Science.gov

Sample records for 9-1-1 checkpoint clamp

  1. The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint

    PubMed Central

    Majka, Jerzy; Niedziela-Majka, Anita; Burgers, Peter M. J.

    2007-01-01

    SUMMARY Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA-damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a non-specific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1. PMID:17189191

  2. The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint.

    PubMed

    Majka, Jerzy; Niedziela-Majka, Anita; Burgers, Peter M J

    2006-12-28

    Yeast Mec1/Ddc2 protein kinase, the ortholog of human ATR/ATRIP, plays a central role in the DNA damage checkpoint. The PCNA-like clamp Rad17/Mec3/Ddc1 (the 9-1-1 complex in human) and its loader Rad24-RFC are also essential components of this signal transduction pathway. Here we have studied the role of the clamp in regulating Mec1, and we delineate how the signal generated by DNA lesions is transduced to the Rad53 effector kinase. The checkpoint clamp greatly activates the kinase activity of Mec1, but only if the clamp is appropriately loaded upon partial duplex DNA. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, and the Rpa1 and Rpa2 subunits of RPA. Phosphorylation of Rad53, and of human PHAS-1, a nonspecific target, also requires a properly loaded clamp. Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1. PMID:17189191

  3. Genome Protection by the 9-1-1 Complex Subunit HUS1 Requires Clamp Formation, DNA Contacts, and ATR Signaling-independent Effector Functions.

    PubMed

    Lim, Pei Xin; Patel, Darshil R; Poisson, Kelsey E; Basuita, Manpreet; Tsai, Charlton; Lyndaker, Amy M; Hwang, Bor-Jang; Lu, A-Lien; Weiss, Robert S

    2015-06-12

    The RAD9A-HUS1-RAD1 (9-1-1) complex is a heterotrimeric clamp that promotes checkpoint signaling and repair at DNA damage sites. In this study, we elucidated HUS1 functional residues that drive clamp assembly, DNA interactions, and downstream effector functions. First, we mapped a HUS1-RAD9A interface residue that was critical for 9-1-1 assembly and DNA loading. Next, we identified multiple positively charged residues in the inner ring of HUS1 that were crucial for genotoxin-induced 9-1-1 chromatin localization and ATR signaling. Finally, we found two hydrophobic pockets on the HUS1 outer surface that were important for cell survival after DNA damage. Interestingly, these pockets were not required for 9-1-1 chromatin localization or ATR-mediated CHK1 activation but were necessary for interactions between HUS1 and its binding partner MYH, suggesting that they serve as interaction domains for the recruitment and coordination of downstream effectors at damage sites. Together, these results indicate that, once properly loaded onto damaged DNA, the 9-1-1 complex executes multiple, separable functions that promote genome maintenance. PMID:25911100

  4. Genome Protection by the 9-1-1 Complex Subunit HUS1 Requires Clamp Formation, DNA Contacts, and ATR Signaling-independent Effector Functions*

    PubMed Central

    Lim, Pei Xin; Patel, Darshil R.; Poisson, Kelsey E.; Basuita, Manpreet; Tsai, Charlton; Lyndaker, Amy M.; Hwang, Bor-Jang; Lu, A-Lien; Weiss, Robert S.

    2015-01-01

    The RAD9A-HUS1-RAD1 (9-1-1) complex is a heterotrimeric clamp that promotes checkpoint signaling and repair at DNA damage sites. In this study, we elucidated HUS1 functional residues that drive clamp assembly, DNA interactions, and downstream effector functions. First, we mapped a HUS1-RAD9A interface residue that was critical for 9-1-1 assembly and DNA loading. Next, we identified multiple positively charged residues in the inner ring of HUS1 that were crucial for genotoxin-induced 9-1-1 chromatin localization and ATR signaling. Finally, we found two hydrophobic pockets on the HUS1 outer surface that were important for cell survival after DNA damage. Interestingly, these pockets were not required for 9-1-1 chromatin localization or ATR-mediated CHK1 activation but were necessary for interactions between HUS1 and its binding partner MYH, suggesting that they serve as interaction domains for the recruitment and coordination of downstream effectors at damage sites. Together, these results indicate that, once properly loaded onto damaged DNA, the 9-1-1 complex executes multiple, separable functions that promote genome maintenance. PMID:25911100

