due to lack of the ESC-specific Filia-Floped scaffold. Thus, our study reveals that ESCs utilize an additional and unique regulatory layer to efficiently promote the stalled fork restart and maintain genomic stability.

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

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks*

PubMed Central

Kim, Hyun-Suk; Nickoloff, Jac A.; Wu, Yuehan; Williamson, Elizabeth A.; Sidhu, Gurjit Singh; Reinert, Brian L.; Jaiswal, Aruna S.; Srinivasan, Gayathri; Patel, Bhavita; Kong, Kimi; Burma, Sandeep; Lee, Suk-Hee; Hromas, Robert A.

2017-01-01

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5′ end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5′-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5′ end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks. PMID:28049724

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

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

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.

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.

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.

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.

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

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

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

Methods for sampling geographically mobile female traders in an East African market setting

PubMed Central

Achiro, Lillian; Kwena, Zachary A.; McFarland, Willi; Neilands, Torsten B.; Cohen, Craig R.; Bukusi, Elizabeth A.; Camlin, Carol S.

2018-01-01

Background The role of migration in the spread of HIV in sub-Saharan Africa is well-documented. Yet migration and HIV research have often focused on HIV risks to male migrants and their partners, or migrants overall, often failing to measure the risks to women via their direct involvement in migration. Inconsistent measures of mobility, gender biases in those measures, and limited data sources for sex-specific population-based estimates of mobility have contributed to a paucity of research on the HIV prevention and care needs of migrant and highly mobile women. This study addresses an urgent need for novel methods for developing probability-based, systematic samples of highly mobile women, focusing on a population of female traders operating out of one of the largest open air markets in East Africa. Our method involves three stages: 1.) identification and mapping of all market stall locations using Global Positioning System (GPS) coordinates; 2.) using female market vendor stall GPS coordinates to build the sampling frame using replicates; and 3.) using maps and GPS data for recruitment of study participants. Results The location of 6,390 vendor stalls were mapped using GPS. Of these, 4,064 stalls occupied by women (63.6%) were used to draw four replicates of 128 stalls each, and a fifth replicate of 15 pre-selected random alternates for a total of 527 stalls assigned to one of five replicates. Staff visited 323 stalls from the first three replicates and from these successfully recruited 306 female vendors into the study for a participation rate of 94.7%. Mobilization strategies and involving traders association representatives in participant recruitment were critical to the study’s success. Conclusion The study’s high participation rate suggests that this geospatial sampling method holds promise for development of probability-based samples in other settings that serve as transport hubs for highly mobile populations. PMID:29324780

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

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

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.

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

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

Structure-function analysis of ribonucleotide bypass by B family DNA replicases

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

Clausen, Anders R.; Murray, Michael S.; Passer, Andrew R.

2013-11-01

Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and ε increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol ε. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal thatmore » increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structure–function studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.« less

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

Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery

PubMed Central

Murphy, Anar K.; Fitzgerald, Michael; Ro, Teresa; Kim, Jee Hyun; Rabinowitsch, Ariana I.; Chowdhury, Dipanjan; Schildkraut, Carl L.

2014-01-01

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress. PMID:25113031

Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.

PubMed

Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa; Kim, Jee Hyun; Rabinowitsch, Ariana I; Chowdhury, Dipanjan; Schildkraut, Carl L; Borowiec, James A

2014-08-18

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress. © 2014 Murphy et al.

Recovery from the DNA Replication Checkpoint

PubMed Central

Chaudhury, Indrajit; Koepp, Deanna M.

2016-01-01

Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838

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.

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

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.

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.

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

Replication fork reversal triggers fork degradation in BRCA2-defective cells.

PubMed

Mijic, Sofija; Zellweger, Ralph; Chappidi, Nagaraja; Berti, Matteo; Jacobs, Kurt; Mutreja, Karun; Ursich, Sebastian; Ray Chaudhuri, Arnab; Nussenzweig, Andre; Janscak, Pavel; Lopes, Massimo

2017-10-16

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.BRCA2 is involved in both homologous recombination (HR) and the protection of stalled replication forks from degradation. Here the authors reveal how HR factors cooperate in fork remodeling, showing that BRCA2 supports RAD51 loading on the regressed arms of reversed replication forks to protect them from degradation.

THE FORK AND THE KINASE: A DNA REPLICATION TALE FROM A CHK1 PERSPECTIVE

PubMed Central

González Besteiro, Marina A.; Gottifredi, Vanesa

2014-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. The checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged-DNA. Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Such findings unveil a puzzling connection between Chk1 and DNA-lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, the multifaceted and versatile functions of Chk1 at ongoing forks and replication origins determine the extent and quality of the cellular response to replication stress. PMID:25795119

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.

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.

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.

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.

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.

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.

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.

The fork and the kinase: a DNA replication tale from a CHK1 perspective.

PubMed

González Besteiro, Marina A; Gottifredi, Vanesa

2015-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. Checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged DNA. Subsequently, Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Indeed, such findings have unveiled a puzzling connection between Chk1 and DNA lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, Chk1 is a multifaceted and versatile signaling factor that acts at ongoing forks and replication origins to determine the extent and quality of the cellular response to replication stress. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.

PubMed

Barnes, Ryan P; Hile, Suzanne E; Lee, Marietta Y; Eckert, Kristin A

2017-09-01

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA. Copyright © 2017 Elsevier B.V. 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 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.

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

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.

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.

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.

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

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.

Stop Stalling: Mus81 Required for Efficient Replication | Center for Cancer Research

Cancer.gov

DNA replication is precisely controlled to ensure that daughter cells receive intact, accurate genetic information. Each segment of DNA must be copied only once, and the rate of replication coordinated genome-wide. Mild replication stress slows DNA synthesis and activates a pathway involving the Mus81 endonuclease, which generates a series of DNA breaks that are rapidly

27nt-RNAs guide histone variant deposition via 'RNA-induced DNA replication interference' and thus transmit parental genome partitioning in Stylonychia.

PubMed

Postberg, Jan; Jönsson, Franziska; Weil, Patrick Philipp; Bulic, Aneta; Juranek, Stefan Andreas; Lipps, Hans-Joachim

2018-06-12

During sexual reproduction in the unicellular ciliate Stylonychia somatic macronuclei differentiate from germline micronuclei. Thereby, programmed sequence reduction takes place, leading to the elimination of > 95% of germline sequences, which priorly adopt heterochromatin structure via H3K27me3. Simultaneously, 27nt-ncRNAs become synthesized from parental transcripts and are bound by the Argonaute protein PIWI1. These 27nt-ncRNAs cover sequences destined to the developing macronucleus and are thought to protect them from degradation. We provide evidence and propose that RNA/DNA base-pairing guides PIWI1/27nt-RNA complexes to complementary macronucleus-destined DNA target sequences, hence transiently causing locally stalled replication during polytene chromosome formation. This spatiotemporal delay enables the selective deposition of temporarily available histone H3.4K27me3 nucleosomes at all other sequences being continuously replicated, thus dictating their prospective heterochromatin structure before becoming developmentally eliminated. Concomitantly, 27nt-RNA-covered sites remain protected. We introduce the concept of 'RNA-induced DNA replication interference' and explain how the parental functional genome partition could become transmitted to the progeny.

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

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

ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress*

PubMed Central

Kemp, Michael G.; Sancar, Aziz

2016-01-01

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase that maintains genome stability and halts cell cycle phase transitions in response to DNA lesions that block DNA polymerase movement. These DNA replication-associated features of ATR function have led to the emergence of ATR kinase inhibitors as potential adjuvants for DNA-damaging cancer chemotherapeutics. However, whether ATR affects the genotoxic stress response in non-replicating, non-cycling cells is currently unknown. We therefore used chemical inhibition of ATR kinase activity to examine the role of ATR in quiescent human cells. Although ATR inhibition had no obvious effects on the viability of non-cycling cells, inhibition of ATR partially protected non-replicating cells from the lethal effects of UV and UV mimetics. Analyses of various DNA damage response signaling pathways demonstrated that ATR inhibition reduced the activation of apoptotic signaling by these agents in non-cycling cells. The pro-apoptosis/cell death function of ATR is likely due to transcription stress because the lethal effects of compounds that block RNA polymerase movement were reduced in the presence of an ATR inhibitor. These results therefore suggest that whereas DNA polymerase stalling at DNA lesions activates ATR to protect cell viability and prevent apoptosis, the stalling of RNA polymerases instead activates ATR to induce an apoptotic form of cell death in non-cycling cells. These results have important implications regarding the use of ATR inhibitors in cancer chemotherapy regimens. PMID:26940878

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.

UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, γ-H2AX formation, and Mre11 relocalization

PubMed Central

Limoli, Charles L.; Giedzinski, Erich; Bonner, William M.; Cleaver, James E.

2002-01-01

UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (γ-H2AX) in large nuclear foci at sites of stalled replication forks. These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs). This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest. After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of γ-H2AX foci that paralleled the production of Mre11 foci. The percentage of foci-positive cells increased rapidly (10–15%) up to fluences of 10 J⋅m−2 before saturating at higher fluences. Frequencies of γ-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation. This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of γ-H2AX foci were found to precede the formation of Mre11 foci by several hours. The nuclear distributions of γ-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation. UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and γ-H2AX foci-positive cells. These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest. These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation. PMID:11756691

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.

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

Causes and Consequences of Replication Stress

PubMed Central

Zeman, Michelle K.; Cimprich, Karlene A.

2015-01-01

Replication stress is a complex phenomenon which has serious implications for genome stability, cell survival, and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATM- and Rad3-related (ATR). ATR and its downstream effectors stabilize and help to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding these pathways may be key to diagnosis and treatment of human diseases caused by defective responses to replication stress. PMID:24366029

Allyl isothiocyanate induces replication-associated DNA damage response in NSCLC cells and sensitizes to ionizing radiation.

PubMed

Tripathi, Kaushlendra; Hussein, Usama K; Anupalli, Roja; Barnett, Reagan; Bachaboina, Lavanya; Scalici, Jennifer; Rocconi, Rodney P; Owen, Laurie B; Piazza, Gary A; Palle, Komaraiah

2015-03-10

Allyl isothiocyanate (AITC), a constituent of many cruciferous vegetables exhibits significant anticancer activities in many cancer models. Our studies provide novel insights into AITC-induced anticancer mechanisms in human A549 and H1299 non-small cell lung cancer (NSCLC) cells. AITC exposure induced replication stress in NSCLC cells as evidenced by γH2AX and FANCD2 foci, ATM/ATR-mediated checkpoint responses and S and G2/M cell cycle arrest. Furthermore, AITC-induced FANCD2 foci displayed co-localization with BrdU foci, indicating stalled or collapsed replication forks in these cells. Although PITC (phenyl isothiocyanate) exhibited concentration-dependent cytotoxic effects, treatment was less effective compared to AITC. Previously, agents that induce cell cycle arrest in S and G2/M phases were shown to sensitize tumor cells to radiation. Similar to these observations, combination therapy involving AITC followed by radiation treatment exhibited increased DDR and cell killing in NSCLC cells compared to single agent treatment. Combination index (CI) analysis revealed synergistic effects at multiple doses of AITC and radiation, resulting in CI values of less than 0.7 at Fa of 0.5 (50% reduction in survival). Collectively, these studies identify an important anticancer mechanism displayed by AITC, and suggest that the combination of AITC and radiation could be an effective therapy for NSCLC.

A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair.

PubMed

Bharti, Sanjay Kumar; Sommers, Joshua A; Awate, Sanket; Bellani, Marina A; Khan, Irfan; Bradley, Lynda; King, Graeme A; Seol, Yeonee; Vidhyasagar, Venkatasubramanian; Wu, Yuliang; Abe, Takuye; Kobayashi, Koji; Shin-Ya, Kazuo; Kitao, Hiroyuki; Wold, Marc S; Branzei, Dana; Neuman, Keir C; Brosh, Robert M

2018-05-21

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

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.

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

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

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

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

Dynamics of Leading-strand Lesion Skipping by the Replisome

PubMed Central

Yeeles, Joseph T.P.; Marians, Kenneth J.

2013-01-01

SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

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.

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.

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 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.

Evaluation of a stall-flutter spring-damper pushrod in the rotating control system of a CH-54B helicopter

NASA Technical Reports Server (NTRS)

Nettles, W. E.; Paul, W. F.; Adams, D. O.

1974-01-01

Results of a design and flight test program conducted to define the effect of rotating pushrod damping on stall-flutter induced control loads are presented. The CH-54B helicopter was chosen as the test aircraft because it exhibited stall induced control loads. Damping was introduced into the CH-54B control system by replacing the standard pushrod with spring-damper assemblies. Design features of the spring-damper are described and the results of a dynamic analysis are shown which define the pushrod stiffness and damping requirements. Flight test measurements taken at 47,000 lb gross weight with and without the damper are presented. The results indicate that the spring-damper pushrods reduced high frequency, stall-induced rotating control loads by almost 50%. Fixed system control loads were reduced by 40%. Handling qualities in stall were unchanged, as expected.

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication

PubMed Central

Slaine, Patrick D.; Kleer, Mariel; Smith, Nathan K.; Khaperskyy, Denys A.

2017-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5′ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication. PMID:29258238

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

PubMed

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

2017-09-07

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

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.

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.

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

PubMed Central

Żabka, Aneta; Polit, Justyna Teresa; Maszewski, Janusz

2012-01-01

Background and Aims Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis. Methods Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H2O2 production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation). Key Results Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H2O2, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants. Conclusions The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus. PMID:23087128

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

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.

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

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.

Stop Stalling: Mus81 Required for Efficient Replication | Center for Cancer Research

Cancer.gov

DNA replication is precisely controlled to ensure that daughter cells receive intact, accurate genetic information. Each segment of DNA must be copied only once, and the rate of replication coordinated genome-wide. Mild replication stress slows DNA synthesis and activates a pathway involving the Mus81 endonuclease, which generates a series of DNA breaks that are rapidly repaired, allowing the cell to avoid activating the S-phase checkpoint and its potentially damaging outcomes of apoptosis or error-prone repair. Mirit Aladjem, Ph.D., of CCR’s Developmental Therapeutics Branch, and her colleagues wondered whether Mus81 also plays a role in regulating the replication rate during growth in the absence of stress.

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.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

PubMed Central

Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel MA; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin AM; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-01-01

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability. PMID:28191891

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.

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

Drug-Sensing by the Ribosome Induces Translational Arrest via Active Site Perturbation

PubMed Central

Arenz, Stefan; Meydan, Sezen; Starosta, Agata L.; Berninghausen, Otto; Beckmann, Roland; Vázquez-Laslop, Nora; Wilson, Daniel N.

2014-01-01

SUMMARY During protein synthesis, nascent polypeptide chains within the ribosomal tunnel can act in cis to induce ribosome stalling and regulate expression of downstream genes. The Staphylococcus aureus ErmCL leader peptide induces stalling in the presence of clinically important macrolide antibiotics, such as erythromycin, leading to the induction of the downstream macrolide resistance methyltransferase ErmC. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmCL-stalled ribosome at 3.9 Å resolution. The structure reveals how the ErmCL nascent chain directly senses the presence of the tunnel-bound drug and thereby induces allosteric conformational rearrangements at the peptidyltransferase center (PTC) of the ribosome. ErmCL-induced perturbations of the PTC prevent stable binding and accommodation of the aminoacyl-tRNA at the A-site leading to inhibition of peptide bond formation and translation arrest. PMID:25306253

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

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.

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

Topic 1.1.2, Unsteady Aerodynamics: Time-Varying Compressible Dynamic Stall Mechanisms Due to Freestream Mach Oscillations

DTIC Science & Technology

2014-12-31

separation during the pitch-up motion – thus interrupting the vortex shedding that is characteristic of deep dynamic stall (Ericsson and Reding , 1984). The...Aircraft, Vol. 31, No. 4, pp. 782-786. Ericsson, L. E. and Reding , J. P., (1971) “Dynamic Stall Simulation Problems,” Journal of Aircraft, Vol. 8, No...7, pp. 579-583. Ericsson, L. E. and Reding , J. P., (1984) “Shock-Induced Dynamic Stall,” Journal of Aircraft, Vol. 21, No. 5, pp. 316-321. Favier

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.

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

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.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

PubMed

Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel M A; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin A M; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-04-01

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

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.

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.

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.

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

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.

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

PubMed Central

Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

2015-01-01

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

PubMed Central

Adeyemi, Richard O.

2014-01-01

ABSTRACT The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. IMPORTANCE Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. PMID:24965470

The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-09-01

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

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

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

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

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 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

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

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle.

PubMed

Daigaku, Yasukazu; Mashiko, Satsuki; Mishiba, Keiichiro; Yamamura, Saburo; Ui, Ayako; Enomoto, Takemi; Yamamoto, Kazuo

2006-08-30

A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

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

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

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

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.

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

Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

PubMed Central

Sareen, Archana; Chaudhury, Indrajit; Adams, Nicole; Sobeck, Alexandra

2012-01-01

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase. PMID:22753026

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.

Force-induced rupture of double-stranded DNA in the absence and presence of covalently bonded anti-tumor drugs: Insights from molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Upadhyaya, Anurag; Nath, Shesh; Kumar, Sanjay

2018-06-01

DNA intra-strand cross-link (ICL) agents are widely used in the treatment of cancer. ICLs are thought to form a link between the same strand (intra-strand) or complimentary strand (inter-strand) and thereby increase the stability of DNA, which forbids the processes like replication and transcription. As a result, cell death occurs. In this work, we have studied the enhanced stability of a double stranded DNA in the presence of ICLs and compared our findings with the results obtained in the absence of these links. Using atomistic simulations with explicit solvent, a force is applied along and perpendicular to the direction of the helix and we measured the rupture force and the unzipping force of DNA-ICL complexes. Our results show that the rupture and the unzipping forces increase significantly in the presence of these links. The ICLs bind to the minor groove of DNA, which enhance the DNA stabilisation. Such information may be used to design alternative drugs that can stall replication and transcription that are critical to a growing number of anticancer drug discovery efforts.

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.

A Two-Year Follow-Up of a Staff Development Program Designed to Change Teacher Behavior

ERIC Educational Resources Information Center

Schaffer, Eugene; Stringfield, Samuel; Devlin-Scherer, Roberta

2017-01-01

Two years after participating in a replication of the Stallings Effective Use of Time (EUOT) Program, ten teachers were re-observed and interviewed to determine the extent to which they had maintained the measured changes in their behavior patterns. Subjects were selected for the follow-up from a 27 EUOT teacher sample based on having exhibited…

Analysis of an unswept propfan blade with a semiempirical dynamic stall model

NASA Technical Reports Server (NTRS)

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

1989-01-01

The time history response of a propfan wind tunnel model with dynamic stall is studied analytically. The response obtained from the analysis is compared with available experimental data. The governing equations of motion are formulated in terms of blade normal modes which are calculated using the COSMIC-NASTRAN computer code. The response analysis considered the blade plunging and pitching motions. The lift, drag and moment coefficients for angles of attack below the static stall angle are obtained from a quasi-steady theory. For angles above static stall angles, a semiempirical dynamic stall model based on a correction to angle of attack is used to obtain lift, drag and moment coefficients. Using these coefficients, the aerodynamic forces are calculated at a selected number of strips, and integrated to obtain the total generalized forces. The combined momentum-blade element theory is used to calculate the induced velocity. The semiempirical stall model predicted a limit cycle oscillation near the setting angle at which large vibratory stresses were observed in an experiment. The predicted mode and frequency of oscillation also agreed with those measured in the experiment near the setting angle.

A Comparative Study of Three Methodologies for Modeling Dynamic Stall

NASA Technical Reports Server (NTRS)

Sankar, L.; Rhee, M.; Tung, C.; ZibiBailly, J.; LeBalleur, J. C.; Blaise, D.; Rouzaud, O.

2002-01-01

During the past two decades, there has been an increased reliance on the use of computational fluid dynamics methods for modeling rotors in high speed forward flight. Computational methods are being developed for modeling the shock induced loads on the advancing side, first-principles based modeling of the trailing wake evolution, and for retreating blade stall. The retreating blade dynamic stall problem has received particular attention, because the large variations in lift and pitching moments encountered in dynamic stall can lead to blade vibrations and pitch link fatigue. Restricting to aerodynamics, the numerical prediction of dynamic stall is still a complex and challenging CFD problem, that, even in two dimensions at low speed, gathers the major difficulties of aerodynamics, such as the grid resolution requirements for the viscous phenomena at leading-edge bubbles or in mixing-layers, the bias of the numerical viscosity, and the major difficulties of the physical modeling, such as the turbulence models, the transition models, whose both determinant influences, already present in static maximal-lift or stall computations, are emphasized by the dynamic aspect of the phenomena.

A method for detecting genetic toxicity using the RNA synthesis response to DNA damage.

PubMed

Morita, Yoko; Iwai, Shigenori; Kuraoka, Isao

2011-10-01

To date, biological risk assessment studies of chemicals that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication but also with transcription. Therefore, detecting the damaging effects of DNA lesions during transcription might be important for estimating the safety of chemical mutagens and carcinogens. However, methods to address these effects have not been developed. Here, we report a simple, non-isotopic method for determining the toxicity of chemical agents by visualizing transcription in a mammalian cell system. The method is based on the measurement of the incorporation of bromouridine (as the uridine analogue) into the nascent RNA during RNA synthesis inhibition (RSI) induced by the stalling of RNA polymerases at DNA lesions on the transcribed DNA strand, which triggers transcription-coupled nucleotide excision repair (TC-NER). When we tested chemical agents (camptothecin, etoposide, 4-nitroquinoline-1-oxide, mitomycin C, methyl methanesulfonate, and cisplatin) in HeLa cells by the method, RSI indicative of genomic toxicity was observed in the nucleoli of the tested cells. This procedure provides the following advantages: 1) it uses common, affordable mammalian cells (HeLa cells, WI38VA13 cells, human dermal fibroblasts, or Chinese hamster ovary cells) rather than genetically modified microorganisms; 2) it can be completed within approximately 8 hr after the cells are prepared because RNA polymerase responses during TC-NER are faster than other DNA damage responses (replication, recombination, and apoptosis); and 3) it is safe because it uses non-radioactive bromouridine and antibodies to detect RNA synthesis on undamaged transcribed DNA strands.

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.

Propeller swirl effect on single-engine general-aviation aircraft stall-spin tendencies

NASA Technical Reports Server (NTRS)

Katz, Joseph; Feistel, Terry W.

1987-01-01

An investigation is conducted of the effect of a single engine, untapered low wing general aviation aircraft propeller's swirl on the craft's stall pattern. The asymmetrical character of the propeller's swirl can trigger an early stall of one of the wings, aggravating the spin-entry condition. It is shown that the combination of this propeller-induced effect with adverse sideslip can result in large and abrupt changes in the rolling moment, in such conditions as uncoordinated low speed turning maneuvers where the pilot yaws the aircraft with wings level, rather than rolling it.

MMS Exposure Promotes Increased MtDNA Mutagenesis in the Presence of Replication-Defective Disease-Associated DNA Polymerase γ Variants

PubMed Central

Stumpf, Jeffrey D.; Copeland, William C.

2014-01-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities. PMID:25340760

MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

PubMed

Stumpf, Jeffrey D; Copeland, William C

2014-10-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities.

Pilot evaluation of sailplane handling qualities

NASA Technical Reports Server (NTRS)

Bennett, A. G., Jr.

1978-01-01

The evaluation sailplanes were found generally deficient in the area of cockpit layout. The pilots indicated general dissatisfaction with high pitch sensitivity especially when coupled with inertially induced stick forces. While all sailplanes were judged satisfactory for centering thermals and in the ease of speed control in circling flight, pilot opinions diverged on the maneuvering response, pull-out characteristics from a dive, and on phugoid damping. Lateral-directional control problems were noted mainly during takeoff and landing for most sailplanes with the landing wheel ahead of center of gravity. Pilot opinion of in-flight lateral-directional stability and control was generally satisfactory. Five of the evaluation sailplanes exhibited a very narrow airspeed band in which perceptible stall warning buffet occurred. However, this characteristic was considered not objectionable when stall recovery was easy. The pilots objected to the characteristics of a wide airspeed band of stall warning followed by a stall with yawing and rolling tendency and substantial loss of altitude during the stall. Glide path control for the evaluation sailplanes was found to be generally objectionable.

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.

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

Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

DTIC Science & Technology

2014-09-01

Watters, K. M., Das, S., Bryan, K., Bernas, T., Prehn , J. H., and Stallings, R. L. (2011) MicroRNAs 10a and 10b are potent inducers of neuroblastoma...Cell Biol 2009;29(19):5290-5305. 30. Foley NH, Bray I, Watters KM, Das S, Bryan K, Bernas T, Prehn JH, Stallings RL. MicroRNAs 10a and 10b are potent

Flow and Performance Calculations of Axial Compressor near Stall Margin

NASA Astrophysics Data System (ADS)

Hwang, Yoojun; Kang, Shin-Hyoung

2010-06-01

Three-dimensional flows through a Low Speed Research Axial Compressor were numerically conducted in order to estimate the performance through unsteady and steady-state simulations. The first stage with the inlet guide vane was investigated at the design point to confirm that the rotor blade induced periodicity exists. Special attention was paid to the flow near the stall condition to inspect the flow behavior in the vicinity of the stall margin. The performance predicted under the steady-state assumption is in good agreement with the measured data. However, the steady-state calculations induce more blockage through the blade passage. Flow separations on the blade surface and end-walls are reduced when unsteady simulation is conducted. The negative jet due to the wake of the rotor blade periodically distorts the boundary layer on the surface of the stator blade and improves the performance of the compressor in terms of the pressure rise. The advantage of the unsteadiness increases as the flow rate reduces. In addition, the rotor tip leakage flow is forced downstream by the unsteadiness. Consequently, the behavior contributes to extending the range of operation by preventing the leakage flow from proceeding upstream near the stall margin.

A novel variant of DNA polymerase ζ, Rev3ΔC, highlights differential regulation of Pol32 as a subunit of polymerase δ versus ζ in Saccharomyces cerevisiae

PubMed Central

Siebler, Hollie M.; Lada, Artem G.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Pavlov, Youri I.

2014-01-01

Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe–4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3–Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches. PMID:24819597

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.

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.

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

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.

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway.

PubMed

Nakada, Daisuke; Hirano, Yukinori; Sugimoto, Katsunori

2004-11-01

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

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 computation of the post-stall behavior of a circulation controlled airfoil

NASA Technical Reports Server (NTRS)

Linton, Samuel W.

1993-01-01

The physics of the circulation controlled airfoil is complex and poorly understood, particularly with regards to jet stall, which is the eventual breakdown of lift augmentation by the jet at some sufficiently high blowing rate. The present paper describes the numerical simulation of stalled and unstalled flows over a two-dimensional circulation controlled airfoil using a fully implicit Navier-Stokes code, and the comparison with experimental results. Mach numbers of 0.3 and 0.5 and jet total to freestream pressure ratios of 1.4 and 1.8 are investigated. The Baldwin-Lomax and k-epsilon turbulence models are used, each modified to include the effect of strong streamline curvature. The numerical solutions of the post-stall circulation controlled airfoil show a highly regular unsteady periodic flowfield. This is the result of an alternation between adverse pressure gradient and shock induced separation of the boundary layer on the airfoil trailing edge.

An experimental study of mushroom shaped stall cells. [on finite wings with separated flow

NASA Technical Reports Server (NTRS)

Winkelmann, A. E.

1982-01-01

Surface patterns characterized by a pair of counter-rotating swirls have been observed in connection with the conduction of surface flow visualization experiments involving test geometries with separated flows. An example of this phenomenon occurring on a finite wing with trailing edge stall has been referred to by Winkelmann and Barlow (1980) as 'mushroom shaped'. A description is presented of a collection of experimental results which show or suggest the occurrence of mushroom shaped stall cells on a variety of test geometries. Investigations conducted with finite wings, airfoil models, and flat plates are considered, and attention is given to studies involving the use of bluff models, investigations of shock induced boundary layer separation, and mushroom shaped patterns observed in a number of miscellaneous cases. It is concluded that the mushroom shaped stall cell appears commonly in separated flow regions.

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.

Plasma-based Compressor Stall Control

NASA Astrophysics Data System (ADS)

McGowan, Ryan; Corke, Thomas

2017-11-01

The use of dielectric barrier discharge (DBD) plasma actuator casing treatment to prevent or delay stall inception in an axial fan is examined. The actuators are powered by a pulsed-DC waveform which induces a larger peak velocity than a purely AC waveform such as a sine or sawtooth wave. With this system, a high-voltage DC source is supplied to both electrodes, remaining constant in time for the exposed electrode. Meanwhile, the covered electrode is periodically grounded for several microseconds and allowed to rise back to the source DC level. To test the actuators' ability to interact with and modify the formation of stall cells, a facility has been designed and constructed around nonconductive fan blades. The actuators are installed in the fan casing near the blade tips. The instrumentation allows for the measurement of rotating pressure disturbances (traveling stall cells) in this tip gap region as well as fan performance characteristics including pressure rise and flow rate. The casing plasma actuation is found to reduce the correlation of the rotating stall cells, thereby extending the stall margin of the fan. Various azimuthal arrangements of the plasma actuator casing treatment is explored, as well as input voltage levels to the actuator to determine optimum conditions. NASA SBIR Contract NNX14CC12C.

Induced Power of the Helicopter Rotor

NASA Technical Reports Server (NTRS)

Ormiston, Robert A.

2004-01-01

A simplified rotor model was used to explore fundamental behavior of lifting rotor induced power at moderate and high advance ratios. Several rotor inflow theories, including dynamic inflow theory and prescribed-wake vortex theory, together with idealized notional airfoil stall models were employed. A number of unusual results were encountered at high advance ratios including trim control reversal and multiple trim solutions. Significant increases in rotor induced power (torque) above the ideal minimum were observed for moderately high advance ratio. Very high induced power was observed near and above unity advance ratio. The results were sensitive to the stall characteristics of the airfoil models used. An equivalent wing analysis was developed to determine induced power from Prandtl lifting line theory and help interpret the rotor induced power behavior in terms of the spanwise airload distribution. The equivalent wing approach was successful in capturing the principal variations of induced power for different configurations and operating conditions. The effects blade root cutout were found to have a significant effect on rotor trim and induced power at high advance ratios.

Stalled RNAP-II molecules bound to non-coding rDNA spacers are required for normal nucleolus architecture.

PubMed

Freire-Picos, M A; Landeira-Ameijeiras, V; Mayán, María D

2013-07-01

The correct distribution of nuclear domains is critical for the maintenance of normal cellular processes such as transcription and replication, which are regulated depending on their location and surroundings. The most well-characterized nuclear domain, the nucleolus, is essential for cell survival and metabolism. Alterations in nucleolar structure affect nuclear dynamics; however, how the nucleolus and the rest of the nuclear domains are interconnected is largely unknown. In this report, we demonstrate that RNAP-II is vital for the maintenance of the typical crescent-shaped structure of the nucleolar rDNA repeats and rRNA transcription. When stalled RNAP-II molecules are not bound to the chromatin, the nucleolus loses its typical crescent-shaped structure. However, the RNAP-II interaction with Seh1p, or cryptic transcription by RNAP-II, is not critical for morphological changes. Copyright © 2013 John Wiley & Sons, Ltd.

Aeroelastic Stability of Idling Wind Turbines

NASA Astrophysics Data System (ADS)

Wang, Kai; Riziotis, Vasilis A.; Voutsinas, Spyros G.

2016-09-01

Wind turbine rotors in idling operation mode can experience high angles of attack, within the post stall region that are capable of triggering stall-induced vibrations. In the present paper rotor stability in slow idling operation is assessed on the basis of non-linear time domain and linear eigenvalue analysis. Analysis is performed for a 10 MW conceptual wind turbine designed by DTU. First the flow conditions that are likely to favour stall induced instabilities are identified through non-linear time domain aeroelastic analysis. Next, for the above specified conditions, eigenvalue stability simulations are performed aiming at identifying the low damped modes of the turbine. Finally the results of the eigenvalue analysis are evaluated through computations of the work of the aerodynamic forces by imposing harmonic vibrations following the shape and frequency of the various modes. Eigenvalue analysis indicates that the asymmetric and symmetric out-of-plane modes have the lowest damping. The results of the eigenvalue analysis agree well with those of the time domain analysis.

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions

PubMed Central

Markkanen, Enni; Castrec, Benoît; Villani, Giuseppe; Hübscher, Ulrich

2012-01-01

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, β, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols β and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ−/− mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ. PMID:23175785

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.

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.

Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress

PubMed Central

Bi, Xin; Yu, Qun; Siler, Jasmine; Li, Chong; Khan, Ali

2015-01-01

The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5. PMID:25806814

NEDDylation promotes stress granule assembly.

PubMed

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

2016-07-06

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1.

PubMed

Vogt, Julia; Wernstedt, Annekatrin; Ripperger, Tim; Pabst, Brigitte; Zschocke, Johannes; Kratz, Christian; Wimmer, Katharina

2016-11-01

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5'-part from the 3'-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms.

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1

PubMed Central

Vogt, Julia; Wernstedt, Annekatrin; Ripperger, Tim; Pabst, Brigitte; Zschocke, Johannes; Kratz, Christian; Wimmer, Katharina

2016-01-01

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5′-part from the 3′-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms. PMID:27329736

Rapid activity-induced transcription of arc and other IEGs relies on poised RNA polymerase II

PubMed Central

Saha, Ramendra N.; Wissink, Erin M.; Bailey, Emma R.; Zhao, Meilan; Fargo, David C.; Hwang, Ji-yeon; Daigle, Kelly R.; Fenn, J. Daniel; Adelman, Karen; Dudek, Serena M.

2011-01-01

Summary Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We demonstrate that several IEGs such as arc/arg3.1 are poised for near-instantaneous transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site in rat neurons. RNAi-depletion of Negative Elongation Factor, a mediator of Pol II stalling, reduces the Pol II occupancy of the arc promoter and compromises the rapid induction of arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that are expressed later and largely lack promoter proximal Pol II stalling. Together, our data strongly indicate that rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory. PMID:21623364

Simulator study of stall/post-stall characteristics of a fighter airplane with relaxed longitudinal static stability. [F-16

NASA Technical Reports Server (NTRS)

Nguyen, L. T.; Ogburn, M. E.; Gilbert, W. P.; Kibler, K. S.; Brown, P. W.; Deal, P. L.

1979-01-01

A real-time piloted simulation was conducted to evaluate the high-angle-of-attack characteristics of a fighter configuration based on wind-tunnel testing of the F-16, with particular emphasis on the effects of various levels of relaxed longitudinal static stability. The aerodynamic data used in the simulation was conducted on the Langley differential maneuvering simulator, and the evaluation involved representative low-speed combat maneuvering. Results of the investigation show that the airplane with the basic control system was resistant to the classical yaw departure; however, it was susceptible to pitch departures induced by inertia coupling during rapid, large-amplitude rolls at low airspeed. The airplane also exhibited a deep-stall trim which could be flown into and from which it was difficult to recover. Control-system modifications were developed which greatly decreased the airplane susceptibility to the inertia-coupling departure and which provided a reliable means for recovering from the deep stall.

Aerodynamic study of a blade with sine variation of chord length along the height for Darrieus wind turbine

NASA Astrophysics Data System (ADS)

Crunteanu, D. E.; Constantinescu, S. G.; 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. In contrast to these wind turbines, the Darrieus wind turbines are simpler and their price is lower. Unfortunately, their blades run at high variations of angles of attack, in stall and post-stall regimes, which can induce significant vibrations, fatigue and even the wind turbine failure. For this reason, the present paper deals with a blade with sine variation of chord length along the height because it has better behavior in stall and post-stall regimes than the classic blade with constant chord length.

A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest

NASA Astrophysics Data System (ADS)

Arenz, Stefan; Bock, Lars V.; Graf, Michael; Innis, C. Axel; Beckmann, Roland; Grubmüller, Helmut; Vaiana, Andrea C.; Wilson, Daniel N.

2016-07-01

Nascent polypeptides can induce ribosome stalling, regulating downstream genes. Stalling of ErmBL peptide translation in the presence of the macrolide antibiotic erythromycin leads to resistance in Streptococcus sanguis. To reveal this stalling mechanism we obtained 3.6-Å-resolution cryo-EM structures of ErmBL-stalled ribosomes with erythromycin. The nascent peptide adopts an unusual conformation with the C-terminal Asp10 side chain in a previously unseen rotated position. Together with molecular dynamics simulations, the structures indicate that peptide-bond formation is inhibited by displacement of the peptidyl-tRNA A76 ribose from its canonical position, and by non-productive interactions of the A-tRNA Lys11 side chain with the A-site crevice. These two effects combine to perturb peptide-bond formation by increasing the distance between the attacking Lys11 amine and the Asp10 carbonyl carbon. The interplay between drug, peptide and ribosome uncovered here also provides insight into the fundamental mechanism of peptide-bond formation.

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.

Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya.

PubMed

Wardle, Josephine; Burgers, Peter M J; Cann, Isaac K O; Darley, Kate; Heslop, Pauline; Johansson, Erik; Lin, Li-Jung; McGlynn, Peter; Sanvoisin, Jonathan; Stith, Carrie M; Connolly, Bernard A

2008-02-01

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures approximately 100 degrees C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases delta and epsilon for nuclear DNA and polymerase gamma for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.

Stall induced instability of a teetered rotor

NASA Astrophysics Data System (ADS)

Glasgow, J. C.; Corrigan, R. D.

Recent tests on the 38m Mod-0 horizontal experimental wind turbine yielded quantitative information on stall induced instability of a teetered rotor. Tests were conducted on rotor blades with NACA 230 series and NACA 643-618 airfoils at low rotor speeds to produce high angles of attack at relatively low wind speeds and power levels. The behavior of the rotor shows good agreement with predicted rotor response based on blade angle of attack calculations and airfoil section properties. The untwisted blades with the 64 series airfoil sections had a slower rate of onset of rotor instability when compared with the twisted 230 series blades, but high teeter angles and teeter stop impacts were experienced with both rotors as wind speeds increased to produce high angles of attack on the outboard portion of the blade. The relative importance of blade twist and airfoil section stall characteristics on the rate of onset of rotor unstability with increasing wind speed was not established however. Blade pitch was shown to be effective in eliminating rotor instability at the expense of some loss in rotor performance near rated wind speed.

The scaffold protein Nde1 safeguards the brain genome during S phase of early neural progenitor differentiation

PubMed Central

Houlihan, Shauna L; Feng, Yuanyi

2014-01-01

Successfully completing the S phase of each cell cycle ensures genome integrity. Impediment of DNA replication can lead to DNA damage and genomic disorders. In this study, we show a novel function for NDE1, whose mutations cause brain developmental disorders, in safeguarding the genome through S phase during early steps of neural progenitor fate restrictive differentiation. Nde1 mutant neural progenitors showed catastrophic DNA double strand breaks concurrent with the DNA replication. This evoked DNA damage responses, led to the activation of p53-dependent apoptosis, and resulted in the reduction of neurons in cortical layer II/III. We discovered a nuclear pool of Nde1, identified the interaction of Nde1 with cohesin and its associated chromatin remodeler, and showed that stalled DNA replication in Nde1 mutants specifically occurred in mid-late S phase at heterochromatin domains. These findings suggest that NDE1-mediated heterochromatin replication is indispensible for neuronal differentiation, and that the loss of NDE1 function may lead to genomic neurological disorders. DOI: http://dx.doi.org/10.7554/eLife.03297.001 PMID:25245017

Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

PubMed

Appleby, Todd C; Perry, Jason K; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David; Wetmore, Diana R; McGrath, Mary E; Ray, Adrian S; Sofia, Michael J; Swaminathan, S; Edwards, Thomas E

2015-02-13

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.

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.

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

NEDDylation promotes stress granule assembly

PubMed Central

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

2016-01-01

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly. PMID:27381497

Fluid mechanics of dynamic stall. II - Prediction of full scale characteristics

NASA Technical Reports Server (NTRS)

Ericsson, L. E.; Reding, J. P.

1988-01-01

Analytical extrapolations are made from experimental subscale dynamics to predict full scale characteristics of dynamic stall. The method proceeds by establishing analytic relationships between dynamic and static aerodynamic characteristics induced by viscous flow effects. The method is then validated by predicting dynamic test results on the basis of corresponding static test data obtained at the same subscale flow conditions, and the effect of Reynolds number on the static aerodynamic characteristics are determined from subscale to full scale flow conditions.

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

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.

Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.

PubMed

Jõers, Priit; Lewis, Samantha C; Fukuoh, Atsushi; Parhiala, Mikael; Ellilä, Simo; Holt, Ian J; Jacobs, Howard T

2013-01-01

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.

Mitochondrial Transcription Terminator Family Members mTTF and mTerf5 Have Opposing Roles in Coordination of mtDNA Synthesis

PubMed Central

Jõers, Priit; Lewis, Samantha C.; Fukuoh, Atsushi; Parhiala, Mikael; Ellilä, Simo; Holt, Ian J.; Jacobs, Howard T.

2013-01-01

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis. PMID:24068965

DNA Replication Dynamics of the GGGGCC Repeat of the C9orf72 Gene.

PubMed

Thys, Ryan Griffin; Wang, Yuh-Hwa

2015-11-27

DNA has the ability to form a variety of secondary structures in addition to the normal B-form DNA, including hairpins and quadruplexes. These structures are implicated in a number of neurological diseases and cancer. Expansion of a GGGGCC repeat located at C9orf72 is associated with familial amyotrophic lateral sclerosis and frontotemporal dementia. This repeat expands from two to 24 copies in normal individuals to several hundreds or thousands of repeats in individuals with the disease. Biochemical studies have demonstrated that as little as four repeats have the ability to form a stable DNA secondary structure known as a G-quadruplex. Quadruplex structures have the ability to disrupt normal DNA processes such as DNA replication and transcription. Here we examine the role of GGGGCC repeat length and orientation on DNA replication using an SV40 replication system in human cells. Replication through GGGGCC repeats leads to a decrease in overall replication efficiency and an increase in instability in a length-dependent manner. Both repeat expansions and contractions are observed, and replication orientation is found to influence the propensity for expansions or contractions. The presence of replication stress, such as low-dose aphidicolin, diminishes replication efficiency but has no effect on instability. Two-dimensional gel electrophoresis analysis demonstrates a replication stall with as few as 20 GGGGCC repeats. These results suggest that replication of the GGGGCC repeat at C9orf72 is perturbed by the presence of expanded repeats, which has the potential to result in further expansion, leading to disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

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

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain

PubMed Central

Koch, Miriam; Willi, Jessica; Pradère, Ugo; Hall, Jonathan

2017-01-01

Abstract The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation. PMID:28369621

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.