  5. Clamping the Mec1/ATR checkpoint kinase into action.

    PubMed

    Majka, Jerzy; Burgers, Peter M J

    2007-05-15

    The yeast checkpoint protein kinase Mec1, the ortholog of human ATR, is the essential upstream regulator of the cell cycle checkpoint in response to DNA damage and to stalling of DNA replication forks. The activity of Mec1/ATR is not directly regulated by the DNA substrates that signal checkpoint activation. Rather the signal appears to be transduced to Mec1 by factors that interact with the signaling DNA substrates. One of these factors, the DNA damage checkpoint clamp Rad17-Mec3-Ddc1 (human 9-1-1) is loaded onto gapped DNA resulting from the partial repair of DNA damage, and the Ddc1 subunit of this complex activates Mec1. In vertebrate cells, the TopBP1 protein (Cut5 in S. pombe and Dpb11 in S. cervisiae) that is also required for establishment of the replication fork, functions during replication fork dysfunction to activate ATR. Both mechanisms of activation generally upregulate the kinase activity towards all downstream targets. PMID:17495536

  6. The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage

    PubMed Central

    Alver, Robert C.; Zhang, Tianji; Josephrajan, Ajeetha; Fultz, Brandy L.; Hendrix, Chance J.; Das-Bradoo, Sapna; Bielinsky, Anja-Katrin

    2014-01-01

    Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair. PMID:24972833

  7. Yeast Rad17/Mec3/Ddc1: A sliding clamp for the DNA damage checkpoint

    PubMed Central

    Majka, Jerzy; Burgers, Peter M. J.

    2003-01-01

    The Saccharomyces cerevisiae Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad17/3/1) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad17/3/1 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad17/3/1 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad17/3/1 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad17/3/1 showed no detectable exonuclease activity. PMID:12604797

  8. Association of the Rad9-Rad1-Hus1 checkpoint clamp with MYH DNA glycosylase and DNA.

    PubMed

    Hwang, Bor-Jang; Jin, Jin; Gunther, Randall; Madabushi, Amrita; Shi, Guoli; Wilson, Gerald M; Lu, A-Lien

    2015-07-01

    Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9-1-1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9-1-1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134-155) is a key determinant of MYH binding. Both the N-(residues 1-146) and C-terminal (residues 147-280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1(K136A) mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9(1-266) (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9(1-266)-1-1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9(1-266)-1-1 to 5'-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9-1-1 subunits to MYH-directed BER based on subunit asymmetry in protein-protein interactions and DNA binding events. PMID:26021743

  9. Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding.

    PubMed

    Takeishi, Yukimasa; Iwaya-Omi, Rie; Ohashi, Eiji; Tsurimoto, Toshiki

    2015-08-01

    The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9(ΔC)-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9(ΔC)-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism. PMID:26088138

  10. Checkpointing filesystem

    DOEpatents

    Gara, Alan G.; Giampapa, Mark E.; Steinmacher-Burow, Burkhard D.

    2005-05-17

    The present in invention is directed to a checkpointing filesystem of a distributed-memory parallel supercomputer comprising a node that accesses user data on the filesystem, the filesystem comprising an interface that is associated with a disk for storing the user data. The checkpointing filesystem provides for taking and checkpoint of the filesystem and rolling back to a previously taken checkpoint, as well as for writing user data to and deleting user data from the checkpointing filesystem. The checkpointing filesystem provides a recently written file allocation table (WFAT) for maintaining information regarding the user data written since a previously taken checkpoint and a recently deleted file allocation table (DFAT) for maintaining information regarding user data deleted from since the previously taken checkpoint, both of which are utilized by the checkpointing filesystem to take a checkpoint of the filesystem and rollback the filesystem to a previously taken checkpoint, as well as to write and delete user data from the checkpointing filesystem.

  11. Post clamp

    NASA Technical Reports Server (NTRS)

    Ramsey, John K. (Inventor); Meyn, Erwin H. (Inventor)

    1990-01-01

    A pair of spaced collars are mounted at right angles on a clamp body by retaining rings which enable the collars to rotate with respect to the clamp body. Mounting posts extend through aligned holes in the collars and clamp body. Each collar can be clamped onto the inserted post while the clamp body remains free to rotate about the post and collar. The clamp body is selectively clamped onto each post.