The effect of leading edge tubercles on dynamic stall

NASA Astrophysics Data System (ADS)

Hrynuk, John

The effect of the leading edge tubercles of humpback whales has been heavily studied for their static benefits. These studies have shown that tubercles inhibit flow separation, limit spanwise flow, and extend the operating angle of a wing beyond the static stall point while maintaining lift, all while having a comparatively low negative impact on drag. The current study extends the prior work to investigating the effect of tubercles on dynamic stall, a fundamental flow phenomenon that occurs when wings undergo dynamic pitching motions. Flow fields around the wing models tested were studied using Laser Induced Fluorescence (LIF) and Molecular Tagging Velocimetry (MTV).Resulting velocity fields show that the dynamics of the formation and separation of the leading edge vortex were fundamentally different between the straight wing and the tubercled wing. Tracking of the Dynamic Stall Vortex (DSV) and Shear Layer Vortices (SLVs), which may have a significant impact on the overall flow behavior, was done along with calculations of vortex circulation. Proximity to the wing surface and total circulation were used to evaluate potential dynamic lift increases provided by the tubercles. The effects of pitch rate on the formation process and benefits of the tubercles were also studied and were generally consistent with prior dynamic stall studies. However, tubercles were shown to affect the SLV formation and the circulation differently at higher pitch rates.

Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53

PubMed Central

Chen, Bo; Simpson, Dennis A.; Zhou, Yingchun; Mitra, Amritava; Mitchell, David L.; Cordeiro-Stone, Marila; Kaufmann, William K.

2015-01-01

After treatment with ultraviolet radiation (UV), human fibroblasts that express the HPV type 16 E6 oncoprotein display defects in repair of cyclobutane pyrimidine dimers, hypersensitivity to inactivation of clonogenic survival and an inability to sustain DNA replication. To determine whether these effects are specific to depletion of p53 or inactivation of its function, fibroblast lines were constructed with ectopic expression of a dominant-negative p53 allele (p53-H179Q) to inactivate function or a short-hairpin RNA (p53-RNAi) to deplete expression of p53. Only the expression of HPV16E6 sensitized fibroblasts to UV or the chemical carcinogen, benzo[a]pyrene diolepoxide I (BPDE). Carcinogen-treated cells expressing p53-H179Q or p53-RNAi were resistant to inactivation of colony formation and did not suffer replication arrest. CHK1 is a key checkpoint kinase in the response to carcinogen-induced DNA damage. Control and p53-RNAi-expressing fibroblasts displayed phosphorylation of Ser345 on CHK1 45–120 min after carcinogen treatment with a return to near baseline phosphorylation by 6 h after treatment. HPV16E6-expressing fibroblasts displayed enhanced and sustained phosphorylation of CHK1. This was associated with enhanced phosphorylation of Thr68 on CHK2 and Ser139 on H2AX, both markers of severe replication stress and DNA double strand breaks. Incubation with the phosphatase inhibitor okadaic acid produced more phosphorylation of CHK1 in UV-treated HPV16E6-expressing cells than in p53-H179Q-expressing cells suggesting that HPV16E6 may interfere with the recovery of coupled DNA replication at replication forks that are stalled at [6-4]pyrimidine-pyrimidone photoproducts and BPDE-DNA adducts. The results indicate that HPV16E6 targets a protein or proteins other than p53 to deregulate the activity of CHK1 in carcinogen-damaged cells. PMID:19411857

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

PubMed Central

Khaperskyy, Denys A.; Emara, Mohamed M.; Johnston, Benjamin P.; Anderson, Paul; Hatchette, Todd F.; McCormick, Craig

2014-01-01

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication. PMID:25010204

Domain structure, localization, and function of DNA polymerase η, defective in xeroderma pigmentosum variant cells

PubMed Central

Kannouche, Patricia; Broughton, Bernard C.; Volker, Marcel; Hanaoka, Fumio; Mullenders, Leon H.F.; Lehmann, Alan R.

2001-01-01

DNA polymerase η carries out translesion synthesis past UV photoproducts and is deficient in xeroderma pigmentosum (XP) variants. We report that polη is mostly localized uniformly in the nucleus but is associated with replication foci during S phase. Following treatment of cells with UV irradiation or carcinogens, it accumulates at replication foci stalled at DNA damage. The C-terminal third of polη is not required for polymerase activity. However, the C-terminal 70 aa are needed for nuclear localization and a further 50 aa for relocalization into foci. Polη truncations lacking these domains fail to correct the defects in XP-variant cells. Furthermore, we have identified mutations in two XP variant patients that leave the polymerase motifs intact but cause loss of the localization domains. PMID:11157773

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

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

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.

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

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain.

PubMed

Koch, Miriam; Willi, Jessica; Pradère, Ugo; Hall, Jonathan; Polacek, Norbert

2017-06-20

The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson-Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

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.

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.

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.

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

Sequential replication-coupled destruction at G1/S ensures genome stability

PubMed Central

Coleman, Kate E.; Grant, Gavin D.; Haggerty, Rachel A.; Brantley, Kristen; Shibata, Etsuko; Workman, Benjamin D.; Dutta, Anindya; Varma, Dileep; Purvis, Jeremy E.; Cook, Jeanette Gowen

2015-01-01

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4Cdt2, which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4Cdt2 targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNADNA) and recruits CRL4Cdt2. Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNADNA. Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability. PMID:26272819

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

PubMed Central

Yang, Yang; Durando, Michael; Smith-Roe, Stephanie L.; Sproul, Chris; Greenwalt, Alicia M.; Kaufmann, William; Oh, Sehyun; Hendrickson, Eric A.; Vaziri, Cyrus

2013-01-01

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase. PMID:23295675

Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription

PubMed Central

Aydin, Özge Z.; Marteijn, Jurgen A.; Ribeiro-Silva, Cristina; Rodríguez López, Aida; Wijgers, Nils; Smeenk, Godelieve; van Attikum, Haico; Poot, Raymond A.; Vermeulen, Wim; Lans, Hannes

2014-01-01

Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA. PMID:24990377

Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

NASA Astrophysics Data System (ADS)

Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

2015-12-01

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling.

PubMed

Su, Ting; Cheng, Jingdong; Sohmen, Daniel; Hedman, Rickard; Berninghausen, Otto; von Heijne, Gunnar; Wilson, Daniel N; Beckmann, Roland

2017-05-30

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50-55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.

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

Helicopter gust response characteristics including unsteady aerodynamic stall effects

NASA Technical Reports Server (NTRS)

Arcidiacono, P. J.; Bergquist, R. R.; Alexander, W. T., Jr.

1974-01-01

The results of an analytical study to evaluate the general response characteristics of a helicopter subjected to various types of discrete gust encounters are presented. The analysis employed was a nonlinear coupled, multi-blade rotorfuselage analysis including the effects of blade flexibility and unsteady aerodynamic stall. Only the controls-fixed response of the basic aircraft without any aircraft stability augmentation was considered. A discussion of the basic differences between gust sensitivity of fixed and rotary wing aircraft is presented. The effects of several rotor configuration and aircraft operating parameters on initial gust-induced load factor and blade vibratory stress and pushrod loads are discussed.

Molecular basis for erythromycin-dependent ribosome-stalling during translation of the ErmBL leader peptide

PubMed Central

Arenz, Stefan; Ramu, Haripriya; Gupta, Pulkit; Berninghausen, Otto; Beckmann, Roland; Vázquez-Laslop, Nora; Mankin, Alexander S.; Wilson, Daniel N.

2014-01-01

In bacteria, ribosome-stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase center (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural bases for understanding how the drug and the nascent peptide cooperate to inhibit peptide-bond formation and induce translation arrest. PMID:24662426

Stall behavior of a scaled three-dimensional wind turbine blade

NASA Astrophysics Data System (ADS)

Mulleners, Karen; Melius, Matthew; Cal, Raul Bayoan

2014-11-01

The power generation of a wind turbine is influenced by many factors including the unsteady incoming flow characteristics, pitch regulation, and the geometry of the various turbine components. Within the framework of maximizing energy extraction, it is important to understand and tailor the aerodynamics of a wind turbine. In the interest of seeking further understanding into the complex flow over wind turbine blades, a three-dimensional scaled blade model has been designed and manufactured to be dynamically similar to a rotating full-scale NREL 5MW wind turbine blade. A wind tunnel experiment has been carried out in the 2.2 m × 1.8 m cross-section closed loop wind tunnel at DLR in Göttingen by means of time-resolved stereoscopic PIV. An extensive coherent structure analysis of the time-resolved velocity field over the suction side of the blade was performed to study stall characteristics under a geometrically induced pressure gradient. In particular, the radial extent and propagation of stalled flow regions were characterized for various static angles of attack.

The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion▿

PubMed Central

Saberi, Alihossein; Nakahara, Makoto; Sale, Julian E.; Kikuchi, Koji; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Yamamoto, Kenichi; Takeda, Shunichi; Sonoda, Eiichiro

2008-01-01

Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9−/− and RAD17−/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9−/− and RAD17−/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair. PMID:18662998

Control of unsteady separated flow associated with the dynamic stall of airfoils

NASA Technical Reports Server (NTRS)

Wilder, M. C.

1995-01-01

An effort to understand and control the unsteady separated flow associated with the dynamic stall of airfoils was funded for three years through the NASA cooperative agreement program. As part of this effort a substantial data base was compiled detailing the effects various parameters have on the development of the dynamic stall flow field. Parameters studied include Mach number, pitch rate, and pitch history, as well as Reynolds number (through two different model chord lengths) and the condition of the boundary layer at the leading edge of the airfoil (through application of surface roughness). It was found for free stream Mach numbers as low as 0.4 that a region of supersonic flow forms on the leading edge of the suction surface of the airfoil at moderate angles of attack. The shocks which form in this supersonic region induce boundary-layer separation and advance the dynamic stall process. Under such conditions a supercritical airfoil profile is called for to produce a flow field having a weaker leading-edge pressure gradient and no leading-edge shocks. An airfoil having an adaptive-geometry, or dynamically deformable leading edge (DDLE), is under development as a unique active flow-control device. The DDLE, formed of carbon-fiber composite and fiberglass, can be flexed between a NACA 0012 profile and a supercritical profile in a controllable fashion while the airfoil is executing an angle-of-attack pitch-up maneuver. The dynamic stall data were recorded using point diffraction interferometry (PDI), a noninvasive measurement technique. A new high-speed cinematography system was developed for recording interferometric images. The system is capable of phase-locking with the pitching airfoil motion for real-time documentation of the development of the dynamic stall flow field. Computer-aided image analysis algorithms were developed for fast and accurate reduction of the images, improving interpretation of the results.

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.

The formation mechanism and impact of streamwise vortices on NACA 0021 airfoil's performance with undulating leading edge modification

NASA Astrophysics Data System (ADS)

Rostamzadeh, N.; Hansen, K. L.; Kelso, R. M.; Dally, B. B.

2014-10-01

Wings with tubercles have been shown to display advantageous loading behavior at high attack angles compared to their unmodified counterparts. In an earlier study by the authors, it was shown that an undulating leading-edge configuration, including but not limited to a tubercled model, induces a cyclic variation in circulation along the span that gives rise to the formation of counter-rotating streamwise vortices. While the aerodynamic benefits of full-span tubercled wings have been associated with the presence of such vortices, their formation mechanism and influence on wing performance are still in question. In the present work, experimental and numerical tests were conducted to further investigate the effect of tubercles on the flow structure over full-span modified wings based on the NACA 0021 profile, in the transitional flow regime. It is found that a skew-induced mechanism accounts for the formation of streamwise vortices whose development is accompanied by flow separation in delta-shaped regions near the trailing edge. The presence of vortices is detrimental to the performance of full-span wings pre-stall, however renders benefits post-stall as demonstrated by wind tunnel pressure measurement tests. Finally, primary and secondary vortices are identified post-stall that produce an enhanced momentum transfer effect that reduces flow separation, thus increasing the generated amount of lift.

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.

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.

Performance of a 1.57 pressure-ratio transonic fan stage with a screen-induced 90 deg circumferential inlet flow distortion

NASA Technical Reports Server (NTRS)

Sanger, N. L.

1976-01-01

A transonic fan stage having a design pressure ratio of 1.57 was tested with a 90 degree circumferential distortion imposed on the inlet flow. The rotor diameter was approximately 50.8 cm, and the design pressure ratio was 1.60 at a tip speed of 425 m/sec. Overall performance at 70 and 100 percent of design speed showed a loss of stall pressure ratio and flow range at design speed and no significant loss in stall pressure ratio at 70 percent of design speed. Detailed flow measurements are presented to show the rotor-upstream flow interactions and the attenuation and amplification properties through the stage.

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

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

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.

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.

A Survey of Reynolds Number and Wing Geometry Effects on Lift Characteristics in the Low Speed Stall Region

NASA Technical Reports Server (NTRS)

Polhamus, Edward C.

1996-01-01

This paper presents a survey of the effects of Reynolds number on the low- speed lift characteristics of wings encountering separated flows at their leading and side edges, with emphasis on the region near the stall. The influence of leading-edge profile and Reynolds number on the stall characteristics of two- dimensional airfoils are reviewed first to provide a basis for evaluating three- dimensional effects associated with various wing planforms. This is followed by examples of the effects of Reynolds number and geometry on the lift characteristics near the stall for a series of three-dimensional wings typical of those suitable for high-speed aircraft and missiles. Included are examples of the effects of wing geometry on the onset and spanwise progression of turbulent reseparation near the leading edge and illustrations of the degree to which simplified theoretical approaches can be useful in defining the influence of the various geometric parameters. Also illustrated is the manner in which the Reynolds number and wing geometry parameters influence whether the turbulent reseparation near the leading edge results in a sudden loss of lift, as in the two-dimensional case, or the formation of a leading-edge vortex with Rs increase in lift followed by a gentle stall as in the highly swept wing case. Particular emphasis is placed on the strong influence of 'induced camber' on the development of turbulent reseparation. R is believed that the examples selected for this report may be useful in evaluating viscous flow solutions by the new computational methods based on the Navier-Stokes equations as well as defining fruitful research areas for the high-Reynolds-number wind tunnels.

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.

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

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.

(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.

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

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...

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling

PubMed Central

Su, Ting; Cheng, Jingdong; Sohmen, Daniel; Hedman, Rickard; Berninghausen, Otto; von Heijne, Gunnar; Wilson, Daniel N; Beckmann, Roland

2017-01-01

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50–55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome. DOI: http://dx.doi.org/10.7554/eLife.25642.001 PMID:28556777

Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

PubMed Central

Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

2015-01-01

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer. PMID:26681117

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.

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.

A numerical investigation into the effects of Reynolds number on the flow mechanism induced by a tubercled leading edge

NASA Astrophysics Data System (ADS)

Rostamzadeh, Nikan; Kelso, Richard M.; Dally, Bassam

2017-02-01

Leading-edge modifications based on designs inspired by the protrusions on the pectoral flippers of the humpback whale (tubercles) have been the subject of research for the past decade primarily due to their flow control potential in ameliorating stall characteristics. Previous studies have demonstrated that, in the transitional flow regime, full-span wings with tubercled leading edges outperform unmodified wings at high attack angles. The flow mechanism associated with such enhanced loading traits is, however, still being investigated. Also, the performance of full-span tubercled wings in the turbulent regime is largely unexplored. The present study aims to investigate Reynolds number effects on the flow mechanism induced by a full-span tubercled wing with the NACA-0021 cross-sectional profile in the transitional and near-turbulent regimes using computational fluid dynamics. The analysis of the flow field suggests that, with the exception of a few different flow features, the same underlying flow mechanism, involving the presence of transverse and streamwise vorticity, is at play in both cases. With regard to lift-generation characteristics, the numerical simulation results indicate that in contrast to the transitional flow regime, where the unmodified NACA-0021 undergoes a sudden loss of lift, in the turbulent regime, the baseline foil experiences gradual stall and produces more lift than the tubercled foil. This observation highlights the importance of considerations regarding the Reynolds number effects and the stall characteristics of the baseline foil, in the industrial applications of tubercled lifting bodies.

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

Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase

PubMed Central

Baharoglu, Zeynep; Babosan, Anamaria; Mazel, Didier

2014-01-01

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated. PMID:24319148

Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase.

PubMed

Baharoglu, Zeynep; Babosan, Anamaria; Mazel, Didier

2014-02-01

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.

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.

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase*

PubMed Central

Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.

2014-01-01

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

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

Cabazitaxel operates anti-metastatic and cytotoxic via apoptosis induction and stalls brain tumor angiogenesis.

PubMed

Ghoochani, Ali; Hatipoglu Majernik, Gökce; Sehm, Tina; Wach, Sven; Buchfelder, Michael; Taubert, Helge; Eyupoglu, Ilker Y; Savaskan, Nicolai

2016-06-21

Taxanes target microtubules and are clinically established chemotherapeutic agents with proven efficacy in human cancers. Cabazitaxel (XRP-6258, Jevtana®) is a second generation semisynthetic taxane with high chemotherapeutic potential in prostate cancer. There, cabazitaxel can overcome docetaxel-resistant prostate cancer. Here, we tested the effects of cabazitaxel on glioma cells, and non-transformed cells such as neurons and astrocytes. Cabazitaxel operates highly toxic in various human glioma cells at nanomolar concentrations. In contrast, primary astrocytes and neurons are not affected by this agent. Cabazitaxel disrupts cytoskeletal F-actin fibers and induces apoptotic cell death in gliomas. Moreover, cabazitaxel displayed highest efficacy in inhibiting glioma cell migration and invasion. Here we demonstrate that cabazitaxel inhibited tumor migration already at 1 nM. We also tested cabazitaxel in the ex vivo VOGiM assay. Cabazitaxel stalled glioma growth and at the same time inhibited tumor-induced angiogenesis. In summary, we found that cabazitaxel operates as an apoptosis-inducing gliomatoxic agent with strongest effects on migration and invasive growth. Thus, our report uncovered cabazitaxel actions on gliomas and on the brain tumor microenvironment. These data reveal novel aspects for adjuvant approaches when applied to brain tumor patients.

Cabazitaxel operates anti-metastatic and cytotoxic via apoptosis induction and stalls brain tumor angiogenesis

PubMed Central

Ghoochani, Ali; Majernik, Gökce Hatipoglu; Sehm, Tina; Wach, Sven; Buchfelder, Michael; Taubert, Helge

2016-01-01

Taxanes target microtubules and are clinically established chemotherapeutic agents with proven efficacy in human cancers. Cabazitaxel (XRP-6258, Jevtana®) is a second generation semisynthetic taxane with high chemotherapeutic potential in prostate cancer. There, cabazitaxel can overcome docetaxel-resistant prostate cancer. Here, we tested the effects of cabazitaxel on glioma cells, and non-transformed cells such as neurons and astrocytes. Cabazitaxel operates highly toxic in various human glioma cells at nanomolar concentrations. In contrast, primary astrocytes and neurons are not affected by this agent. Cabazitaxel disrupts cytoskeletal F-actin fibers and induces apoptotic cell death in gliomas. Moreover, cabazitaxel displayed highest efficacy in inhibiting glioma cell migration and invasion. Here we demonstrate that cabazitaxel inhibited tumor migration already at 1 nM. We also tested cabazitaxel in the ex vivo VOGiM assay. Cabazitaxel stalled glioma growth and at the same time inhibited tumor-induced angiogenesis. In summary, we found that cabazitaxel operates as an apoptosis-inducing gliomatoxic agent with strongest effects on migration and invasive growth. Thus, our report uncovered cabazitaxel actions on gliomas and on the brain tumor microenvironment. These data reveal novel aspects for adjuvant approaches when applied to brain tumor patients. PMID:27203678

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.

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

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.

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.

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.

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...

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2006-04-01

replication in yeast cells. In the prior reporting period we demonstrated that re-replication induces a rapid and significant decrease in cell viability...repair, DNA replication, checkpoint, cell cycle, yeast , RAD9 16. SECURITY CLASSIFICATION OF: 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON...initiation, our laboratory has been able to conditionally induce varying amounts of re- replication in yeast cells. Effectively, cells enter, but do not

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.

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.

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.

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain

PubMed Central

Lintner, Nathanael G.; McClure, Kim F.; Petersen, Donna; Londregan, Allyn T.; Piotrowski, David W.; Wei, Liuqing; Xiao, Jun; Bolt, Michael; Loria, Paula M.; Maguire, Bruce; Geoghegan, Kieran F.; Huang, Austin; Rolph, Tim; Liras, Spiros; Doudna, Jennifer A.; Dullea, Robert G.

2017-01-01

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts. PMID:28323820

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

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...

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 A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription

PubMed Central

Shimi, Takeshi; Pfleghaar, Katrin; Kojima, Shin-ichiro; Pack, Chan-Gi; Solovei, Irina; Goldman, Anne E.; Adam, Stephen A.; Shumaker, Dale K.; Kinjo, Masataka; Cremer, Thomas; Goldman, Robert D.

2008-01-01

The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A- and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS). Silencing lamin B1 (LB1) expression dramatically increases the lamina meshwork size and the mobility of nucleoplasmic lamin A (LA). The changes in lamina mesh size are coupled to the formation of LA/C-rich nuclear envelope blebs deficient in LB2. Comparative genomic hybridization (CGH) analyses of microdissected blebs, fluorescence in situ hybridization (FISH), and immunofluorescence localization of modified histones demonstrate that gene-rich euchromatin associates with the LA/C blebs. Enrichment of hyperphosphorylated RNA polymerase II (Pol II) and histone marks for active transcription suggest that blebs are transcriptionally active. However, in vivo labeling of RNA indicates that transcription is decreased, suggesting that the LA/C-rich microenvironment induces promoter proximal stalling of Pol II. We propose that different lamins are organized into separate, but interacting, microdomains and that LB1 is essential for their organization. Our evidence suggests that the organization and regulation of chromatin are influenced by interconnections between these lamin microdomains. PMID:19141474

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.

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...

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.

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

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.

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.

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

Mms1 is an assistant for regulating G-quadruplex DNA structures.

PubMed

Schwindt, Eike; Paeschke, Katrin

2018-06-01

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

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.

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

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

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.

An experimental study of static and oscillating rotor blade sections in reverse flow

NASA Astrophysics Data System (ADS)

Lind, Andrew Hume

The rotorcraft community has a growing interest in the development of high-speed helicopters to replace outdated fleets. One barrier to the design of such helicopters is the lack of understanding of the aerodynamic behavior of retreating rotor blades in the reverse flow region. This work considers two fundamental models of this complex unsteady flow regime: static and oscillating (i.e., pitching) airfoils in reverse flow. Wind tunnel tests have been performed at the University of Maryland (UMD) and the United States Naval Academy (USNA). Four rotor blade sections are considered: two featuring a sharp geometric trailing edge (NACA 0012 and NACA 0024) and two featuring a blunt geometric trailing edge (ellipse and cambered ellipse). Static airfoil experiments were performed at angles of attack through 180 deg and Reynolds numbers up to one million, representative of the conditions found in the reverse flow region of a full-scale high-speed helicopter. Time-resolved velocity field measurements were used to identify three unsteady flow regimes: slender body vortex shedding, turbulent wake, and deep stall vortex shedding. Unsteady airloads were measured in these three regimes using unsteady pressure transducers. The magnitude of the unsteady airloads is high in the turbulent wake regime when the separated shear layer is close to the airfoil surface and in deep stall due to periodic vortex-induced flow. Oscillating airfoil experiments were performed on a NACA 0012 and cambered ellipse to investigate reverse flow dynamic stall characteristics by modeling cyclic pitching kinematics. The parameter space spanned three Reynolds numbers (165,000; 330,000; and 500,000), five reduced frequencies between 0.100 and 0.511, three mean pitch angles (5,10, and 15 deg), and two pitch amplitudes (5 deg and 10 deg). The sharp aerodynamic leading edge of the NACA 0012 airfoil forces flow separation resulting in deep dynamic stall. The number of associated vortex structures depends strongly on pitching kinematics. The cambered ellipse exhibits light reverse flow dynamic stall for a wide range of pitching kinematics. Deep dynamic stall over the cambered ellipse airfoil is observed for high mean pitch angles and pitch amplitudes. The detailed results and analysis in this work contributes to the development of a new generation of high-speed helicopters.

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

PubMed Central

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

2015-01-01

ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. PMID:26699641

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.

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2001-05-01

We are interested in the molecular mechanisms involved in DNA replication arrest by the S phase DNA damage checkpoints. Using in vitro simian virus...40 DNA replication assays, we have found three factors that directly contribute to DNA damage-induced DNA replication arrest: Replication Protein A...trans-acting inhibitors. RPA is the major eukaryotic single-stranded DNA binding protein required for DNA replication , repair and recombination. Upon DNA

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.

Quiet airfoils for small and large wind turbines

DOEpatents

Tangler, James L [Boulder, CO; Somers, Dan L [Port Matilda, PA

2012-06-12

Thick airfoil families with desirable aerodynamic performance with minimal airfoil induced noise. The airfoil families are suitable for a variety of wind turbine designs and are particularly well-suited for use with horizontal axis wind turbines (HAWTs) with constant or variable speed using pitch and/or stall control. In exemplary embodiments, a first family of three thick airfoils is provided for use with small wind turbines and second family of three thick airfoils is provided for use with very large machines, e.g., an airfoil defined for each of three blade radial stations or blade portions defined along the length of a blade. Each of the families is designed to provide a high maximum lift coefficient or high lift, to exhibit docile stalls, to be relatively insensitive to roughness, and to achieve a low profile drag.

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.

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2018-03-01

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

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.

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.

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.

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.

Correlated waves of actin filaments and PIP3 in Dictyostelium cells.

PubMed

Asano, Yukako; Nagasaki, Akira; Uyeda, Taro Q P

2008-12-01

Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity. Copyright 2008 Wiley-Liss, Inc.

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.

The Role of the Transcriptional Response to DNA Replication Stress

PubMed Central

Herlihy, Anna E.; de Bruin, Robertus A.M.

2017-01-01

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage. PMID:28257104

The Role of the Transcriptional Response to DNA Replication Stress.

PubMed

Herlihy, Anna E; de Bruin, Robertus A M

2017-03-02

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage.

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.

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

PubMed

Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

2016-01-01

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

Increased global transcription activity as a mechanism of replication stress in cancer

PubMed Central

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M.; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-01-01

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer. PMID:27725641

Increased global transcription activity as a mechanism of replication stress in cancer.

PubMed

Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

2016-10-11

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRAS V12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRAS V12 , elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

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.

Biomimetics and Tubercles on Flippers for Hydrodynamic Flow Control

NASA Astrophysics Data System (ADS)

Fish, Frank E.

2011-11-01

The biomimetic approach seeks to incorporate designs based on biological organisms into engineered technologies. Biomimetics can be used to engineer machines that emulate the performance of organisms, particularly in instances where the organism's performance exceeds current mechanical technology or provides new directions to solve existing problems. The ability to control the flow of water around the body dictates the performance of marine mammals in the aquatic environment. Morphological specializations of marine mammals afford mechanisms for passive flow control. Aside from the design of the body, which minimizes drag, the morphology of the appendages provide hydrodynamic advantages with respect to drag, lift, thrust, and stall. Of particular interest are the pectoral flippers of the humpback whale (Megaptera novaeangliae). These flippers act as wing-like structures to provide hydrodynamic lift for maneuvering. The use of any such wing-like structure in making small radius turns to enhance both agility and maneuverability is constrained by performance associated with stall. Delay of stall can be accomplished passively by modification of the flipper leading edge. The design of the flippers includes prominent leading edge bumps or tubercles. Such a design is exhibited by the leading edge tubercles on the flippers of humpback whales. These novel morphological structures induce a spanwise flow field of separated vortices alternating with regions of accelerated flow. The coupled flow regions maintain areas of attached flow and delay stall to high angles of attack. The morphological features of humpback whales for flow control can be utilized in the biomimetic design of engineered structures and commercial products for increased hydrodynamic performance. Nature retains a store of untouched knowledge, which would be beneficial in advancing technology.

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.

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

PubMed Central

Armentrout, Richard W.; Rutberg, Lars

1971-01-01

A temperature-inducible mutant of temperate Bacillus bacteriophage φ105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in “mock-induced” wild-type φ105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome. PMID:5002012

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.

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.

SeMet attenuates OTA-induced PCV2 replication promotion by inhibiting autophagy by activating the AKT/mTOR signaling pathway.

PubMed

Qian, Gang; Liu, Dandan; Hu, Junfa; Gan, Fang; Hou, Lili; Zhai, Nianhui; Chen, Xingxiang; Huang, Kehe

2018-02-13

Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. PCV2 replication could be promoted by low doses of ochratoxin A (OTA) as in our previous study and selenium has been shown to attenuate PCV2 replication. However, the underlying mechanism remains unclear. The aim of the study was to investigate the effects of selenomethionine (SeMet), the major component of organic selenium, on OTA-induced PCV2 replication promotion and its potential mechanism. The present study demonstrates that OTA could promote PCV2 replication as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. In addition, OTA could activate autophagy as indicated by up-regulated light chain 3 (LC3)-II and autophagy-related protein 5 expressions and autophagosome formation. Further, OTA could down-regulate p-AKT and p-mTOR expressions and OTA-induced autophagy was inhibited when insulin was applied. SeMet at 2, 4 and 6 μM had significant inhibiting effects against OTA-induced PCV2 replication promotion. Furthermore, SeMet could attenuate OTA-induced autophagy and up-regulate OTA-induced p-AKT and p-mTOR expression inhibition. Rapamycin, an inhibitor of AKT/mTOR, could reverse the effects of SeMet on OTA-induced autophagy and the PCV2 replication promotion. In conclusion, SeMet could block OTA-induced PCV2 replication promotion by inhibiting autophagy by activating the AKT/mTOR pathway. Therefore, SeMet supplementation could be an effective prophylactic strategy against PCV2 infections and autophagy may be a potential marker to develop novel anti-PCV2 drugs.

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.

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 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.

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%.

Collapsing white dwarfs

NASA Technical Reports Server (NTRS)

Baron, E.; Cooperstein, J.; Kahana, S.; Nomoto, K.

1987-01-01

The results of the hydrodynamic collapse of an accreting C + O white dwarf are presented. Collapse is induced by electron captures in the iron core behind a conductive deflagration front. The shock wave produced by the hydrodynamic bounce of the iron core stalls at about 115 km, and thus a neutron star formed in such a model would be formed as an optically quiet event.

Predicted and experimental aerodynamic forces on the Darrieus rotor

NASA Astrophysics Data System (ADS)

Paraschivoiu, I.

1983-12-01

The present paper compares the aerodynamic loads predicted by a double-multiple-streamtube model with wind tunnel measurements for a straight-bladed Darrieus rotor. Thus the CARDAA computer code uses two constant-interference factors in the induced velocity for estimating the aerodynamic loads. This code has been improved by considering the variation in the upwind and downwind induced velocities as a function of the blade position, and, in this case, the CARDAAV code is used. The Boeing-Vertol dynamic-stall model is incorporated in both the CARDAA and CARDAAV codes, and a better approach is obtained. The transient normal- and tangential-force coefficients predicted with and without dynamic-stall effects are compared with wind tunnel data for one and two NACA 0018 straight-bladed rotors. The results are given for a rotor with a large solidity (chord-to-radius ratio of 0.20) at two tip-speed ratios (X = 1.5 and 3.0) and at a low Reynolds number of 3.8 x 10 to the 4th. The comparisons between experimental data and theoretical results show the CARDAAV predictions to be more accurate than those estimated by the CARDAA code.

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

Pirrung, Georg; Madsen, Helge; Schreck, Scott

Current fast aeroelastic wind turbine codes suitable for certification lack an induction model for standstill conditions. A trailed vorticity model previously used as addition to a blade element momentum theory based aerodynamic model in normal operation has been extended to allow computing the induced velocities in standstill. The model is validated against analytical results for an elliptical wing in constant inflow and against stand still measurements from the NREL/NASA Phase VI unsteady experiment. The extended model obtains good results in case of the elliptical wing, but underpredicts the steady loading for the Phase VI blade in attached flow. The predictionmore » of the dynamic force coefficient loops from the Phase VI experiment is improved by the trailed vorticity modeling in both attached flow and stall in most cases. The exception is the tangential force coefficient in stall, where the codes and measurements deviate and no clear improvement is visible.« less

Trailed vorticity modeling for aeroelastic wind turbine simulations in stand still

DOE PAGES

Pirrung, Georg; Madsen, Helge; Schreck, Scott

2016-10-03

Current fast aeroelastic wind turbine codes suitable for certification lack an induction model for standstill conditions. A trailed vorticity model previously used as addition to a blade element momentum theory based aerodynamic model in normal operation has been extended to allow computing the induced velocities in standstill. The model is validated against analytical results for an elliptical wing in constant inflow and against stand still measurements from the NREL/NASA Phase VI unsteady experiment. The extended model obtains good results in case of the elliptical wing, but underpredicts the steady loading for the Phase VI blade in attached flow. The predictionmore » of the dynamic force coefficient loops from the Phase VI experiment is improved by the trailed vorticity modeling in both attached flow and stall in most cases. The exception is the tangential force coefficient in stall, where the codes and measurements deviate and no clear improvement is visible.« less

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

PubMed Central

Baars, Destiny L.; Pelegri, Francisco

2016-01-01

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species. PMID:28060351

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.

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.

Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Inducible Factor-Dependent Extension of the Replicative Life Span during Hypoxia▿

PubMed Central

Bell, Eric L.; Klimova, Tatyana A.; Eisenbart, James; Schumacker, Paul T.; Chandel, Navdeep S.

2007-01-01

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span. PMID:17562866

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.

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

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.

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

PubMed

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

2015-12-23

High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Ursolic Acid-Regulated Energy Metabolism—Reliever or Propeller of Ultraviolet-Induced Oxidative Stress and DNA Damage?

PubMed Central

Lee, Yuan-Hao; Sun, Youping; Glickman, Randolph D.

2014-01-01

Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts (e.g., pyrimidine dimers) and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR (mammalian target of rapamycin), whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic acid (UA), which results in the metabolic adaptation of normal cells against UV-induced ROS, and the metabolic switch of tumor cells subject to UV-induced damage. The multifaceted natural compound, UA, specifically inhibits photo-oxidative DNA damage in retinal pigment epithelial cells while enhancing that in skin melanoma. Considering the UA-mediated differential effects on cell bioenergetics, this article reviews the disparities in glucose metabolism between tumor and normal cells, along with (peroxisome proliferator-activated receptor-γ coactivator 1α)-dependent mitochondrial metabolism and redox (reduction-oxidation) control to demonstrate UA-induced synthetic lethality in tumor cells. PMID:28250388

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...

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

Journal of Engineering Thermophysics (Selected Articles),

DTIC Science & Technology

1983-05-13

compressor, prediction of unsteady vibration , and prevention of unsteady vibration . This test was undergone on a turbojet engine. The paper stresses the...induce unsteady engine vibration . While studying the effect of inlet anomaly and variation of the first stage nozzle area of the turbine, the engine...constant revolution speed curve until unsteady vibration or stall appeared. In studying the influence of the starting sequence, starting was

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...

Effect of timing of relocation of replacement gilts from group pens to individual stalls before breeding on fertility and well-being.

PubMed

Knox, R V; Shen, J; Greiner, L L; Connor, J F

2016-12-01

Variation in gilt fertility is associated with increased replacement and reduced longevity. Stress before breeding is hypothesized to be involved in reduced fertility. This study tested the timing of gilt relocation from pens to individual stalls before breeding on fertility and well-being. The experiment was performed in replicates on a commercial research farm. After detection of first estrus, gilts ( = 563) were assigned to treatment for relocation into stalls 3 wk (REL3wk), 2 wk (REL2wk), or 1 wk (REL1wk) before breeding at second estrus. Subsets of gilts from each treatment ( = 60) were selected for assessment of follicles at second estrus. Data included interestrus interval, number of services, conception, farrowing, total born, and wean to service interval. Piglet birth weight was obtained on subsets of litters ( = 42/treatment). Measures of well-being included BW, backfat, BCS, lesions, and lameness from wk 1 after first estrus until wk 16. Gilt BW at wk 5 (158.4 kg) was not affected ( > 0.10) by treatment. Measures of BCS, lameness, and lesions at breeding and throughout gestation did not differ ( > 0.10). Treatment did not affect ( > 0.10) gilts expressing a normal interestrus interval of 18 to 24 d (83.4%) but did influence ( < 0.05) the proportion expressing shorter ( < 0.001) and longer ( < 0.001) intervals. Gilts in REL3wk had a shorter ( < 0.001) interestrus interval (20.7 d) than those in REL2wk and REL1wk (22.6 d). Gilts with shorter intervals ( = 24) had fewer total born while gilts expressing longer cycles ( = 65) had reduced farrowing rates. The number of services (1.9) and number of follicles (19.7) at breeding were not affected ( > 0.10) by relocation. There was no effect of treatment on farrowing rate (85.2%), born alive (12.6), or any litter birth weight measures ( > 0.10). The percentage of sows bred within 7 d after weaning (94.4%) was also not affected by treatment ( > 0.10). These results suggest that the timing of relocation before breeding had no effect on well-being or on the majority of gilts with normal estrous cycles and their subsequent fertility. However, a smaller proportion of the gilts exhibited shorter and longer interestrus intervals in response to relocation 1 or 3 wk before breeding. In cases where gilt fertility may be less than optimal, producers that relocate gilts from pens to stalls before breeding should evaluate interestrus interval as a response criterion.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

PubMed

Alejo, Jose L; Blanchard, Scott C

2017-10-10

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome

PubMed Central

Alejo, Jose L.; Blanchard, Scott C.

2017-01-01

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

DNA damage mediated transcription arrest: Step back to go forward.

PubMed

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Selenizing astragalus polysaccharide attenuates PCV2 replication promotion caused by oxidative stress through autophagy inhibition via PI3K/AKT activation.

PubMed

Liu, Dandan; Xu, Jing; Qian, Gang; Hamid, Mohammed; Gan, Fang; Chen, Xingxiang; Huang, Kehe

2018-03-01

Our previous studies have shown that oxidative stress could promote the porcine circovirus type 2 (PCV2) replication, and astragalus polysaccharide (APS)/selenium could suppress PCV2 replication. However, whether selenizing astragalus polysaccharide (sAPS) provides protection against oxidative stress-induced PCV2 replication promotion and the mechanism involved remain unclear. The present study aimed to explore the mechanism of the PCV2 replication promotion induced by oxidative stress and a novel pharmacotherapeutic approach involving the regulation of autophagy of sAPS. Our results showed that H 2 O 2 promoted PCV2 replication via enhancing autophagy by using 3-methyladenine (3-MA) and autophagy-related gene 5 (ATG5) knockdown. Sodium selenite, APS, the mixture of sodium selenite and APS, and sAPS significantly inhibited H 2 O 2 -induced PCV2 replication promotion, respectively. Among these, sAPS exerted maximal inhibitory effect. sAPS could also significantly inhibit autophagy activated by H 2 O 2 and increase the Akt and mTOR phosphorylation. Moreover, LY294002, the specific phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor, significantly alleviated the effects of sAPS on autophagy and PCV2 replication. Taken together, we conclude that H 2 O 2 promotes PCV2 replication by inducing autophagy and sAPS attenuates the PCV2 replication promotion through autophagy inhibition via PI3K/AKT activation. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

PubMed Central

Vyas, Rajan; Efthimiopoulos, Georgia; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2015-01-01

1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism. PMID:26327169

Stress-induced premature senescence (SIPS)--influence of SIPS on radiotherapy.

PubMed

Suzuki, Masatoshi; Boothman, David A

2008-03-01

Replicative senescence is a fundamental feature in normal human diploid cells and results from dysfunctional telomeres at the Hayflick cell division limit. Ionizing radiation (IR) prematurely induces the same phenotypes as replicative senescence prior to the Hayflick limit. This process is known as stress-induced premature senescence (SIPS). Since the cell cycle is irreversibly arrested in SIPS-induced cells, even if they are stimulated by various growth factors, it is thought that SIPS is a form of cell death, irreversibly eliminating replicating cells. IR-induced-focus formation of DNA repair proteins, a marker of DNA damage, is detected in SIPS as well as replicative senescent cells. Furthermore, both processes persistently induce cell cycle checkpoint mechanisms, indicating DNA damage created by ionizing radiation induces SIPS in normal cells, possibly by the same mechanisms as those occurring in replicative senescence. Interestingly, IR induces SIPS not only in normal cells, but also in tumor cells. Due to the expression of telomerase in tumor cells, telomere-dependent replicative senescence does not occur. However, SIPS is induced under certain conditions after IR exposure. Thus, cell death triggered by IR can be attributed to apoptosis or SIPS in tumor cells. However, metabolic function remains intact in SIPS-induced cancer cells, and recent studies show that senescence eliminate cells undergoing SIPS secrete various kinds of factors outside the cell, changing the microenvironment. Evidence using co-culture systems containing normal senescent stromal cells and epithelial tumor cells show that factors secreted from senescent stroma cells promote the growth of tumor epithelial cells both in vitro and in vivo. Thus, regulation of factors secreted from SIPS-induced stromal cells, as well as tumor cells, may affect radiotherapy.

In Vitro Lesion Bypass Studies of O(4)-Alkylthymidines with Human DNA Polymerase η.

PubMed

Williams, Nicole L; Wang, Pengcheng; Wu, Jiabin; Wang, Yinsheng

2016-04-18

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O(4)-alkylthymidine (O(4)-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O(4)-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O(4)-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O(4)-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O(4)-iPrdT, O(4)-iBudT, and O(4)-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O(4)-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

PubMed Central

Kirouac, Kevin N.; Basu, Ashis K.; Ling, Hong

2013-01-01

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. PMID:23876706

Structural mechanism of replication stalling on a bulky amino-polycyclic aromatic hydrocarbon DNA adduct by a y family DNA polymerase.

PubMed

Kirouac, Kevin N; Basu, Ashis K; Ling, Hong

2013-11-15

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. © 2013.

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

PubMed Central

Kirouac, Kevin N.; Ling, Hong

2011-01-01

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι’s biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O8 atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase. PMID:21300901

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...

Design Information for Civil Works Housing.

DTIC Science & Technology

1984-01-01

tmm GUIDANCE Lawn Mower , Garden Equipment, Bicycles, etc. 20 ft ^ r" T STORAGE 1-STALL OARAGE 2-STALL QARAQE I I c COMMENTARY !A. Al A...Weatherproof 110-V outlets (as required per ap- plicable code). V. J 17 GUIDANCE Lawn Mower , Garden Equipment, and Bicycles 1-STALL CARPORT

Clutch-Starting Stalled Research Students

ERIC Educational Resources Information Center

Ahern, Kathy; Manathunga, Catherine

2004-01-01

Many research students go through periods where their research seems to stall, their motivation drops, and they seem unable to make any progress. As supervisors, we attempt to remain alert to signs that our student's progress has stalled. Drawing on cognitive strategies, this article explores a problem-solving model supervisors can use to identify…

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.

Analysis of Low-Speed Stall Aerodynamics of a Business Jets Wing Using STAR-CCM+

NASA Technical Reports Server (NTRS)

Bui, Trong

2016-01-01

Reynolds-Averaged Navier-Stokes (RANS) computational fluid dynamics (CFD) analysis was conducted: to study the low-speed stall aerodynamics of a GIII aircrafts swept wing modified with (1) a laminar-flow wing glove, or (2) a seamless flap. The stall aerodynamics of these two different wing configurations were analyzed and compared with the unmodified baseline wing for low-speed flight. The Star-CCM+ polyhedral unstructured CFD code was first validated for wing stall predictions using the wing-body geometry from the First AIAA CFD High-Lift Prediction Workshop.