  12. Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.

    PubMed

    Xu, Yong-Jie

    2016-04-01

    DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2(+) sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling are seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside the nucleus, a previously unknown mechanism. PMID:26746798

  13. Rigid clamp

    DOEpatents

    Benavides, G.L.; Burt, J.D.

    1994-07-12

    The invention relates to a clamp mechanism that can be used to attach or temporarily support objects inside of tubular goods. The clamp mechanism can also be modified so that it grips objects. The clamp has a self-centering feature to accommodate out-of-roundness or other internal defections in tubular objects such as pipe. A plurality of clamping shoes are expanded by a linkage which is preferably powered by a motor to contact the inside of a pipe. The motion can be reversed and jaw elements can be connected to the linkage so as to bring the jaws together to grab an object. 12 figs.

  14. Rigid clamp

    DOEpatents

    Benavides, Gilbert L.; Burt, Jack D.

    1994-01-01

    The invention relates to a clamp mechanism that can be used to attach or temporarily support objects inside of tubular goods. The clamp mechanism can also be modified so that it grips objects. The clamp has a self-centering feature to accommodate out-of-roundness or other internal defections in tubular objects such as pipe. A plurality of clamping shoes are expanded by a linkage which is preferably powered by a motor to contact the inside of a pipe. The motion can be reversed and jaw elements can be connected to the linkage so as to bring the jaws together to grab an object.

  15. The KYxxL motif in Rad17 protein is essential for the interaction with the 9-1-1 complex.

    PubMed

    Fukumoto, Yasunori; Ikeuchi, Masayoshi; Nakayama, Yuji; Yamaguchi, Naoto

    2016-09-01

    ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The Rad17-RFC and Rad9-Rad1-Hus1 (9-1-1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of Rad17 protein, and demonstrated that this motif is essential for the interaction with the 9-1-1 complex. We also show that UV-induced Rad17 phosphorylation is increased in the Rad17 KYxxL mutants. These data indicate that the interaction with the 9-1-1 complex is not required for Rad17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9-1-1 complex plays a negative regulatory role in the Rad17 phosphorylation. We also show that the nucleotide-binding activity of Rad17 is required for its nuclear localization. PMID:27387238

  16. DNA damage checkpoints in mammals.

    PubMed

    Niida, Hiroyuki; Nakanishi, Makoto

    2006-01-01

    DNA damage is a common event and probably leads to mutation or deletion within chromosomal DNA, which may cause cancer or premature aging. DNA damage induces several cellular responses including DNA repair, checkpoint activity and the triggering of apoptotic pathways. DNA damage checkpoints are associated with biochemical pathways that end delay or arrest of cell-cycle progression. These checkpoints engage damage sensor proteins, such as the Rad9-Rad1-Hus1 (9-1-1) complex, and the Rad17-RFC complex, in the detection of DNA damage and transduction of signals to ATM, ATR, Chk1 and Chk2 kinases. Chk1 and Chk2 kinases regulate Cdc25, Wee1 and p53 that ultimately inactivate cyclin-dependent kinases (Cdks) which inhibit cell-cycle progression. In this review, we discuss the molecular mechanisms by which DNA damage is recognized by sensor proteins and signals are transmitted to Cdks. We classify the genes involved in checkpoint signaling into four categories, namely sensors, mediators, transducers and effectors, although their proteins have the broad activity, and thus this classification is for convenience and is not definitive. PMID:16314342

  17. Charlie's Clamp.

    ERIC Educational Resources Information Center

    Tarino, Janet Z.

    1998-01-01

    Presents a version of the crush-the-can demonstration which is a hands-on activity in which students use an inexpensive, easily made holder for the can called Charlie's clamp. Includes some suggestions for the follow-up discussion. (DDR)

  18. 78 FR 21879 - Improving 9-1-1 Reliability; Reliability and Continuity of Communications Networks, Including...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-12

    ...The Federal Communications Commission proposes a range of approaches to ensure that providers of 9-1-1 communications services implement best practices and other sound engineering principles to improve the reliability and resiliency of the Nation's 9-1-1 networks. The Notice of Proposed Rulemaking also proposes amendments to the Commission's current rules to clarify and add specificity to......