Post-Stall Aerodynamic Modeling and Gain-Scheduled Control Design

NASA Technical Reports Server (NTRS)

Wu, Fen; Gopalarathnam, Ashok; Kim, Sungwan

2005-01-01

A multidisciplinary research e.ort that combines aerodynamic modeling and gain-scheduled control design for aircraft flight at post-stall conditions is described. The aerodynamic modeling uses a decambering approach for rapid prediction of post-stall aerodynamic characteristics of multiple-wing con.gurations using known section data. The approach is successful in bringing to light multiple solutions at post-stall angles of attack right during the iteration process. The predictions agree fairly well with experimental results from wind tunnel tests. The control research was focused on actuator saturation and .ight transition between low and high angles of attack regions for near- and post-stall aircraft using advanced LPV control techniques. The new control approaches maintain adequate control capability to handle high angle of attack aircraft control with stability and performance guarantee.

Experimental evaluation of a TF30-P-3 turbofan engine in an altitude facility: Effect of steady-state temperature distortion

NASA Technical Reports Server (NTRS)

Braithwaite, W. M.

1973-01-01

The effects of circumferential distortion of the total temperature entering 25, 50, and 75 percent of the inlet circumferential annulus of a turbofan engine were determined. Complete compressor stall resulted from distortions of from 14 to 20 percent of the face averaged temperature. Increasing the temperature level in one sector resulted in that sector moving toward stall by decreasing the equivalent rotor speeds while the pressure ratio remained approximately constant. Stall originated as a rotating zone in the low-pressure compressor which resulted as a terminal stall in the high-pressure compressor. Decreasing the Reynolds number index to 0.25 from 0.5 reduced the required distortion for stall by 50 percent for the conditions investigated.

Analysis of the cycle-to-cycle pressure distribution variations in dynamic stall

NASA Astrophysics Data System (ADS)

Harms, Tanner; Nikoueeyan, Pourya; Naughton, Jonathan

2017-11-01

Dynamic stall is an unsteady flow phenomenon observed on blades and wings that, despite decades of focused study, remains a challenging problem for rotorcraft and wind turbine applications. Traditionally, dynamic stall has been studied on pitch-oscillating airfoils by measuring the unsteady pressure distribution that is phase-averaged, by which the typical flow pattern may be observed and quantified. In cases where light to deep dynamic stall are observed, pressure distributions with high levels of variance are present in regions of separation. It was recently observed that, under certain conditions, this scatter may be the result of a two-state flow solution - as if there were a bifurcation in the unsteady pressure distribution behavior on the suction side of the airfoil. This is significant since phase-averaged dynamic stall data are often used to tune dynamic stall models and for validation of simulations of dynamic stall. In order to better understand this phenomenon, statistical analysis of the pressure data using probability density functions (PDFs) and other statistical approaches has been carried out for the SC 1094R8, DU97-W-300, and NACA 0015 airfoil geometries. 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.

Unsteady aerodynamics of reverse flow dynamic stall on an oscillating blade section

NASA Astrophysics Data System (ADS)

Lind, Andrew H.; Jones, Anya R.

2016-07-01

Wind tunnel experiments were performed on a sinusoidally oscillating NACA 0012 blade section in reverse flow. Time-resolved particle image velocimetry and unsteady surface pressure measurements were used to characterize the evolution of reverse flow dynamic stall and its sensitivity to pitch and flow parameters. The effects of a sharp aerodynamic leading edge on the fundamental flow physics of reverse flow dynamic stall are explored in depth. Reynolds number was varied up to Re = 5 × 105, reduced frequency was varied up to k = 0.511, mean pitch angle was varied up to 15∘, and two pitch amplitudes of 5∘ and 10∘ were studied. It was found that reverse flow dynamic stall of the NACA 0012 airfoil is weakly sensitive to the Reynolds numbers tested due to flow separation at the sharp aerodynamic leading edge. Reduced frequency strongly affects the onset and persistence of dynamic stall vortices. The type of dynamic stall observed (i.e., number of vortex structures) increases with a decrease in reduced frequency and increase in maximum pitch angle. The characterization and parameter sensitivity of reverse flow dynamic stall given in the present work will enable the development of a physics-based analytical model of this unsteady aerodynamic phenomenon.

The Error-Prone DNA Polymerase κ Promotes Temozolomide Resistance in Glioblastoma through Rad17-Dependent Activation of ATR-Chk1 Signaling.

PubMed

Peng, Chenghao; Chen, Zhengxin; Wang, Shuai; Wang, Hong-Wei; Qiu, Wenjin; Zhao, Lin; Xu, Ran; Luo, Hui; Chen, Yuanyuan; Chen, Dan; You, Yongping; Liu, Ning; Wang, Huibo

2016-04-15

The acquisition of drug resistance is a persistent clinical problem limiting the successful treatment of human cancers, including glioblastoma (GBM). However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. In this study, we report that Pol κ, an error-prone polymerase that participates in translesion DNA synthesis, was significantly upregulated in GBM cell lines and tumor tissues following temozolomide treatment. Overexpression of Pol κ in temozolomide-sensitive GBM cells conferred resistance to temozolomide, whereas its inhibition markedly sensitized resistant cells to temozolomide in vitro and in orthotopic xenograft mouse models. Mechanistically, depletion of Pol κ disrupted homologous recombination (HR)-mediated repair and restart of stalled replication forks, impaired the activation of ATR-Chk1 signaling, and delayed cell-cycle re-entry and progression. Further investigation of the relationship between Pol κ and temozolomide revealed that Pol κ inactivation facilitated temozolomide-induced Rad17 ubiquitination and proteasomal degradation, subsequently silencing ATR-Chk1 signaling and leading to defective HR repair and the reversal of temozolomide resistance. Moreover, overexpression of Rad17 in Pol κ-depleted GBM cells restored HR efficiency, promoted the clearance of temozolomide-induced DNA breaks, and desensitized cells to the cytotoxic effects of temozolomide observed in the absence of Pol κ. Finally, we found that Pol κ overexpression correlated with poor prognosis in GBM patients undergoing temozolomide therapy. Collectively, our findings identify a potential mechanism by which GBM cells develop resistance to temozolomide and suggest that targeting the DNA damage tolerance pathway may be beneficial for overcoming resistance. Cancer Res; 76(8); 2340-53. ©2016 AACR. ©2016 American Association for Cancer Research.

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.

PubMed

Ireno, Ivanildce C; Baumann, Cindy; Stöber, Regina; Hengstler, Jan G; Wiesmüller, Lisa

2014-05-01

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

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.

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

PubMed

Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

2016-04-07

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

Optimism and education buffer the effects of syndemic conditions on HIV status among African American men who have sex with men.

PubMed

O'Leary, Ann; Jemmott, John B; Stevens, Robin; Rutledge, Scott Edward; Icard, Larry D

2014-11-01

The present study sought to replicate effects of the number of syndemic psychosocial health conditions on sexual risk behavior and HIV infection among a sample of high-risk African American men who have sex with men (MSM) and to identify resilience factors that may buffer these effects. We used baseline data from an HIV risk-reduction trial to examine whether a higher number of syndemic conditions was associated with higher rates of self-reported sexual risk behavior and HIV infection. Using logistic regression models, we tested for interactions between number of syndemic conditions and several potential resilience factors to identify buffering effects. Replicating previous studies, we found significant associations between numbers of syndemic conditions and higher rates of sexual risk behavior and HIV infection. Surprisingly, we also replicated a previous finding (Stall et al., Am J Public Health, 93(6):939-942, 2003) that the effects of syndemic burden on HIV status fell off at the highest levels of syndemic conditions. Among a variety of potential resilience factors, two-optimism and education-buffered the syndemic effect on HIV prevalence. This is, to our knowledge, the first paper to identify resilience factors buffering against syndemic effects among MSM. It also constitutes a significant contribution to the literature regarding prevention among black MSM. These results point to the need to identify HIV-positive black MSM and provide effective treatment for them and to develop interventions addressing both syndemic and resilience factors.

pelo Is Required for High Efficiency Viral Replication

PubMed Central

Wu, Xiurong; He, Wan-Ting; Tian, Shuye; Meng, Dan; Li, Yuanyue; Chen, Wanze; Li, Lisheng; Tian, Lili; Zhong, Chuan-Qi; Han, Felicia; Chen, Jianming; Han, Jiahuai

2014-01-01

Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection. PMID:24722736

Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes.

PubMed

Zhang, Zhenfeng; Filzmayer, Christina; Ni, Yi; Sültmann, Holger; Mutz, Pascal; Hiet, Marie-Sophie; Vondran, Florian W R; Bartenschlager, Ralf; Urban, Stephan

2018-07-01

Hepatitis B virus (HBV) and D virus (HDV) co-infections cause the most severe form of viral hepatitis. HDV induces an innate immune response, but it is unknown how the host cell senses HDV and if this defense affects HDV replication. We aim to characterize interferon (IFN) activation by HDV, identify the responsible sensor and evaluate the effect of IFN on HDV replication. HDV and HBV susceptible hepatoma cell lines and primary human hepatocytes (PHH) were used for infection studies. Viral markers and cellular gene expression were analyzed at different time points after infection. Pattern recognition receptors (PRRs) required for HDV-mediated IFN activation and the impact on HDV replication were studied using stable knock-down or overexpression of the PRRs. Microarray analysis revealed that HDV but not HBV infection activated a broad range of interferon stimulated genes (ISGs) in HepG2 NTCP cells. HDV strongly activated IFN-β and IFN-λ in cell lines and PHH. HDV induced IFN levels remained unaltered upon RIG-I (DDX58) or TLR3 knock-down, but were almost completely abolished upon MDA5 (IFIH1) depletion. Conversely, overexpression of MDA5 but not RIG-I and TLR3 in HuH7.5 NTCP cells partially restored ISG induction. During long-term infection, IFN levels gradually diminished in both HepG2 NTCP and HepaRG NTCP cell lines. MDA5 depletion had little effect on HDV replication despite dampening HDV-induced IFN response. Moreover, treatment with type I or type III IFNs did not abolish HDV replication. Active replication of HDV induces an IFN-β/λ response, which is predominantly mediated by MDA5. This IFN response and exogenous IFN treatment have only a moderate effect on HDV replication in vitro indicating the adaption of HDV replication to an IFN-activated state. In contrast to hepatitis B virus, infection with hepatitis D virus induces a strong IFN-β/λ response in innate immune competent cell lines. MDA5 is the key sensor for the recognition of hepatitis D virus replicative intermediates. An IFN-activated state did not prevent hepatitis D virus replication in vitro, indicating that hepatitis D virus is resistant to self-induced innate immune responses and therapeutic IFN treatment. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Bedding on geotextile mattresses: how much is needed to improve cow comfort?

PubMed

Tucker, C B; Weary, D M

2004-09-01

The objective of our study was to evaluate how the amount of sawdust bedding on mattresses affects dairy cattle behavior and preferences. Eleven nonlactating, multiparous cows were housed individually in pens with access to 3 free stalls. Each stall was fitted with a geotextile mattress covered with either 0, 1, or 7.5 kg of kiln-dried sawdust. The experiment began with 7 d of acclimatization to all 3 stalls. Cows were then allowed access to only 1 of the 3 stalls at a time, each for 3 d (restriction phase). At the end of this restriction phase, cows were allowed free access to all 3 stalls for 3 d (free-choice phase). Time spent lying and the number of lying bouts increased significantly with the amount of bedding, from 12.3 +/- 0.53 h lying and 8.5 +/- 0.62 bouts per 24 h on bare mattresses to 13.8 +/- 0.53 h lying and 10.0 +/- 0.62 bouts per 24 h on mattresses with 7.5 kg of sawdust. In addition, the animals spent less time standing with only the front hooves in the stalls when more sawdust was present. When allowed free access to all 3 options, all 11 animals spent a majority of their time lying and standing in the 7.5-kg option. In conclusion, cows preferred mattresses bedded with 7.5 kg of sawdust, on which they spent more time lying down and less time standing with only the front hooves in stalls. These results indicate that more sawdust bedding improves cow comfort in stalls with geotextile mattresses.

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.

Preliminary Results of the Determination of Inlet-Pressure Distortion Effects on Compressor Stall and Altitude Operating Limits of the J57-P-1 Turbojet Engine

NASA Technical Reports Server (NTRS)

Wallner, L. E.; Lubick, R. J.; Chelko, L. J.

1955-01-01

During an investigation of the J57-P-1 turbojet engine in the Lewis altitude wind tunnel, effects of inlet-flow distortion on engine stall characteristics and operating limits were determined. In addition to a uniform inlet-flow profile, the inlet-pressure distortions imposed included two radial, two circumferential, and one combined radial-circumferential profile. Data were obtained over a range of compressor speeds at an altitude of 50,000 and a flight Mach number of 0.8; in addition, the high- and low-speed engine operating limits were investigated up to the maximum operable altitude. The effect of changing the compressor bleed position on the stall and operating limits was determined for one of the inlet distortions. The circumferential distortions lowered the compressor stall pressure ratios; this resulted in less fuel-flow margin between steady-state operation and compressor stall. Consequently, the altitude operating Limits with circumferential distortions were reduced compared with the uniform inlet profile. Radial inlet-pressure distortions increased the pressure ratio required for compressor stall over that obtained with uniform inlet flow; this resulted in higher altitude operating limits. Likewise, the stall-limit fuel flows required with the radial inlet-pressure distortions were considerably higher than those obtained with the uniform inlet-pressure profile. A combined radial-circumferential inlet distortion had effects on the engine similar to the circumferential distortion. Bleeding air between the two compressors eliminated the low-speed stall limit and thus permitted higher altitude operation than was possible without compressor bleed.

Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

PubMed

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-06-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

14 CFR 25.203 - Stall characteristics.

Code of Federal Regulations, 2010 CFR

2010-01-01

... recovery and to regain control of the airplane. The maximum bank angle that occurs during the recovery may... controls, up to the time the airplane is stalled. No abnormal nose-up pitching may occur. The longitudinal control force must be positive up to and throughout the stall. In addition, it must be possible to...

14 CFR 25.203 - Stall characteristics.

Code of Federal Regulations, 2011 CFR

2011-01-01

... recovery and to regain control of the airplane. The maximum bank angle that occurs during the recovery may... controls, up to the time the airplane is stalled. No abnormal nose-up pitching may occur. The longitudinal control force must be positive up to and throughout the stall. In addition, it must be possible to...

Evaluating Classroom Interaction with the iPad®: An Updated Stalling's Tool

ERIC Educational Resources Information Center

MacKinnon, Gregory; Schep, Lourens; Borden, Lisa Lunney; Murray-Orr, Anne; Orr, Jeff; MacKinnon, Paula

2016-01-01

A large study of classrooms in the Caribbean context necessitated the use of a validated classroom observation tool. In practice, the paper-version Stalling's instrument (Stallings & Kaskowitz 1974) presented specific challenges with respect to (a) facile data collection and (b) qualitative observations of classrooms. In response to these…

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.

A theory of rotating stall of multistage axial compressors

NASA Technical Reports Server (NTRS)

Moore, F. K.

1983-01-01

A theoretical analysis was made of rotating stall in axial compressors of many stages, finding conditions for a permanent, straight-through traveling disturbance, with the steady compressor characteristic assumed known, and with simple lag processes ascribed to the flows in the inlet, blade passages, and exit regions. For weak disturbances, predicted stall propagation speeds agree well with experimental results. For a locally-parabolic compressor characteristic, an exact nonlinear solution is found and discussed. For deep stall, the stall-zone boundary is most abrupt at the trailing edge, as expected. When a complete characteristic having unstalling and reverse-flow features is adopted, limit cycles governed by a Lienard's equation are found. Analysis of these cycles yields predictions of recovery from rotating stall; a relaxation oscillation is found at some limiting flow coefficient, above which no solution exists. Recovery is apparently independent of lag processes in the blade passages, but instead depends on the lags originating in the inlet and exit flows, and also on the shape of the given characteristic diagram. Small external lags and tall diagrams favor early recovery. Implications for future research are discussed.

Analysis of Ribosome Stalling and Translation Elongation Dynamics by Deep Learning.

PubMed

Zhang, Sai; Hu, Hailin; Zhou, Jingtian; He, Xuan; Jiang, Tao; Zeng, Jianyang

2017-09-27

Ribosome stalling is manifested by the local accumulation of ribosomes at specific codon positions of mRNAs. Here, we present ROSE, a deep learning framework to analyze high-throughput ribosome profiling data and estimate the probability of a ribosome stalling event occurring at each genomic location. Extensive validation tests on independent data demonstrated that ROSE possessed higher prediction accuracy than conventional prediction models, with an increase in the area under the receiver operating characteristic curve by up to 18.4%. In addition, genome-wide statistical analyses showed that ROSE predictions can be well correlated with diverse putative regulatory factors of ribosome stalling. Moreover, the genome-wide ribosome stalling landscapes of both human and yeast computed by ROSE recovered the functional interplays between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particles and protein secondary structure formation. Overall, our study provides a novel method to complement the ribosome profiling techniques and further decipher the complex regulatory mechanisms underlying translation elongation dynamics encoded in the mRNA sequence. Copyright © 2017 Elsevier Inc. All rights reserved.

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

PubMed

Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

2016-12-15

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

Lameness and Claw Lesions of the Norwegian Red Dairy Cattle Housed in Free Stalls in Relation to Environment, Parity and Stage of Lactation

PubMed Central

Sogstad, ÅM; Fjeldaas, T; Østerås, O

2005-01-01

Approximately 88% of Norwegian dairy cattle are housed in tie stalls. Free stall housing for all dairy cattle will be implemented within 20 years. This means that the majority of existing stalls will be rebuilt in the near future. Fifty-seven free stall herds of the Norwegian Red breed were randomly selected and 1547 cows and 403 heifers were trimmed by 13 claw trimmers during the late winter and spring of 2002. The claw trimmers had been taught diagnosing and recording of claw lesions. Environment, management- and feeding routines were also recorded. Fifty-three herds had concrete slatted alleys while 4 had solid concrete. Thirty-five herds had concrete as a stall base, while 17 had rubber mats, 2 had wood and 3 had deep litter straw beds. The prevalence of lameness was 1.6% in hind claws. Models for lameness and claw lesions were designed to estimate the influence of different risk factors and to account for the cluster effects within herd and claw trimmer. Detected risk factors for lameness were: parity three and above and narrow cubicles; for heel horn erosions: lactation stage around 5–7 months after calving and solid concrete alleys; for haemorrhages of the white line: lactation stage around 3–5 months after calving and solid concrete alleys; for haemorrhages of the sole: parity one, lactation stage around 5–7 months after calving and short cubicles, for white line fissures: slatted concrete alleys; for asymmetrical claws: parities two and above and for corkscrewed claws: solid concrete alleys. The prevalence of lameness in heifers was low, however 29% had one or more claw lesions. Heifers that were housed in pens or free stalls had more heel-horn erosions, haemorrhages of the sole and white-line fissures than heifers in tie stalls. As new free stalls are being built, it is important to optimise the conditions for claw health. PMID:16398332

Rotating stall investigation of 0.72 hub-tip ratio single-stage compressor

NASA Technical Reports Server (NTRS)

Graham, Robert W; Prian, Vasily D

1954-01-01

The rotating stall characteristics of a 0.72 hub-tip ratio, single-stage compressor were investigated. The stage was a 14-inch-diameter replica of the fourth stage of an experimental multistage compressor. No similarity existed between the frequency and propagation rate of the stall patterns observed in the single-stage replica and those observed in the multistage compressor after the fourth stage. A fatigue failure of the rotor blades occurred during the testing which was attributed to a resonance between the stall frequency and the natural bending frequency of the blades.

Investigation of rotor blade element airloads for a teetering rotor in the blade stall regime

NASA Technical Reports Server (NTRS)

Dadone, L. U.; Fukushima, T.

1974-01-01

A model of a teetering rotor was tested in a low speed wind tunnel. Blade element airloads measured on an articulated model rotor were compared with the teetering rotor and showed that the teetering rotor is subjected to less extensive flow separation. Retreating blade stall was studied. Results show that stall, under the influence of unsteady aerodynamic effects, consists of four separate stall events, each associated with a vortex shed from the leading edge and sweeping over the upper surface of the rotor blade. Current rotor performance prediction methodology was evaluated through computer simulation.

Simulation Modeling Requirements for Loss-of-Control Accident Prevention of Turboprop Transport Aircraft

NASA Technical Reports Server (NTRS)

Crider, Dennis; Foster, John V.

2012-01-01

In-flight loss of control remains the leading contributor to aviation accident fatalities, with stall upsets being the leading causal factor. The February 12, 2009. Colgan Air, Inc., Continental Express flight 3407 accident outside Buffalo, New York, brought this issue to the forefront of public consciousness and resulted in recommendations from the National Transportation Safety Board to conduct training that incorporates stalls that are fully developed and develop simulator standards to support such training. In 2010, Congress responded to this accident with Public Law 11-216 (Section 208), which mandates full stall training for Part 121 flight operations. Efforts are currently in progress to develop recommendations on implementation of stall training for airline pilots. The International Committee on Aviation Training in Extended Envelopes (ICATEE) is currently defining simulator fidelity standards that will be necessary for effective stall training. These recommendations will apply to all civil transport aircraft including straight-wing turboprop aircraft. Government-funded research over the previous decade provides a strong foundation for stall/post-stall simulation for swept-wing, conventional tail jets to respond to this mandate, but turboprops present additional and unique modeling challenges. First among these challenges is the effect of power, which can provide enhanced flow attachment behind the propellers. Furthermore, turboprops tend to operate for longer periods in an environment more susceptible to ice. As a result, there have been a significant number of turboprop accidents as a result of the early (lower angle of attack) stalls in icing. The vulnerability of turboprop configurations to icing has led to studies on ice accumulation and the resulting effects on flight behavior. Piloted simulations of these effects have highlighted the important training needs for recognition and mitigation of icing effects, including the reduction of stall margins. This paper addresses simulation modeling requirements that are unique to turboprop transport aircraft and highlights the growing need for aerodynamic models suitable for stall training for these configurations. A review of prominent accidents that involved aerodynamic stall is used to illustrate various modeling features unique to turboprop configurations and the impact of stall behavior on susceptibility to loss of control that has led to new training requirements. This is followed by an overview of stability and control behavior of straight-wing turboprops, the related aerodynamic characteristics, and a summary of recent experimental studies on icing effects. In addition, differences in flight dynamics behavior between swept-wing jets and straight-wing turboprop configurations are discussed to compare and contrast modeling requirements. Specific recommendations for aerodynamic models along with further research needs and data measurements are also provided. 1

Effects of milking stall dimensions on behavior of dairy cows during milking in different milking parlor types.

PubMed

Gómez, Y; Terranova, M; Zähner, M; Hillmann, E; Savary, P

2017-02-01

Dairy cow body size has increased over time because of breeding selection for higher milk yield, but milking stall dimensions have never been adjusted and are based on the practical experience of milking-machine manufacturers and advisory institutions. Narrow, limited milking stall dimensions might lead to behavioral changes during milking due to lack of comfort. The aim of this study was to examine the current space allowance in milking stalls on dairy farms and assess the effect of space allowance on cow behavior during milking. On 15 Swiss dairy farms, we measured clear milking stall dimensions and cow body dimensions. We calculated space ratios for length (SR length ) and width (SR width ) by dividing the milking stall length or width by cow body length or belly width, respectively. When the space ratio was >1, we assumed that the body length or width of cow was smaller than the milking stall length or width. On each farm, 10 healthy cows were chosen for behavioral observation during 1 evening milking. We recorded rumination, elimination, and latency to enter the milking stall by direct observation. Hind leg activity was recorded using acceleration loggers. Data were analyzed using general linear mixed-effects models with farm as a random effect. Due to a strong collinearity between SR width and SR length , we chose SR length for further analysis, because it is based on skeletal characteristics. The SR length was smallest in side-by-side parlors (1.07 ± 0.01) and largest in tandem parlors (1.18 ± 0.01). More cows had a tendency to ruminate with increasing SR length (odds ratio: 1.8). None of hind leg activity, maximum peaks of hind leg accelerations, or latency to enter the milking stall were significantly affected by SR length . Latency to enter the milking stall was longer for group milking parlors (side-by-side: 44.0 ± 3.2 s; herringbone: 34.3 ± 2.9 s) than for tandem parlors (19.0 ± 2.7 s). Milking parlor type had no effect on hind leg activity, maximum peaks of hind leg accelerations or rumination. The SR length affected rumination behavior to some extent, indicating that cow comfort was positively affected by larger milking stall length. Because cow comfort is important for good milking performance, further investigations of milking stall dimensions for cow comfort and thus welfare are needed. Furthermore, the results showed that parlor type affected cow behavior, irrespective of SR length , making future research necessary to identify the factors leading to this effect of parlor type. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Mink parvoviruses and interferons: in vitro studies.

PubMed Central

Wiedbrauk, D L; Bloom, M E; Lodmell, D L

1986-01-01

Although interferons can inhibit the replication of a number of viruses, little is known about their ability to inhibit parvovirus replication. Therefore, in vitro experiments were done to determine if Aleutian disease virus and mink enteritis virus, two autonomously replicating mink parvoviruses, induced interferon, were sensitive to the effects of interferon, or inhibited the production of interferon. The results indicated that these parvoviruses neither induced nor were sensitive to the effects of interferon. Furthermore, preexisting parvovirus infections did not inhibit poly(I).poly(C)-induced interferon production. This independence from the interferon system may, therefore, be a general property of the autonomously replicating parvoviruses. PMID:2431162

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

PubMed Central

Osterman, Ilya A.; Komarova, Ekaterina S.; Shiryaev, Dmitry I.; Korniltsev, Ilya A.; Khven, Irina M.; Lukyanov, Dmitry A.; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Efremenkova, Olga V.; Ivanenkov, Yan A.; Bogdanov, Alexey A.; Dontsova, Olga A.

2016-01-01

In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals. PMID:27736765

Measurement of Flow Pattern Within a Rotating Stall Cell in an Axial Compressor

NASA Technical Reports Server (NTRS)

Lepicovsky, Jan; Braunscheidel, Edward P.

2006-01-01

Effective active control of rotating stall in axial compressors requires detailed understanding of flow instabilities associated with this compressor regime. Newly designed miniature high frequency response total and static pressure probes as well as commercial thermoanemometric probes are suitable tools for this task. However, during the rotating stall cycle the probes are subjected to flow direction changes that are far larger than the range of probe incidence acceptance, and therefore probe data without a proper correction would misrepresent unsteady variations of flow parameters. A methodology, based on ensemble averaging, is proposed to circumvent this problem. In this approach the ensemble averaged signals acquired for various probe setting angles are segmented, and only the sections for probe setting angles close to the actual flow angle are used for signal recombination. The methodology was verified by excellent agreement between velocity distributions obtained from pressure probe data, and data measured with thermoanemometric probes. Vector plots of unsteady flow behavior during the rotating stall regime indicate reversed flow within the rotating stall cell that spreads over to adjacent rotor blade channels. Results of this study confirmed that the NASA Low Speed Axial Compressor (LSAC) while in a rotating stall regime at rotor design speed exhibits one stall cell that rotates at a speed equal to 50.6 percent of the rotor shaft speed.

KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry.

PubMed

Betz, Karin; Malyshev, Denis A; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Dwyer, Tammy J; Ordoukhanian, Phillip; Romesberg, Floyd E; Marx, Andreas

2012-07-01

Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.

14 CFR 23.201 - Wings level stall.

Code of Federal Regulations, 2013 CFR

2013-01-01

... stall. (a) It must be possible to produce and to correct roll by unreversed use of the rolling control and to produce and to correct yaw by unreversed use of the directional control, up to the time the.... Starting from a speed at least 10 knots above the stall speed, the elevator control must be pulled back so...

14 CFR 23.201 - Wings level stall.

Code of Federal Regulations, 2011 CFR

2011-01-01

... stall. (a) It must be possible to produce and to correct roll by unreversed use of the rolling control and to produce and to correct yaw by unreversed use of the directional control, up to the time the.... Starting from a speed at least 10 knots above the stall speed, the elevator control must be pulled back so...

14 CFR 23.201 - Wings level stall.

Code of Federal Regulations, 2012 CFR

2012-01-01

... produce and to correct roll by unreversed use of the rolling control and to produce and to correct yaw by unreversed use of the directional control, up to the time the airplane stalls. (b) The wings level stall... speed, the elevator control must be pulled back so that the rate of speed reduction will not exceed one...

14 CFR 23.201 - Wings level stall.

Code of Federal Regulations, 2014 CFR

2014-01-01

... stall. (a) It must be possible to produce and to correct roll by unreversed use of the rolling control and to produce and to correct yaw by unreversed use of the directional control, up to the time the.... Starting from a speed at least 10 knots above the stall speed, the elevator control must be pulled back so...

INMATE SOCIETY IN THE ERA OF MASS INCARCERATION*

PubMed Central

Kreager, Derek A.; Kruttschnitt, Candace

2017-01-01

Inmate social organization was once a central area within criminology that stalled just as incarceration rates dramatically climbed. In this review, we return to seminal works on this topic before summarizing the changes that mass incarceration wrought in correctional contexts and the potential impacts of these changes for inmate society. We then review the few recent studies that document contemporary inmate social life and call for increased researcher-practitioner partnerships that embed criminologists within carceral settings. We suggest that network approaches are particularly useful for building on past qualitative and ethnographic insights to provide replicable results that are also easily conveyed to correctional authorities. As the era of mass incarceration peaks, we argue that the time is ripe for renewed interest in inmate society and its connections to prison stability, rehabilitation, and community reintegration. PMID:29542738

Investigation of Unsteady Flow Behavior in Transonic Compressor Rotors with LES and PIV Measurements

NASA Technical Reports Server (NTRS)

Hah, Chunill; Voges, Melanie; Mueller, Martin; Schiffer, Heinz-Peter

2009-01-01

In the present study, unsteady flow behavior in a modern transonic axial compressor rotor is studied in detail with large eddy simulation (LES) and particle image velocimetry (PIV). The main purpose of the study is to advance the current understanding of the flow field near the blade tip in an axial transonic compressor rotor near the stall and peak-efficiency conditions. Flow interaction between the tip leakage vortex and the passage shock is inherently unsteady in a transonic compressor. Casing-mounted unsteady pressure transducers have been widely applied to investigate steady and unsteady flow behavior near the casing. Although many aspects of flow have been revealed, flow structures below the casing cannot be studied with casing-mounted pressure transducers. In the present study, unsteady velocity fields are measured with a PIV system and the measured unsteady flow fields are compared with LES simulations. The currently applied PIV measurements indicate that the flow near the tip region is not steady even at the design condition. This self-induced unsteadiness increases significantly as the compressor rotor operates near the stall condition. Measured data from PIV show that the tip clearance vortex oscillates substantially near stall. The calculated unsteady characteristics of the flow from LES agree well with the PIV measurements. Calculated unsteady flow fields show that the formation of the tip clearance vortex is intermittent and the concept of vortex breakdown from steady flow analysis does not seem to apply in the current flow field. Fluid with low momentum near the pressure side of the blade close to the leading edge periodically spills over into the adjacent blade passage. The present study indicates that stall inception is heavily dependent on unsteady behavior of the flow field near the leading edge of the blade tip section for the present transonic compressor rotor.

Global surface pressure measurements of static and dynamic stall on a wind turbine airfoil at low Reynolds number

NASA Astrophysics Data System (ADS)

Disotell, Kevin J.; Nikoueeyan, Pourya; Naughton, Jonathan W.; Gregory, James W.

2016-05-01

Recognizing the need for global surface measurement techniques to characterize the time-varying, three-dimensional loading encountered on rotating wind turbine blades, fast-responding pressure-sensitive paint (PSP) has been evaluated for resolving unsteady aerodynamic effects in incompressible flow. Results of a study aimed at demonstrating the laser-based, single-shot PSP technique on a low Reynolds number wind turbine airfoil in static and dynamic stall are reported. PSP was applied to the suction side of a Delft DU97-W-300 airfoil (maximum thickness-to-chord ratio of 30 %) at a chord Reynolds number of 225,000 in the University of Wyoming open-return wind tunnel. Static and dynamic stall behaviors are presented using instantaneous and phase-averaged global pressure maps. In particular, a three-dimensional pressure topology driven by a stall cell pattern is detected near the maximum lift condition on the steady airfoil. Trends in the PSP-measured pressure topology on the steady airfoil were confirmed using surface oil visualization. The dynamic stall case was characterized by a sinusoidal pitching motion with mean angle of 15.7°, amplitude of 11.2°, and reduced frequency of 0.106 based on semichord. PSP images were acquired at selected phase positions, capturing the breakdown of nominally two-dimensional flow near lift stall, development of post-stall suction near the trailing edge, and a highly three-dimensional topology as the flow reattaches. Structural patterns in the surface pressure topologies are considered from the analysis of the individual PSP snapshots, enabled by a laser-based excitation system that achieves sufficient signal-to-noise ratio in the single-shot images. The PSP results are found to be in general agreement with observations about the steady and unsteady stall characteristics expected for the airfoil.

Mouse Norovirus infection promotes autophagy induction to facilitate replication but prevents final autophagosome maturation

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

O’Donnell, Tanya B.; Hyde, Jennifer L.; Mintern, Justine D.

Autophagy is a cellular process used to eliminate intracellular pathogens. Many viruses however are able to manipulate this cellular process for their own advantage. Here we demonstrate that Mouse Norovirus (MNV) infection induces autophagy but does not appear to utilise the autophagosomal membrane for establishment and formation of the viral replication complex. We have observed that MNV infection results in lipidation and recruitment of LC3 to the autophagosome membrane but prevents subsequent fusion of the autophagosomes with lysosomes, as SQSTM1 (an autophagy receptor) accumulates and Lysosome-Associated Membrane Protein1 is sequestered to the MNV replication complex (RC) rather than to autophagosomes.more » We have additionally observed that chemical modulation of autophagy differentially affects MNV replication. From this study we can conclude that MNV infection induces autophagy, however suppresses the final maturation step of this response, indicating that autophagy induction contributes to MNV replication independently of RC biogenesis. - Highlights: • MNV induces autophagy in infected murine macrophages. • MNV does not utilise autophagosomal membranes for replication. • The MNV-induced autophagosomes do not fuse with lysosomes. • MNV sequesters SQSTM1 to prevent autophagy degradation and turnover. • Chemical modulation of autophagy enhances MNV replication.« less

Effects of bedding type on compost quality of equine stall waste: implications for small horse farms.

PubMed

Komar, S; Miskewitz, R; Westendorf, M; Williams, C A

2012-03-01

Our objective in this study is to compare 4 of the most common bedding materials used by equine operations on the chemical and physical characteristics of composted equine stall waste. Twelve Standardbred horses were adapted to the barn and surrounding environment for 2 wk before the start of the study. Groups of 3 horses were bedded on 1 of 4 different bedding types (wood shavings, pelletized wood materials, long straw, and pelletized straw) for 16 h per day for 18 d. Stalls were cleaned by trained staff daily, and all contents removed were weighed and stored separately by bedding material on a level covered concrete pad for the duration of the study. Compost piles were constructed using 3 replicate piles of each bedding type in a randomized complete block design. Each pile was equipped with a temperature sensor and data logger. Water was added and piles were turned weekly throughout the 100-d compost process. Initial and final samples were taken, dried, and analyzed for DM mass, OM, inorganic nitrogen (nitrate-N and ammonium-N), electrical conductivity, and soluble (plant-available) nutrients. Data were analyzed using the GLM procedure, and means were separated using Fischer's protected LSD test (P < 0.05). No significant temperature differences were observed among the bedding materials. The composting process resulted in significant reductions (P < 0.05) in DM mass for each of the 4 bedding materials. The composting process resulted in significant reductions (P < 0.05) in OM and C:N ratio for all 4 bedding materials. The composted long straw material had greater concentrations of total Kjeldahl nitrogen (P < 0.05), nitrate-N (P < 0.05), and ammonium-N (P < 0.05) than the composted wood shavings. This study demonstrated that incorporating a simple aerobic composting system may greatly reduce the overall volume of manure and yield a material that is beneficial for land application in pasture-based systems. The straw-based materials may be better suited for composting and subsequent land application; however, factors such as suitability of the bedding material for equine use, material cost, labor, and availability must be considered when selecting a bedding material.

An experimental and analytical investigation of stall effects on flap-lag stability in forward flight

NASA Technical Reports Server (NTRS)

Nagabhushanam, J.; Gaonkar, Gopal H.; Mcnulty, Michael J.

1987-01-01

Experiments have been performed with a 1.62 m diameter hingeless rotor in a wind tunnel to investigate flap-lag stability of isolated rotors in forward flight. The three-bladed rotor model closely approaches the simple theoretical concept of a hingeless rotor as a set of rigid, articulated flap-lag blades with offset and spring restrained flap and lag hinges. Lag regressing mode stability data was obtained for advance ratios as high as 0.55 for various combinations of collective pitch and shaft angle. The prediction includes quasi-steady stall effects on rotor trim and Floquet stability analyses. Correlation between data and prediction is presented and is compared with that of an earlier study based on a linear theory without stall effects. While the results with stall effects show marked differences from the linear theory results, the stall theory still falls short of adequate agreement with the experimental data.

Turbofan compressor dynamics during afterburner transients

NASA Technical Reports Server (NTRS)

Kurkov, A. P.

1975-01-01

The effects of afterburner light-off and shut-down transients on compressor stability were investigated. Experimental results are based on detailed high-response pressure and temperature measurements on the Tf30-p-3 turbofan engine. The tests were performed in an altitude test chamber simulating high-altitude engine operation. It is shown that during both types of transients, flow breaks down in the forward part of the fan-bypass duct. At a sufficiently low engine inlet pressure this resulted in a compressor stall. Complete flow breakdown within the compressor was preceded by a rotating stall. At some locations in the compressor, rotating stall cells initially extended only through part of the blade span. For the shutdown transient, the time between first and last detected occurrence of rotating stall is related to the flow Reynolds number. An attempt was made to deduce the number and speed of propagation of rotating stall cells.

A theory of rotating stall of multistage axial compressors. III - Limit cycles

NASA Technical Reports Server (NTRS)

Moore, F. K.

1983-01-01

A theory of rotating stall, based on single parameters for blade-passage lag and external-flow lag and a given compressor characteristic yields limit cycles in velocity space. These limit cycles are governed by Lienard's equation with the characteristic playing the role of nonlinear damping function. Cyclic integrals of the solution determine stall propagation speed and the effect of rotating stall on average performance. Solution with various line-segment characteristics and various throttle settings are found and discussed. There is generally a limiting flow coefficient beyond which no solution is possible; this probably represents stall recovery. This recovery point is independent of internal compressor lag, but does depend on external lags and on the height-to-width ratio of the diagram. Tall diagrams and small external lags (inlet and diffusor) favor recovery. Suggestions for future theoretical and experimental research are discussed.

Control of unsteady separated flow associated with the dynamic stall of airfoils

NASA Technical Reports Server (NTRS)

Wilder, M. C.

1994-01-01

A unique active flow-control device is proposed for the control of unsteady separated flow associated with the dynamic stall of airfoils. The device is an adaptive-geometry leading-edge which will allow controlled, dynamic modification of the leading-edge profile of an airfoil while the airfoil is executing an angle-of-attack pitch-up maneuver. A carbon-fiber composite skin has been bench tested, and a wind tunnel model is under construction. A baseline parameter study of compressible dynamic stall was performed for flow over an NACA 0012 airfoil. Parameters included Mach number, pitch rate, pitch history, and boundary layer tripping. Dynamic stall data were recorded via point-diffraction interferometry and the interferograms were analyzed with in-house developed image processing software. A new high-speed phase-locked photographic image recording system was developed for real-time documentation of dynamic stall.

High angle-of-attack aerodynamic characteristics of crescent and elliptic wings

NASA Technical Reports Server (NTRS)

Vandam, C. P.

1989-01-01

Static longitudinal and lateral-directional forces and moments were measured for elliptic- and crescent-wing models at high angles-of-attack in the NASA Langley 14 by 22-Ft Subsonic Tunnel. The forces and moments were obtained for an angle-of-attack range including stall and post-stall conditions at a Reynolds number based on the average wing chord of about 1.8 million. Flow-visualization photographs using a mixture of oil and titanium-dioxide were also taken for several incidence angles. The force and moment data and the flow-visualization results indicated that the crescent wing model with its highly swept tips produced much better high angle-of-attack aerodynamic characteristics than the elliptic model. Leading-edge separation-induced vortex flow over the highly swept tips of the crescent wing is thought to produce this improved behavior at high angles-of-attack. The unique planform design could result in safer and more efficient low-speed airplanes.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2007-04-01

Conway, A., Lockhart, D. J., Davis, R. W., Brewer , B. J., and Fangman, W. L. (2001). Replication dynamics of the yeast genome. Science 294, 115–121... Brewer , B. J. (2001). An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Mol. Cell 7, 705–713. Vas, A., Mok, W., and...replication in yeast cells. We have demonstrated that re-replication induces a rapid and significant decrease in cell viability and a cellular DNA damage

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

PubMed Central

Löb, D.; Lengert, N.; Chagin, V. O.; Reinhart, M.; Casas-Delucchi, C. S.; Cardoso, M. C.; Drossel, B.

2016-01-01

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

PubMed Central

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-01-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ− mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ− cells and increased deletion mutagenesis at the ori5 region in ρ+ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5. PMID:23598996

Ultrastructure of the replication sites of positive-strand RNA viruses

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

Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

2015-05-15

Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. Inmore » this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.« less

Control of unsteady separated flow associated with the dynamic stall of airfoils

NASA Technical Reports Server (NTRS)

Wilder, Michael C.

1992-01-01

The two principal objectives of this research were to achieve an improved understanding of the mechanisms involved in the onset and development of dynamic stall under compressible flow conditions, and to investigate the feasibility of employing adaptive airfoil geometry as an active flow control device in the dynamic stall engine. Presented here are the results of a quantitative (PDI) study of the compressibility effects on dynamic stall over the transiently pitching airfoil, as well as a discussion of a preliminary technique developed to measure the deformation produced by the adaptive geometry control device, and bench test results obtained using an airfoil equipped with the device.

Flow Observations with Tufts and Lampblack of the Stalling of Four Typical Airfoil Sections in the NACA Variable-density Tunnel

NASA Technical Reports Server (NTRS)

Abbott, Ira H; Sherman, Albert

1938-01-01

A preliminary investigation of the stalling processes of four typical airfoil sections was made over the critical range of the Reynolds Number. Motion pictures were taken of the movements of small silk tufts on the airfoil surface as the angle of attack increased through a range of angles including the stall. The boundary-layer flow also at certain angles of attack was indicated by the patterns formed by a suspension of lampblack in oil brushed onto the airfoil surface. These observations were analyzed together with corresponding force-test measurements to derive a picture of the stalling processes of airfoils.

Airfoil Dynamic Stall and Rotorcraft Maneuverability

NASA Technical Reports Server (NTRS)

Bousman, William G.