  19. Colocalization of Sensors Is Sufficient to Activate the DNA Damage Checkpoint in the Absence of Damage

    PubMed Central

    Bonilla, Carla Yaneth; Melo, Justine Amy

    2010-01-01

    Summary Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether colocalization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially colocalized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation, and metaphase delay, demonstrating that colocalization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to establish that CDK functions in the checkpoint pathway downstream of damage processing and checkpoint protein recruitment. This CDK dependence is likely, at least in part, through Rad9, since mutation of CDK consensus sites compromised its checkpoint function. PMID:18471973

  20. Clamp usable as jig and lifting clamp

    DOEpatents

    Tsuyama, Yoshizo

    1976-01-01

    There is provided a clamp which is well suited for use as a lifting clamp for lifting and moving materials of assembly in a shipyard, etc. and as a pulling jig in welding and other operations. The clamp comprises a clamp body including a shackle for engagement with a pulling device and a slot for receiving an article, and a pair of jaws provided on the leg portions of the clamp body on the opposite sides of the slot to grip the article in the slot, one of said jaws consisting of a screw rod and the other jaw consisting of a swivel jaw with a spherical surface, whereby when the article clamped in the slot by the pair of jaws tends to slide in any direction with respect to the clamp body, the article is more positively gripped by the pair of jaws.

  1. A Strained 9-1-1 System and Threats to Public Health.

    PubMed

    Cannuscio, Carolyn C; Davis, Andrea L; Kermis, Amelia D; Khan, Yasin; Dupuis, Roxanne; Taylor, Jennifer A

    2016-06-01

    The goal of this study was to understand safety climate in the United States (U.S.) fire service, which responded to more than 31 million calls to the 9-1-1 emergency response system in 2013. The majority of those calls (68 %) were for medical assistance, while only 4 % of calls were fire-related, highlighting that the 9-1-1 system serves as a critical public health safety net. We conducted focus groups and interviews with 123 firefighters from 12 fire departments across the United States. Using an iterative analytic approach supported by NVivo 10 software, we developed consensus regarding key themes. Firefighters concurred that the 9-1-1 system is strained and increasingly called upon to deliver Emergency Medical Services (EMS) in the community. Much like the hospital emergency department, EMS frequently assists low-income and elderly populations who have few alternative sources of support. Firefighters highlighted the high volume of low-acuity calls that occupy much of their workload, divert resources from true emergencies, and lead to unwarranted occupational hazards like speeding to respond to non-serious calls. As a result, firefighters reported high occupational stress, low morale, and desensitization to community needs. Firefighters' called for improvements to the 9-1-1 system-the backbone of emergency response in the U.S.-including better systems of triage, more targeted use of EMS resources, continuing education to align with job demands, and a strengthened social safety net to address the persistent needs of poor and elderly populations. PMID:26704911

  2. Coordinating the Global Information Grid Initiative with the NG9-1-1 Initiative

    SciTech Connect

    Michael Schmitt

    2008-05-01

    As the Department of Defense develops the Global Information Grid, the Department of Transportation develops the Next Generation 9-1-1 system. Close examinations of these initiatives show that the two are similar in architectures, applications, and communications interoperability. These similarities are extracted from the lowest user level to the highest commander rank that will be involved in each network. Once the similarities are brought into perspective, efforts should be made to collaborate between the two departments.

  3. Force-Measuring Clamp

    NASA Technical Reports Server (NTRS)

    Nunnelee, Mark (Inventor)

    2004-01-01

    A precision clamp that accurately measures force over a wide range of conditions is described. Using a full bridge or other strain gage configuration. the elastic deformation of the clamp is measured or detected by the strain gages. Thc strain gages transmit a signal that corresponds to the degree of stress upon the clamp. Thc strain gage signal is converted to a numeric display. Calibration is achieved by ero and span potentiometers which enable accurate measurements by the force-measuring clamp.