2000-01-01

The loading of an airfoil during dynamic stall is examined in terms of the augmented lift and the associated penalties in pitching moment and drag. It is shown that once stall occurs and a leading-edge vortex is shed from the airfoil there is a unique relationship between the augmented lift, the negative pitching moment, and the increase in drag. This relationship, referred to here as the dynamic stall function, shows limited sensitivity to effects such as the airfoil section profile and Mach number, and appears to be independent of such parameters as Reynolds number, reduced frequency, and blade sweep. For single-element airfoils there is little that can be done to improve rotorcraft maneuverability except to provide good static C(l(max)) characteristics and the chord or blade number that is required to provide the necessary rotor thrust. However, multi-element airfoils or airfoils with variable geometry features can provide augmented lift in some cases that exceeds that available from a single-element airfoil. The dynamic stall function is shown to be a useful tool for the evaluation of both measured and calculated dynamic stall characteristics of single element, multi-element, and variable geometry airfoils.

Dynamic Stall Control Using Plasma Actuators

NASA Astrophysics Data System (ADS)

Webb, Nathan; Singhal, Achal; Castaneda, David; Samimy, Mo

2017-11-01

Dynamic stall occurs in many applications, including sharp maneuvers of fixed wing aircraft, wind turbines, and rotorcraft and produces large unsteady aerodynamic loads that can lead to flutter and mechanical failure. This work uses flow control to reduce the unsteady loads by excitation of instabilities in the shear layer over the separated region using nanosecond pulse driven dielectric barrier discharge (NS-DBD) plasma actuators. These actuators have been shown to effectively delay or mitigate static stall. A wide range of flow parameters were explored in the current work: Reynolds number (Re = 167,000 to 500,000), reduced frequency (k = 0.025 to 0.075), and excitation Strouhal number (Ste = 0 to 10). Based on the results, three major conclusions were drawn: (a) Low Strouhal number excitation (Ste <0.5) results in oscillatory aerodynamic loads in the stalled stage of dynamic stall; (b) All excitation resulted in earlier flow reattachment; and (c) Excitation at progressively higher Ste weakened and eventually eliminated the dynamic stall vortex (DSV), thereby dramatically reducing the unsteady loading. The decrease in the strength of the DSV is achieved by the formation of shear layer coherent structures that bleed the leading-edge vorticity prior to the ejection of the DSV.

An Experimental Investigation of Compressible Dynamic Stall on a Pitching Airfoil

NASA Astrophysics Data System (ADS)

Thorne, Katie; Bowles, Patrick

2009-11-01

A new facility has been designed and constructed at the University of Notre Dame to investigate dynamic stall on a 2-D pitching airfoil at high subsonic Mach numbers. This work is motivated by the need to investigate dynamic stall at conditions relevant to military helicopters. One focus of the experiments is to characterize the role of shock/boundary layer interactions during the pitching cycle. The new dynamic stall facility is integrated into a closed-loop, low turbulence wind tunnel capable of achieving test section Mach numbers in excess of M = 0.6. The design of the dynamic stall test section was focused on achieving reduced pitching frequencies of up to k = 0.2 and chord Reynolds numbers up to 5 x10^6. The facility has the unique ability to execute non-harmonic pitching motions through the use of an actuated pitch link mechanism. Optical access is provided to allow the use of high-speed and Schlieren imaging. Thirty-one flush mounted Kulite dynamic pressure transducers provide the instantaneous unsteady surface pressure distribution over the airfoil. Initial dynamic stall measurements obtained in the new facility will be described.

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

PubMed Central

Adeyemi, Richard O.; Landry, Sebastien; Davis, Meredith E.; Weitzman, Matthew D.; Pintel, David J.

2010-01-01

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells. PMID:20949077

Reversible stalling of transcription elongation complexes by high pressure.

PubMed

Erijman, L; Clegg, R M

1998-07-01

We have investigated the effect of high hydrostatic pressure on the stability of RNA polymerase molecules during transcription. RNA polymerase molecules participating in stalled or active ternary transcribing complexes do not dissociate from the template DNA and nascent RNA at pressures up to 180 MPa. A lower limit for the free energy of stabilization of an elongating ternary complex relative to the quaternary structure of the free RNAP molecules is estimated to be 20 kcal/mol. The rate of elongation decreases at high pressure; transcription completely halts at sufficiently high pressure. The overall rate of elongation has an apparent activation volume (DeltaVdouble dagger) of 55-65 ml . mol-1 (at 35 degrees C). The pressure-stalled transcripts are stable and resume elongation at the prepressure rate upon decompression. The efficiency of termination decreases at the rho-independent terminator tR2 after the transcription reaction has been exposed to high pressure. This suggests that high pressure modifies the ternary complex such that termination is affected in a manner different from that of elongation. The solvent and temperature dependence of the pressure-induced inhibition show evidence for major conformational changes in the core polymerase enzyme during RNA synthesis. It is proposed that the inhibition of the elongation phase of the transcription reaction at elevated pressures is related to a reduction of the partial specific volume of the RNA polymerase molecule; under high pressure, the RNA polymerase molecule does not have the necessary structural flexibility required for the protein to translocate.

Comments on potential health effects of MRI-induced DNA lesions: quality is more important to consider than quantity

PubMed Central

Hill, M.A.; O'Neill, P.; McKenna, W.G.

2016-01-01

Magnetic resonance imaging (MRI) is increasingly being used in cardiology to detect heart disease and guide therapy. It is mooted to be a safer alternative to imaging techniques, such as computed tomography (CT) or coronary angiographic imaging. However, there has recently been an increased interest in the potential long-term health risks of MRI, especially in the light of the controversy resulting from a small number of research studies reporting an increase in DNA damage following exposure, with calls to limit its use and avoid unnecessary examination, according to the precautionary principle. Overall the published data are somewhat limited and inconsistent; the ability of MRI to produce DNA lesions has yet to be robustly demonstrated and future experiments should be carefully designed to optimize sensitivity and benchmarked to validate and assess reproducibility. The majority of the current studies have focussed on the initial induction of DNA damage, and this has led to comparisons between the reported induction of γH2AX and implied double-strand break (DSB) yields produced following MRI with induction by imaging techniques using ionizing radiation. However, γH2AX is not only a marker of classical double-ended DSB, but also a marker of stalled replication forks and in certain circumstances stalled DNA transcription. Additionally, ionizing radiation is efficient at producing complex DNA damage, unique to ionizing radiation, with an associated reduction in repairability. Even if the fields associated with MRI are capable of producing DNA damage, the lesions produced will in general be simple, similar to those produced by endogenous processes. It is therefore inappropriate to try and infer cancer risk by simply comparing the yields of γH2AX foci or DNA lesions potentially produced by MRI to those produced by a given exposure of ionizing radiation, which will generally be more biologically effective and have a greater probability of leading to long-term health effects. As a result, it is important to concentrate on more relevant downstream end points (e.g. chromosome aberration production), along with potential mechanisms by which MRI may lead to DNA lesions. This could potentially involve a perturbation in homeostasis of oxidative stress, modifying the background rate of endogenous DNA damage induction. In summary, what the field needs at the moment is more research and less fear mongering. PMID:27550664

Both High-Fidelity Replicative and Low-Fidelity Y-Family Polymerases Are Involved in DNA Rereplication

PubMed Central

Sekimoto, Takayuki; Oda, Tsukasa; Kurashima, Kiminori; Hanaoka, Fumio

2014-01-01

DNA rereplication is a major form of aberrant replication that causes genomic instabilities, such as gene amplification. However, little is known about which DNA polymerases are involved in the process. Here, we report that low-fidelity Y-family polymerases (Y-Pols), Pol η, Pol ι, Pol κ, and REV1, significantly contribute to DNA synthesis during rereplication, while the replicative polymerases, Pol δ and Pol ε, play an important role in rereplication, as expected. When rereplication was induced by depletion of geminin, these polymerases were recruited to rereplication sites in human cell lines. This finding was supported by RNA interference (RNAi)-mediated knockdown of the polymerases, which suppressed rereplication induced by geminin depletion. Interestingly, epistatic analysis indicated that Y-Pols collaborate in a common pathway, independently of replicative polymerases. We also provide evidence for a catalytic role for Pol η and the involvement of Pol η and Pol κ in cyclin E-induced rereplication. Collectively, our findings indicate that, unlike normal S-phase replication, rereplication induced by geminin depletion and oncogene activation requires significant contributions of both Y-Pols and replicative polymerases. These findings offer important mechanistic insights into cancer genomic instability. PMID:25487575

Viral Replication Complexes Are Targeted by LC3-Guided Interferon-Inducible GTPases.

PubMed

Biering, Scott B; Choi, Jayoung; Halstrom, Rachel A; Brown, Hailey M; Beatty, Wandy L; Lee, Sanghyun; McCune, Broc T; Dominici, Erin; Williams, Lelia E; Orchard, Robert C; Wilen, Craig B; Yamamoto, Masahiro; Coers, Jörn; Taylor, Gregory A; Hwang, Seungmin

2017-07-12

All viruses with positive-sense RNA genomes replicate on membranous structures in the cytoplasm called replication complexes (RCs). RCs provide an advantageous microenvironment for viral replication, but it is unknown how the host immune system counteracts these structures. Here we show that interferon-gamma (IFNG) disrupts the RC of murine norovirus (MNV) via evolutionarily conserved autophagy proteins and the induction of IFN-inducible GTPases, which are known to destroy the membrane of vacuoles containing bacteria, protists, or fungi. The MNV RC was marked by the microtubule-associated-protein-1-light-chain-3 (LC3) conjugation system of autophagy and then targeted by immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs) upon their induction by IFNG. Further, the LC3 conjugation system and the IFN-inducible GTPases were necessary to inhibit MNV replication in mice and human cells. These data suggest that viral RCs can be marked and antagonized by a universal immune defense mechanism targeting diverse pathogens replicating in cytosolic membrane structures. Copyright © 2017 Elsevier Inc. All rights reserved.

Compressibility effects on dynamic stall of airfoils undergoing rapid transient pitching motion

NASA Technical Reports Server (NTRS)

Chandrasekhara, M. S.; Platzer, M. F.

1992-01-01

The research was carried out in the Compressible Dynamic Stall Facility, CDSF, at the Fluid Mechanics Laboratory (FML) of NASA Ames Research Center. The facility can produce realistic nondimensional pitch rates experienced by fighter aircraft, which on model scale could be as high as 3600/sec. Nonintrusive optical techniques were used for the measurements. The highlight of the effort was the development of a new real time interferometry method known as Point Diffraction Interferometry - PDI, for use in unsteady separated flows. This can yield instantaneous flow density information (and hence pressure distributions in isentropic flows) over the airfoil. A key finding is that the dynamic stall vortex forms just as the airfoil leading edge separation bubble opens-up. A major result is the observation and quantification of multiple shocks over the airfoil near the leading edge. A quantitative analysis of the PDI images shows that pitching airfoils produce larger suction peaks than steady airfoils at the same Mach number prior to stall. The peak suction level reached just before stall develops is the same at all unsteady rates and decreases with increase in Mach number. The suction is lost once the dynamic stall vortex or vortical structure begins to convect. Based on the knowledge gained from this preliminary analysis of the data, efforts to control dynamic stall were initiated. The focus of this work was to arrive at a dynamically changing leading edge shape that produces only 'acceptable' airfoil pressure distributions over a large angle of attack range.

Group housing during gestation affects the behaviour of sows and the physiological indices of offspring piglets at weaning

USDA-ARS?s Scientific Manuscript database

In order to compare the behaviour of sows in stalls and group housing systems, and the physiological indices of their offspring, 28 sows were randomly distributed into 2 systems with 16 sows in stalls, and the other 12 sows were divided into 3 groups with 4 sows per pen. The area per sow in stalls a...

Influence of free-stall flooring on comfort and hygiene of dairy cows during warm climatic conditions.

PubMed

De Palo, P; Tateo, A; Zezza, F; Corrente, M; Centoducati, P

2006-12-01

An evaluation of behavioral and hygienic conditions was carried out with 4 materials used as free-stall flooring for dairy cows: polyethylene vinyl acetate (EVA) and polypropylene vinyl acetate (PVA) mats, wood shavings, and solid manure. The free-stall type selected by cows was evaluated in response to changes in environmental temperature and humidity. Two tests were used: 1) a preference test, in which 8 cows were housed in a pen with 32 free stalls and 4 types of flooring; and 2) an aversion test, in which 32 cows were placed in 4 pens, each with 8 free stalls. The free stalls in each pen had a single type of bedding material. These tests showed that the comfort of dairy cows was predominantly influenced by environmental conditions. The preference test for lying showed that cows preferred free-stall floors with EVA mats over those with PVA mats, wood shavings, and solid manure (332.4 +/- 24.0 vs. 130.8 +/- 6.2, 160.9 +/- 23.7, and 102.6 +/- 23.2 min/d, respectively), but under conditions of heat stress, with a temperature-humidity index > 80, they chose wood shavings and solid manure lying areas. These results were confirmed by the aversion test. In all experimental and environmental conditions, the PVA mats were the least suitable. The mats contaminated with organic manure and the free stalls bedded with wood shavings and organic solids did not differ in either the coliform load on the lying surfaces (EVA mats: 290 +/- 25; PVA mats: 306 +/- 33; wood shavings: 290 +/- 39; and solid manure: 305 +/- 23 log(10) cfu/mL) or the total bacterial count in the raw milk (EVA mats: 232 +/- 22; PVA mats: 233 + 24; wood shavings: 221 +/- 24; and solid manure: 220 +/- 25 log(10) cfu/mL). These results demonstrate that the comfort of dairy cows housed in barns with free stalls as resting areas does not depend only on the material used, but also on the value of the material in microenvironmental conditions.

Global Aerodynamic Modeling for Stall/Upset Recovery Training Using Efficient Piloted Flight Test Techniques

NASA Technical Reports Server (NTRS)

Morelli, Eugene A.; Cunningham, Kevin; Hill, Melissa A.

2013-01-01

Flight test and modeling techniques were developed for efficiently identifying global aerodynamic models that can be used to accurately simulate stall, upset, and recovery on large transport airplanes. The techniques were developed and validated in a high-fidelity fixed-base flight simulator using a wind-tunnel aerodynamic database, realistic sensor characteristics, and a realistic flight deck representative of a large transport aircraft. Results demonstrated that aerodynamic models for stall, upset, and recovery can be identified rapidly and accurately using relatively simple piloted flight test maneuvers. Stall maneuver predictions and comparisons of identified aerodynamic models with data from the underlying simulation aerodynamic database were used to validate the techniques.

Instrumentation and control system for an F-15 stall/spin

NASA Technical Reports Server (NTRS)

Pitts, F. L.; Holmes, D. C. E.; Zaepfel, K. P.

1974-01-01

An instrumentation and control system is described that was used for radio-controlled F-15 airplane model stall/spin research at the NASA-Langley Research Center. This stall/spin research technique, using scale model aircraft, provides information on the post-stall and spin-entry characteristics of full-scale aircraft. The instrumentation described provides measurements of flight parameters such as angle of attack and sideslip, airspeed, control-surface position, and three-axis rotation rates; these data are recorded on an onboard magnetic tape recorder. The proportional radio control system, which utilizes analog potentiometric signals generated from ground-based pilot inputs, and the ground-based system used in the flight operation are also described.

Large-Vortex Capture by a Wing at Very High Angles of Attack

NASA Technical Reports Server (NTRS)

Wu, J. M.; Wu, J. Z.; Denny, G. A.; Lu, X. Y.

1996-01-01

In generating the lift on a wing, the static stall is a severe barrier. As the angle of attack, alpha, increases to the stall angle, alpha(sub stall) the flow separation point on the upper surface of the wing moves to the leading edge, so that on a two-dimensional airfoil or a large-aspect-ratio wing, the lift abruptly drops to a very low level. Therefore, the first generation of aeronautical flow type, i.e., the attached steady flow, has been limited to alpha less than alpha(sub stall). Owing to the obvious importance in applications, therefore, a great effort has been made in the past two decades to enlarge the range of usable angles of attack by various flow controls for a large-aspect-ratio wing. Basically, relevant works fall into two categories. The first category is usually refereed to as separation control, which concentrates on partially separated flow at alpha less than alpha(sub stall). Since the first experimental study of Collins and Zelenevitz, there has been ample literature showing that a partially separated flow can be turned to almost fully attached by flow controls, so that the lift is recovered and the stall is delayed (for a recent work see Seifert et al.). It has been well established that, in this category, unsteady controls are much more effective than steady ones and can be realized at a very low power-input level (Wu et al.; Seifert et al.). The second and more ambitious category of relevant efforts is the post-stall lift enhancement. Its possibility roots at the existence of a second lift peak at a very high angle of attack. In fact, As alpha further increases from alpha(sub stall), the completely separated flow develops and gradually becomes a bluff-body flow. This flow gives a normal force to the airfoil with a lift component, which reaches a peak at a maximum utilizable angle of attack, alpha(sub m) approx.= 40 deg. This second peak is of the same level as the first lift peak at alpha(sub stall). Meanwhile, the drag is also quickly increased (e.g., Fage and Johansen ; Critzos et al.). Figure 1 shows a typical experimental lift and drag coefficients of NACA-0012 airfoil in this whole range of angle of attack. Obviously, without overcoming the lift crisis at alpha(sub stall) the second lift peak is completely useless. Thus, the ultimate goal of post-stall lift enhancement is to fill the lift valley after stall by flow controls, so that a wing and/or flap can work at the whole range of 0 deg less than alpha less than alpha(sub m). Relevant early experimental studies have been extensively reviewed by Wu et al., who concluded that, first, similar to the leading-edge vortex on a slender wing, the lift enhancement on a large-aspect-ratio wing should be the result of capturing a vortex on the upper surface of the wing; and, second, using steady controls cannot reach the goal, and one must rely on unsteady controls with low-level power input as well. Wu et al. also conjectured that the underlying physics of post-stall lift enhancement by unsteady controls consists of a chain of mechanisms: vortex layer instability - receptivity resonance - nonlinear streaming.

Shaping the Flavivirus Replication Complex: It's Curvaceous!

PubMed

Aktepe, Turgut E; Mackenzie, Jason M

2018-06-22

Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus-induced organelles characteristic of infection. This article is protected by copyright. All rights reserved.

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

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

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.

A theory of post-stall transients in multistage axial compression systems

NASA Technical Reports Server (NTRS)

Moore, F. K.; Greitzer, E. M.

1985-01-01

A theory is presented for post stall transients in multistage axial compressors. The theory leads to a set of coupled first-order ordinary differential equations capable of describing the growth and possible decay of a rotating-stall cell during a compressor mass-flow transient. These changing flow features are shown to have a significant effect on the instantaneous compressor pumping characteristic during unsteady operation, and henace on the overall system behavior. It is also found from the theory that the ultimate mode of system response, stable rotating stall or surge, depends not only on the B parameter but also on other parameters, such as the compressor length-to-diameter ratio. Small values of this latter quantity tend to favor the occurrence of surge, as do large values of B. A limited parametric study is carried out to show the impact of the different system features on transient behavior. Based on analytical and numerical results, several specific topics are suggested for future research on post-stall transients.

Prediction of active control of subsonic centrifugal compressor rotating stall

NASA Technical Reports Server (NTRS)

Lawless, Patrick B.; Fleeter, Sanford

1993-01-01

A mathematical model is developed to predict the suppression of rotating stall in a centrifugal compressor with a vaned diffuser. This model is based on the employment of a control vortical waveform generated upstream of the impeller inlet to damp weak potential disturbances that are the early stages of rotating stall. The control system is analyzed by matching the perturbation pressure in the compressor inlet and exit flow fields with a model for the unsteady behavior of the compressor. The model was effective at predicting the stalling behavior of the Purdue Low Speed Centrifugal Compressor for two distinctly different stall patterns. Predictions made for the effect of a controlled inlet vorticity wave on the stability of the compressor show that for minimum control wave magnitudes, on the order of the total inlet disturbance magnitude, significant damping of the instability can be achieved. For control waves of sufficient amplitude, the control phase angle appears to be the most important factor in maintaining a stable condition in the compressor.

An experimental investigation of the helicopter rotor blade element airloads on a model rotor in the blade stall regime

NASA Technical Reports Server (NTRS)

Fisher, R. K., Jr.; Tompkins, J. E.; Bobo, C. J.; Child, R. F.

1971-01-01

A wind tunnel test program was conducted on an eight foot diameter model rotor system to determine blade element airloads characteristics in the unstalled and stalled flight regimes. The fully articulated model rotor system utilized three blades with a Vertol 23010-1.58 airfoil section, the blades being 1/7.5 scale models of the Ch-47C rotor blades. Instrumentation was incorporated at the blade 75% radial station to measure pressure and skin friction distributions, surface streamline directions and local angle of attack. The test program was conducted in three phases; non-rotating, hover and forward flight at advance ratios of 0.15, 0.35 and 0.60. Test data were analyzed with respect to providing insight to the mechanisms affecting blade stall, particularly retreating blade stall during forward flight conditions. From such data, an assessment was made as to the applicability of current theoretical analyses used for the prediction of blade element airloads in the stall regime.

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.

Study of casing treatment stall margin improvement phenomena. [for compressor rotor blade tips compressor blades rotating stalls

NASA Technical Reports Server (NTRS)

Prince, D. C., Jr.; Wisler, D. C.; Hilvers, D. E.

1974-01-01

The results of a program of experimental and analytical research in casing treatments over axial compressor rotor blade tips are presented. Circumferential groove, axial-skewed slot, and blade angle slot treatments were tested. These yielded, for reduction in stalling flow and loss in peak efficiency, 5.8% and 0 points, 15.3% and 2.0 points, and 15.0% and 1.2 points, respectively. These values are consistent with other experience. The favorable stalling flow situations correlated well with observations of higher-then-normal surface pressures on the rotor blade pressure surfaces in the tip region, and with increased maximum diffusions on the suction surfaces. Annular wall pressure gradients, especially in the 50-75% chord region, are also increased and blade surface pressure loadings are shifted toward the trailing edge for treated configurations. Rotor blade wakes may be somewhat thinner in the presence of good treatments, particularly under operating conditions close to the baseline stall.