  4. Photovoltaic panel clamp

    DOEpatents

    Mittan, Margaret Birmingham; Miros, Robert H. J.; Brown, Malcolm P.; Stancel, Robert

    2012-06-05

    A photovoltaic panel clamp includes an upper and lower section. The interface between the assembled clamp halves and the module edge is filled by a flexible gasket material, such as EPDM rubber. The gasket preferably has small, finger like protrusions that allow for easy insertion onto the module edge while being reversed makes it more difficult to remove them from the module once installed. The clamp includes mounting posts or an integral axle to engage a bracket. The clamp also may include a locking tongue to secure the clamp to a bracket.

  5. Photovoltaic panel clamp

    DOEpatents

    Brown, Malcolm P.; Mittan, Margaret Birmingham; Miros, Robert H. J.; Stancel, Robert

    2013-03-19

    A photovoltaic panel clamp includes an upper and lower section. The interface between the assembled clamp halves and the module edge is filled by a flexible gasket material, such as EPDM rubber. The gasket preferably has small, finger like protrusions that allow for easy insertion onto the module edge while being reversed makes it more difficult to remove them from the module once installed. The clamp includes mounting posts or an integral axle to engage a bracket. The clamp also may include a locking tongue to secure the clamp to a bracket.

  6. Radial wedge flange clamp

    DOEpatents

    Smith, Karl H.

    2002-01-01

    A radial wedge flange clamp comprising a pair of flanges each comprising a plurality of peripheral flat wedge facets having flat wedge surfaces and opposed and mating flat surfaces attached to or otherwise engaged with two elements to be joined and including a series of generally U-shaped wedge clamps each having flat wedge interior surfaces and engaging one pair of said peripheral flat wedge facets. Each of said generally U-shaped wedge clamps has in its opposing extremities apertures for the tangential insertion of bolts to apply uniform radial force to said wedge clamps when assembled about said wedge segments.

  7. Cell Differentiation and Checkpoint

    PubMed Central

    Sancho, Sara Cuesta; Ouchi, Toru

    2015-01-01

    DNA damage is induced in many types of cells by internal and external cell stress. When DNA is damaged, DNA Damage Response (DDR) programs are activated to repair the DNA lesions in order to preserve genomic integrity and suppress subsequent malignant transformation. Among these programs is cell cycle checkpoint that ensures cell cycle arrest and subsequent repair of the damaged DNA, apoptosis and senescence in various phases of the cell cycle. Moreover, recent studies have established the cell differentiation checkpoint, the other type of the checkpoint that is specifically activated in the course of differentiation. We will discuss the evidences that support the link between DNA damage proteins and C2C12 cell differentiation. PMID:26998525

  8. Reusable thermal cycling clamp

    NASA Technical Reports Server (NTRS)

    Debnam, W. J., Jr.; Fripp, A. L.; Crouch, R. K. (Inventor)

    1985-01-01

    A reusable metal clamp for retaining a fused quartz ampoule during temperature cycling in the range of 20 deg C to 1000 deg C is described. A compressible graphite foil having a high radial coefficient of thermal expansion is interposed between the fused quartz ampoule and metal clamp to maintain a snug fit between these components at all temperature levels in the cycle.

  9. Quick action clamp

    NASA Technical Reports Server (NTRS)

    Calco, Frank S. (Inventor)

    1991-01-01

    A quick release toggle clamp that utilizes a spring that requires a deliberate positive action for disengagement is presented. The clamp has a sliding bolt that provides a latching mechanism. The bolt is moved by a handle that tends to remain in an engaged position while under tension.

  10. What influences a decision to call 9-1-1?: exploring differential correlates for retired versus employed samples.

    PubMed

    Blau, Gary; Gibson, Gregory; Bentley, Melissa

    2012-01-01

    In fall 2010, a phone survey of 318 retired and 362 employed respondents examined the decision to call 9-1-1 in a simulated emergency situation. Our study purpose was to investigate if there were different correlates between these two samples which would affect their decision to call 9-1-1. Different variables were measured to help explain this 9-1-1 call decision. Reliable scales for measuring the following variables were found: decision to call 9-1-1; emergency medical service (EMS) credentials importance; EMS wrongdoing license revocation; and EMS training consistency. Separate stepwise regression analyses, first controlling for a set of five demographic variables, then adding a set of five perceptual scales, were carried out for the retired and employed samples. After controlling for five demographic variables (race, marital status, age, overall health, and total 2009 income), three perceptual scales--EMS credentials importance, EMS wrongdoing license revocation, and EMS service comparison scales--each had a significant positive relationship to the retired sample's decision to call 9-1-1. For the employed sample, the perceived EMS training consistency scale had a positive relationship with the decision to call 9-1-1 beyond the controlled-for demographics. Study limitations included excluding cell phone users and the simulated emergency situation, while contributions included finding a number of reliable scales for future research. PMID:22735818

  11. Laser beam guard clamps

    DOEpatents

    Dickson, Richard K.

    2010-09-07

    A quick insert and release laser beam guard panel clamping apparatus having a base plate mountable on an optical table, a first jaw affixed to the base plate, and a spring-loaded second jaw slidably carried by the base plate to exert a clamping force. The first and second jaws each having a face acutely angled relative to the other face to form a V-shaped, open channel mouth, which enables wedge-action jaw separation by and subsequent clamping of a laser beam guard panel inserted through the open channel mouth. Preferably, the clamping apparatus also includes a support structure having an open slot aperture which is positioned over and parallel with the open channel mouth.

  12. A monogenean without clamps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ectoparasites face a daily challenge: to remain attached to their host. Polyopisthocotylean monogeneans attach to the surface of fish gills by highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 50 years...

  13. Sperm Patch-Clamp

    PubMed Central

    Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

    2014-01-01

    Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

  14. Clamp for arctic pipeline support

    SciTech Connect

    Morton, A.W.

    1988-11-29

    This patent describes a ring clamp for supporting and anchoring a large diameter metallic arctic pipeline comprising substantially rigid, curved clamp portions adapted to completely encircle the pipeline and fastening means connecting the clamp portions, the clamp portions having inner and outer layers of fiber reinforced rigid polymer material and an intermediate core layer of honeycomb-form aramid paper.

  15. Clamping characteristics study on different types of clamping unit

    SciTech Connect

    Jiao, Zhiwei; Liu, Haichao; Xie, Pengcheng; Yang, Weimin

    2015-05-22

    Plastic products are becoming more and more widely used in aerospace, IT, digital electronics and many other fields. With the development of technology, the requirement of product precision is getting higher and higher. However, type and working performance of clamping unit play a decisive role in product precision. Clamping characteristics of different types of clamping unit are discussed in this article, which use finite element numerical analysis method through the software ABAQUS to study the clamping uniformity, and detect the clamping force repeatability precision. The result shows that compared with toggled three-platen clamping unit, clamping characteristics of internal circulation two-platen clamping unit are better, for instance, its mold cavity deformation and force that bars and mold parting surface suffered are more uniform, and its clamping uniformity and repeatability precision is also better.

  16. Clamp for detonating fuze

    NASA Technical Reports Server (NTRS)

    Holderman, E. J.

    1968-01-01

    Quick acting clamp provides physical support for a closely confined detonating fuse in an application requiring removal and replacement at frequent intervals during test. It can be designed with a base of any required strength and configuration to permit the insertion of an object.

  17. Checkpoint inhibition in meningiomas.

    PubMed

    Bi, Wenya Linda; Wu, Winona W; Santagata, Sandro; Reardon, David A; Dunn, Ian F

    2016-06-01

    Meningiomas are increasingly appreciated to share similar features with other intra-axial central nervous system neoplasms as well as systemic cancers. Immune checkpoint inhibition has emerged as a promising therapy in a number of cancers, with durable responses of years in a subset of patients. Several lines of evidence support a role for immune-based therapeutic strategies in the management of meningiomas, especially high-grade subtypes. Meningiomas frequently originate juxtaposed to venous sinuses, where an anatomic conduit for lymphatic drainage resides. Multiple populations of immune cells have been observed in meningiomas. PD-1/PD-L1 mediated immunosuppression has been implicated in high-grade meningiomas, with association between PD-L1 expression with negative prognostic outcome. These data point to the promise of future combinatorial therapeutic strategies in meningioma. PMID:27197540

  18. Requirements for Linux Checkpoint/Restart

    SciTech Connect

    Duell, Jason; Hargrove, Paul H.; Roman, Eric S.