Self-Recirculating Casing Treatment Concept for Enhanced Compressor Performance

NASA Technical Reports Server (NTRS)

Hathaway, Michael D.

2002-01-01

A state-of-the-art CFD code (APNASA) was employed in a computationally based investigation of the impact of casing bleed and injection on the stability and performance of a moderate speed fan rotor wherein the stalling mass flow is controlled by tip flow field breakdown. The investigation was guided by observed trends in endwall flow characteristics (e.g., increasing endwall aerodynamic blockage) as stall is approached and based on the hypothesis that application of bleed or injection can mitigate these trends. The "best" bleed and injection configurations were then combined to yield a self-recirculating casing treatment concept. The results of this investigation yielded: 1) identification of the fluid mechanisms which precipitate stall of tip critical blade rows, and 2) an approach to recirculated casing treatment which results in increased compressor stall range with minimal or no loss in efficiency. Subsequent application of this approach to a high speed transonic rotor successfully yielded significant improvements in stall range with no loss in compressor efficiency.

Group space allowance has little effect on sow health, productivity, or welfare in a free-access stall system

USDA-ARS?s Scientific Manuscript database

Free-access stalls allow sows to choose the protection of a stall or use of a shared group space. This study investigated the effect of group space width: 0.91 (SS), 2.13 (IS), and 3.05 (LS) m on the health, production, behavior, and welfare of gestating sows. At gestational day (GD) 35.4 ± 2.3, 21 ...

Stability Analysis for Rotating Stall Dynamics in Axial Flow Compressors

DTIC Science & Technology

1999-01-01

modes determines collectively local stability of the compressor model. Explicit conditions are obtained for local stability of rotating stall which...critical modes determines the stability for rotating stall collectively . We point out that although in a special case our stability condition for...strict crossing assumption implies that the zero solution changes its stability as ~, crosses ~’c. For instance, odk (yc ) > 0 implies that the zero

75 FR 34956 - Airworthiness Directives; Robert E. Rust, Jr. Model DeHavilland DH.C1 Chipmunk 21, DH.C1 Chipmunk...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-21

... retraction of the flaps. This failure could lead to a stall during a landing approach. DATES: We must receive...-commanded retraction of the flaps. This failure could lead to a stall during a landing approach. Relevant... result in an un-commanded retraction of the flaps. This failure could lead to a stall during a landing...

Flight assessment of the onboard propulsion system model for the Performance Seeking Control algorithm on an F-15 aircraft

NASA Technical Reports Server (NTRS)

Orme, John S.; Schkolnik, Gerard S.

1995-01-01

Performance Seeking Control (PSC), an onboard, adaptive, real-time optimization algorithm, relies upon an onboard propulsion system model. Flight results illustrated propulsion system performance improvements as calculated by the model. These improvements were subject to uncertainty arising from modeling error. Thus to quantify uncertainty in the PSC performance improvements, modeling accuracy must be assessed. A flight test approach to verify PSC-predicted increases in thrust (FNP) and absolute levels of fan stall margin is developed and applied to flight test data. Application of the excess thrust technique shows that increases of FNP agree to within 3 percent of full-scale measurements for most conditions. Accuracy to these levels is significant because uncertainty bands may now be applied to the performance improvements provided by PSC. Assessment of PSC fan stall margin modeling accuracy was completed with analysis of in-flight stall tests. Results indicate that the model overestimates the stall margin by between 5 to 10 percent. Because PSC achieves performance gains by using available stall margin, this overestimation may represent performance improvements to be recovered with increased modeling accuracy. Assessment of thrust and stall margin modeling accuracy provides a critical piece for a comprehensive understanding of PSC's capabilities and limitations.

Public Attitudes to Housing Systems for Pregnant Pigs.

PubMed

Ryan, E B; Fraser, D; Weary, D M

2015-01-01

Understanding concerns about the welfare of farm animals is important for the development of socially sustainable production practices. This study used an online survey to test how views on group versus stall housing for pregnant sows varied when Canadian and US participants were provided information about these systems, including access to scientific papers, YouTube videos, Google images, and a frequently-asked-questions page (S1 Appendix). Initial responses and changes in responses after accessing the information were analyzed from Likert scores of 242 participants and from their written comments. Participants were less willing to accept the use of gestation stalls after viewing information on sow housing. For example, initially 30.4% of respondents indicated that they supported the use of gestation stalls; this declined to 17.8% after participants were provided additional information. Qualitative analysis of comments showed that supporters of gestation stalls expressed concern about the spread of disease and aggression between animals in less confined systems, whereas supporters of group housing placed more emphasis on the sow's ability to interact socially and perform natural behaviors. These results point to public opposition to the use of gestation stalls, and indicate that the more that the public learns about gestation stalls the less willing they will be to accept their use.

Mcm2 deficiency results in short deletions allowing high resolution identification of genes contributing to lymphoblastic lymphoma

PubMed Central

Rusiniak, Michael E.; Kunnev, Dimiter; Freeland, Amy; Cady, Gillian K.; Pruitt, Steven C.

2011-01-01

Mini-chromosome maintenance (Mcm) proteins are part of the replication licensing complex that is loaded onto chromatin during the G1-phase of the cell cycle and required for initiation of DNA replication in the subsequent S-phase. Mcm proteins are typically loaded in excess of the number of locations that are utilized during S-phase. Nonetheless, partial depletion of Mcm proteins leads to cancers and stem cell deficiencies. Mcm2 deficient mice, on a 129Sv genetic background, display a high rate of thymic lymphoblastic lymphoma. Here array comparative genomic hybridization (aCGH) is utilized to characterize the genetic damage accruing in these tumors. The predominant events are deletions averaging less than 0.5 Mb, considerably shorter than observed in prior studies using alternative mouse lymphoma models or human tumors. Such deletions facilitate identification of specific genes and pathways responsible for the tumors. Mutations in many genes that have been implicated in human lymphomas are recapitulated in this mouse model. These features, and the fact that the mutation underlying the accelerated genetic damage does not target a specific gene or pathway a priori, are valuable features of this mouse model for identification of tumor suppressor genes. Genes affected in all tumors include Pten, Tcfe2a, Mbd3 and Setd1b. Notch1 and additional genes are affected in subsets of tumors. The high frequency of relatively short deletions is consistent with elevated recombination between nearby stalled replication forks in Mcm2 deficient mice. PMID:22158038

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.

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

PubMed Central

Min, Jaewon; Wright, Woodring E.

2017-01-01

ABSTRACT Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae. Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer. PMID:28760773

Three-Dimensional Aerodynamic Instabilities In Multi-Stage Axial Compressors

NASA Technical Reports Server (NTRS)

Tan, Choon S.; Gong, Yifang; Suder, Kenneth L. (Technical Monitor)

2001-01-01

This thesis presents the conceptualization and development of a computational model for describing three-dimensional non-linear disturbances associated with instability and inlet distortion in multistage compressors. Specifically, the model is aimed at simulating the non-linear aspects of short wavelength stall inception, part span stall cells, and compressor response to three-dimensional inlet distortions. The computed results demonstrated the first-of-a-kind capability for simulating short wavelength stall inception in multistage compressors. The adequacy of the model is demonstrated by its application to reproduce the following phenomena: (1) response of a compressor to a square-wave total pressure inlet distortion; (2) behavior of long wavelength small amplitude disturbances in compressors; (3) short wavelength stall inception in a multistage compressor and the occurrence of rotating stall inception on the negatively sloped portion of the compressor characteristic; (4) progressive stalling behavior in the first stage in a mismatched multistage compressor; (5) change of stall inception type (from modal to spike and vice versa) due to IGV stagger angle variation, and "unique rotor tip incidence" at these points where the compressor stalls through short wavelength disturbances. The model has been applied to determine the parametric dependence of instability inception behavior in terms of amplitude and spatial distribution of initial disturbance, and intra-blade-row gaps. It is found that reducing the inter-blade row gaps suppresses the growth of short wavelength disturbances. It is also concluded from these parametric investigations that each local component group (rotor and its two adjacent 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. For completeness, the methodology has been extended to describe finite amplitude disturbances in high-speed compressors. Results are presented for the response of a transonic compressor subjected to inlet distortions.

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.).

SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner.

PubMed

Lee, Deokjae; An, Jungeun; Park, Young-Un; Liaw, Hungjiun; Woodgate, Roger; Park, Jun Hong; Myung, Kyungjae

2017-04-25

Many DNA repair proteins have additional functions other than their roles in DNA repair. In addition to catalyzing PCNA polyubiquitylation in response to the stalling of DNA replication, SHPRH has the additional function of facilitating rRNA transcription by localizing to the ribosomal DNA (rDNA) promoter in the nucleoli. SHPRH was recruited to the rDNA promoter using its plant homeodomain (PHD), which interacts with histone H3 when the fourth lysine of H3 is not trimethylated. SHPRH enrichment at the rDNA promoter was inhibited by cell starvation, by treatment with actinomycin D or rapamycin, or by depletion of CHD4. SHPRH also physically interacted with the RNA polymerase I complex. Taken together, we provide evidence that SHPRH functions in rRNA transcription through its interaction with histone H3 in a mammalian target of rapamycin (mTOR)-dependent manner.

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

Separated transonic airfoil flow calculations with a nonequilibrium turbulence model

NASA Technical Reports Server (NTRS)

King, L. S.; Johnson, D. A.

1985-01-01

Navier-Stokes transonic airfoil calculations based on a recently developed nonequilibrium, turbulence closure model are presented for a supercritical airfoil section at transonic cruise conditions and for a conventional airfoil section at shock-induced stall conditions. Comparisons with experimental data are presented which show that this nonequilibrium closure model performs significantly better than the popular Baldwin-Lomax and Cebeci-Smith equilibrium algebraic models when there is boundary-layer separation that results from the inviscid-viscous interactions.

A Method to Predict Compressor Stall in the TF34-100 Turbofan Engine Utilizing Real-Time Performance Data

DTIC Science & Technology

2015-06-01

A METHOD TO PREDICT COMPRESSOR STALL IN THE TF34-100 TURBOFAN ENGINE UTILIZING REAL-TIME PERFORMANCE...THE TF34-100 TURBOFAN ENGINE UTILIZING REAL-TIME PERFORMANCE DATA THESIS Presented to the Faculty Department of Systems Engineering and...036 A METHOD TO PREDICT COMPRESSOR STALL IN THE TF34-100 TURBOFAN ENGINE UTILIZING REAL-TIME PERFORMANCE DATA Shuxiang ‘Albert’ Li, BS

Final Environmental Assessment, Family Camp Facility Buckey Air Force Base, Colorado

DTIC Science & Technology

2008-02-07

Table 2-1 for details. This will provide restrooms, showers , and laundry facilities. The proposed FamCamp originally included ten new tent sites...compliant. Two shower stalls will be provided, one of which meets ADA requirements. The interior finishes will consist of ceramic tile floor...with one of the toilet stalls being ADA compliant. Two shower stalls will be provided, one of which meets ADA requirements. The interior finishes

An Experimental Study of Dynamic Stall on Advanced Airfoil Sections. Volume 1. Summary of the Experiment.

DTIC Science & Technology

1982-07-01

Aeronautics and United States Army Space Administration Aviation Research and Ames Remrch Cente Development Command Moffett Field. California 94035 St...appear to be more important than airfoil shape in determining the dynamic- stall airloads. 1. INTRODUCTION Retreating- blade stall limits the high-speed...12.2% Thick R.A.E. Aerofoil Section. RAE Technical Report 68303, Royal Aircraft Establishment, Farnborough Hants, England, Jan. 1969. 14. Fromme, J. A

Stall-proof Airplanes

NASA Technical Reports Server (NTRS)

Lachmann, G

1927-01-01

My lecture has to do with the following questions. Is the danger of stalling necessarily inherent in the airplane in its present form and structure, or can it be diminished or eliminated by suitable means? Do we possess such means or devices and how must they operate? In this connection I will devote special attention to the exhibition of stall-proof airplanes by Fokker under the auspices of the English Air Ministry, which took place in Croyden last April.

Ch-47C Fixed-System Stall-Flutter Damping

DTIC Science & Technology

1975-08-01

flutter. The steady and vibratory loads in the cyclic-trim linkage are so related that motions across the control system’s mechan- ical free play could...be a significant part of the stall-flutter motion, depending on the magnitude of the free play . For this reason it is recommended that future testing...include the deter- mination of the effects of control-system free play on the stall-flutter responses. , f ,**~ - ,***,- * **4 , - - *. i

Dynamic stall experiments on the NACA 0012 airfoil

NASA Technical Reports Server (NTRS)

Mcalister, K. W.; Carr, L. W.; Mccroskey, W. J.

1978-01-01

The flow over a NACA 0012 airfoil undergoing large oscillations in pitch was experimentally studied at a Reynolds number of and over a range of frequencies and amplitudes. Hot-wire probes and surface-pressure transducers were used to clarify the role of the laminar separation bubble, to delineate the growth and shedding of the stall vortex, and to quantify the resultant aerodynamic loads. In addition to the pressure distributions and normal force and pitching moment data that have often been obtained in previous investigations, estimates of the unsteady drag force during dynamic stall have been derived from the surface pressure measurements. Special characteristics of the pressure response, which are symptomatic of the occurrence and relative severity of moment stall, have also been examined.

Analysis and test evaluation of the dynamic response and stability of three advanced turboprop models at low forward speed

NASA Technical Reports Server (NTRS)

Smith, Arthur F.

1985-01-01

Results of wind tunnel tests at low forward speed for blade dynamic response and stability of three 62.2 cm (24.5 in) diameter models of the Prop-Fan, advanced turboprop, are presented. Measurements of dynamic response were made with the rotors mounted on an isolated nacelle, with varying tilt for nonuniform inflow. Low speed stall flutter tests were conducted at Mach numbers from 0.0 to 0.35. Measurements are compared to Eigen-solution flutter boundaries. Calculated 1P stress response agrees favorably with experiment. Predicted stall flutter boundaries correlate well with measured high stress regions. Stall flutter is significantly reduced by increased blade sweep. Susceptibility to stall flutter decreases rapidly with forward speed.

Stall Recovery Guidance Algorithms Based on Constrained Control Approaches

NASA Technical Reports Server (NTRS)

Stepanyan, Vahram; Krishnakumar, Kalmanje; Kaneshige, John; Acosta, Diana

2016-01-01

Aircraft loss-of-control, in particular approach to stall or fully developed stall, is a major factor contributing to aircraft safety risks, which emphasizes the need to develop algorithms that are capable of assisting the pilots to identify the problem and providing guidance to recover the aircraft. In this paper we present several stall recovery guidance algorithms, which are implemented in the background without interfering with flight control system and altering the pilot's actions. They are using input and state constrained control methods to generate guidance signals, which are provided to the pilot in the form of visual cues. It is the pilot's decision to follow these signals. The algorithms are validated in the pilot-in-the loop medium fidelity simulation experiment.

A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system

PubMed Central

Zeier, Zane; Aguilar, J Santiago; Lopez, Cecilia M; Devi-Rao, G B; Watson, Zachary L; Baker, Henry V; Wagner, Edward K; Bloom, David C

2010-01-01

Herpes simplex virus type 1 (HSV-1)–based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4−). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4− vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the non-replicating vector largely resembles mock infection. PMID:20095947

Replication of Minute Virus of Mice in Murine Cells Is Facilitated by Virally Induced Depletion of p21

PubMed Central

Adeyemi, Richard O.

2012-01-01

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G2/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK. PMID:22623787

Development of High Speed Imaging and Analysis Techniques Compressible Dynamics Stall

NASA Technical Reports Server (NTRS)

Chandrasekhara, M. S.; Carr, L. W.; Wilder, M. C.; Davis, Sanford S. (Technical Monitor)

1996-01-01

Dynamic stall has limited the flight envelope of helicopters for many years. The problem has been studied in the laboratory as well as in flight, but most research, even in the laboratory, has been restricted to surface measurement techniques such as pressure transducers or skin friction gauges, except at low speed. From this research, it became apparent that flow visualization tests performed at Mach numbers representing actual flight conditions were needed if the complex physics associated with dynamic stall was to be properly understood. However, visualization of the flow field during compressible conditions required carefully aligned and meticulously reconstructed holographic interferometry. As part of a long-range effort focused on exposing of the physics of compressible dynamic stall, a research wind tunnel was developed at NASA Ames Research Center which permits visual access to the full flow field surrounding an oscillating airfoil during compressible dynamic stall. Initially, a stroboscopic schlieren technique was used for visualization of the stall process, but the primary research tool has been point diffraction interferometry(PDI), a technique carefully optimized for use in th is project. A review of the process of development of PDI will be presented in the full paper. One of the most valuable aspects of PDI is the fact that interferograms are produced in real time on a continuous basis. The use of a rapidly-pulsed laser makes this practical; a discussion of this approach will be presented in the full paper. This rapid pulsing(up to 40,000 pulses/sec) produces interferograms of the rapidly developing dynamic stall field in sufficient resolution(both in space and time) that the fluid physics of the compressible dynamic stall flowfield can be quantitatively determined, including the gradients of pressure in space and time. This permits analysis of the influence of the effect of pitch rate, Mach number, Reynolds number, amplitude of oscillation, and other parameters on the dynamic stall process. When interferograms can be captured in real time, the potential for real-time mapping of a developing unsteady flow such as dynamic stall becomes a possibility. This has been achieved in the present case through the use of a high-speed drum camera combined with electronic circuitry which has resulted in a series of interferograms obtained during a single cycle of dynamic stall; images obtained at the rate of 20 KHz will be presented as a part of the formal presentation. Interferometry has been available for a long time; however, most of its use has been limited to visualization. The present research has focused on use of interferograms for quantitative mapping of the flow over oscillating airfoils. Instantaneous pressure distributions can now be obtained semi-automatically, making practical the analysis of the thousands of interferograms that are produced in this research. A review of the techniques that have been developed as part of this research effort will be presented in the final paper.

Health risk assessment of occupational exposure to particulate-phase polycyclic aromatic hydrocarbons associated with Chinese, Malay and Indian cooking.

PubMed

Wei See, Siao; Karthikeyan, Sathrugnan; Balasubramanian, Rajasekhar

2006-03-01

Food cooking using liquefied petroleum gas (LPG) has received considerable attention in recent years since it is an important source of particulate air pollution in indoor environments for non-smokers. Exposure to organic compounds such as polycyclic aromatic hydrocarbons (PAHs) contained in particles is of particular health concern since some of these compounds are suspected carcinogens. It is therefore necessary to chemically characterize the airborne particles emitted from gas cooking to assess their possible health impacts. In this work, the levels of fine particulate matter (PM(2.5)) and 16 priority PAHs were determined in three different ethnic commercial kitchens, specifically Chinese, Malay and Indian food stalls, where distinctive cooking methods were employed. The mass concentrations of PM(2.5) and PAHs, and the fraction of PAHs in PM(2.5) were the highest at the Malay stall (245.3 microg m(-3), 609.0 ng m(-3), and 0.25%, respectively), followed by the Chinese stall (201.6 microg m(-3), 141.0 ng m(-3), and 0.07%), and the Indian stall (186.9 microg m(-3), 37.9 ng m(-3), and 0.02%). This difference in the levels of particulate pollution among the three stalls may be attributed to the different cooking methods employed at the food stalls, the amount of food cooked, and the cooking time, although the most sensitive parameter appears to be the predominant cooking method used. Frying processes, especially deep-frying, produce more air pollutants, possibly due to the high oil temperatures used in such operations. Furthermore, it is found that frying, be it deep-frying at the Malay stall or stir-frying at the Chinese stall, gave rise to an abundance of higher molecular weight PAHs such as benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene and benzo[g,h,i]perylene whereas low-temperature cooking, such as simmering at the Indian stall, has a higher concentration of lower molecular weight PAHs. In addition, the correlation matrices and diagnostic ratios of PAHs were calculated to determine the markers of gas cooking. To evaluate the potential health threat due to inhalation exposure from the indoor particulate pollution, excess lifetime cancer risk (ELCR) was also calculated for an exposed individual. The findings suggest that cooking fumes in the three commercial kitchens pose adverse health effects.

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.

Effectiveness of a dynein team in a tug of war helped by reduced load sensitivity of detachment: evidence from the study of bidirectional endosome transport in D. discoideum.

PubMed

Bhat, Deepak; Gopalakrishnan, Manoj

2012-08-01

Bidirectional cargo transport by molecular motors in cells is a complex phenomenon in which the cargo (usually a vesicle) alternately moves in retrograde and anterograde directions. In this case, teams of oppositely pulling motors (e.g., kinesin and dynein) bind to the cargo, simultaneously, and 'coordinate' their activity such that the motion consists of spells of positively and negatively directed segments, separated by pauses of varying duration. A set of recent experiments have analyzed the bidirectional motion of endosomes in the amoeba D. discoideum in detail. It was found that in between directional switches, a team of five to six dyneins stall a cargo against a stronger kinesin in a tug of war, which lasts for almost a second. As the mean detachment time of a kinesin under its stall load was also observed to be ∼1 s, we infer that the collective detachment time of the dynein assembly must also be similar. Here, we analyze this inference from a modeling perspective, using experimentally measured single-molecule parameters as inputs. We find that the commonly assumed exponential load-dependent detachment rate is inconsistent with observations, as it predicts that a five-dynein assembly will detach under its combined stall load in less than a hundredth of a second. A modified model where the load-dependent unbinding rate is assumed to saturate at stall-force level for super-stall loads gives results which are in agreement with experimental data. Our analysis suggests that the load-dependent detachment of a dynein in a team is qualitatively different at sub-stall and super-stall loads, a conclusion which is likely to have implications in other situations involving collective effects of many motors.