    2002-02-26

    This document has 4 main objectives: (1) Describe data to be saved and restored during checkpoint/restart; (2) Describe how checkpoint/restart is used within the context of the Scalable Systems environment, and MPI applications; (3) Identify issues for a checkpoint/restart implementation; and (4) Sketch the architecture of a checkpoint/restart implementation.

  19. Checkpointing in speculative versioning caches

    DOEpatents

    Eichenberger, Alexandre E; Gara, Alan; Gschwind, Michael K; Ohmacht, Martin

    2013-08-27

    Mechanisms for generating checkpoints in a speculative versioning cache of a data processing system are provided. The mechanisms execute code within the data processing system, wherein the code accesses cache lines in the speculative versioning cache. The mechanisms further determine whether a first condition occurs indicating a need to generate a checkpoint in the speculative versioning cache. The checkpoint is a speculative cache line which is made non-speculative in response to a second condition occurring that requires a roll-back of changes to a cache line corresponding to the speculative cache line. The mechanisms also generate the checkpoint in the speculative versioning cache in response to a determination that the first condition has occurred.

  20. Cantilever clamp fitting

    NASA Technical Reports Server (NTRS)

    Melton, Patrick B. (Inventor)

    1989-01-01

    A device is disclosed for sealing and clamping a cylindrical element which is to be attached to an object such as a wall, a pressurized vessel or another cylindrical element. The device includes a gland having an inner cylindrical wall, which is threaded at one end and is attached at a bendable end to a deformable portion, which in turn is attached to one end of a conical cantilever structure. The other end of the cantilever structure connects at a bendable area to one end of an outer cylindrical wall. The opposite end of cylindrical wall terminates in a thickened portion, the radially outer surface of which is adapted to accommodate a tool for rotating the gland. The terminal end of cylindrical wall also includes an abutment surface, which is adapted to engage a seal, which in turn engages a surface of a receiver. The receiver further includes a threaded portion for engagement with the threaded portion of gland whereby a tightening rotation of gland relative to receiver will cause relative movement between cylindrical walls and of gland. This movement causes a rotation of the conical structure and thus a bending action at bending area and at the bending end of the upper end of inner cylindrical wall. These rotational and bending actions result in a forcing of the deformable portion radially inwardly so as to contact and deform a pipe. This forcible contact creates a seal between gland and pipe, and simultaneously clamps the pipe in position.

  1. Energy harvesting under excitation of clamped-clamped beam

    NASA Astrophysics Data System (ADS)

    Batra, Ashok; Alomari, Almuatasim; Aggarwal, Mohan; Bandyopadhyay, Alak

    2016-04-01

    In this article, a piezoelectric energy harvesting has been developed experimentally and theoretically based on Euler- Bernoulli Theory. A PVDF piezoelectric thick film has attached along of clamped-clamped beam under sinusoidal base excitation of shaker. The results showed a good agreement between the experimental and simulation of suggested model. The voltage output frequency response function (FRF), current FRF, and output power has been studied under short and open circuit conditions at first vibration mode. The mode shape of the clamped-clamped beam for first three resonance frequency has been modeled and investigated using COMSOL Multiphysics and MATLAB.

  2. 77 FR 45607 - 9-1-1 Resiliency and Reliability In Wake of, June 29, 2012, Derecho Storm In Central, Mid...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-01

    ... COMMISSION 9-1-1 Resiliency and Reliability In Wake of, June 29, 2012, Derecho Storm In Central, Mid-Atlantic..., 2012. ADDRESSES: Pursuant to sections 1.415 and 1.419 of the Commission's rules, 47 CFR 1.415, 1.419... related to the impact of the storm on emergency and 9-1-1 communications accessed by...

  3. Lazy checkpoint coordination for bounding rollback propagation

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. Kent

    1992-01-01

    Independent checkpointing allows maximum process autonomy but suffers from potential domino effects. Coordinated checkpointing eliminates the domino effect by sacrificing a certain degree of process autonomy. In this paper, we propose the technique of lazy checkpoint coordination which preserves process autonomy while employing communication-induced checkpoint coordination for bounding rollback propagation. The introduction of the notion of laziness allows a flexible trade-off between the cost for checkpoint coordination and the average rollback distance. Worst-case overhead analysis provides a means for estimating the extra checkpoint overhead. Communication trace-driven simulation for several parallel programs is used to evaluate the benefits of the proposed scheme for real applications.

  4. Compiler-assisted static checkpoint insertion

    NASA Technical Reports Server (NTRS)

    Long, Junsheng; Fuchs, W. K.; Abraham, Jacob A.

    1992-01-01

    This paper describes a compiler-assisted approach for static checkpoint insertion. Instead of fixing the checkpoint location before program execution, a compiler enhanced polling mechanism is utilized to maintain both the desired checkpoint intervals and reproducible checkpoint 1ocations. The technique has been implemented in a GNU CC compiler for Sun 3 and Sun 4 (Sparc) processors. Experiments demonstrate that the approach provides for stable checkpoint intervals and reproducible checkpoint placements with performance overhead comparable to a previously presented compiler assisted dynamic scheme (CATCH) utilizing the system clock.

  5. Hand-Held Power Clamp

    NASA Technical Reports Server (NTRS)

    Clancy, J. P.

    1985-01-01

    Tool furnishes large pushing or pulling forces. Device includes two clamping blocks, two clamping plates, and a motor-driven linear actuator with selflocking screw shaft. Power clamp exerts opening or closing force at push of switch. Tool approximately 1 m long. Originally designed to secure payload aboard Space Shuttle, operated with one hand to apply opening or closing force of up to 1,000 lb (4,400 N). Clamp has potential applications as end effector for industrial robots and in rescue work to push or pull wreckage with great force.

  6. Cell Cycle Regulation by Checkpoints

    PubMed Central

    Barnum, Kevin J.; O’Connell, Matthew J.

    2016-01-01

    Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequences of their dysfunction on cell fate. PMID:24906307

  7. Network support for system initiated checkpoints

    DOEpatents

    Chen, Dong; Heidelberger, Philip

    2013-01-29

    A system, method and computer program product for supporting system initiated checkpoints in parallel computing systems. The system and method generates selective control signals to perform checkpointing of system related data in presence of messaging activity associated with a user application running at the node. The checkpointing is initiated by the system such that checkpoint data of a plurality of network nodes may be obtained even in the presence of user applications running on highly parallel computers that include ongoing user messaging activity.

  8. Non-volatile memory for checkpoint storage

    SciTech Connect

    Blumrich, Matthias A.; Chen, Dong; Cipolla, Thomas M.; Coteus, Paul W.; Gara, Alan; Heidelberger, Philip; Jeanson, Mark J.; Kopcsay, Gerard V.; Ohmacht, Martin; Takken, Todd E.

    2014-07-22

    A system, method and computer program product for supporting system initiated checkpoints in high performance parallel computing systems and storing of checkpoint data to a non-volatile memory storage device. The system and method generates selective control signals to perform checkpointing of system related data in presence of messaging activity associated with a user application running at the node. The checkpointing is initiated by the system such that checkpoint data of a plurality of network nodes may be obtained even in the presence of user applications running on highly parallel computers that include ongoing user messaging activity. In one embodiment, the non-volatile memory is a pluggable flash memory card.

  9. Checkpointing for a hybrid computing node

    DOEpatents

    Cher, Chen-Yong

    2016-03-08

    According to an aspect, a method for checkpointing in a hybrid computing node includes executing a task in a processing accelerator of the hybrid computing node. A checkpoint is created in a local memory of the processing accelerator. The checkpoint includes state data to restart execution of the task in the processing accelerator upon a restart operation. Execution of the task is resumed in the processing accelerator after creating the checkpoint. The state data of the checkpoint are transferred from the processing accelerator to a main processor of the hybrid computing node while the processing accelerator is executing the task.

  10. REMEM: REmote MEMory as Checkpointing Storage

    SciTech Connect

    Jin, Hui; Sun, Xian-He; Chen, Yong; Ke, Tao

    2010-01-01

    Checkpointing is a widely used mechanism for supporting fault tolerance, but notorious in its high-cost disk access. The idea of memory-based checkpointing has been extensively studied in research but made little success in practice due to its complexity and potential reliability concerns. In this study we present the design and implementation of REMEM, a REmote MEMory checkpointing system to extend the checkpointing storage from disk to remote memory. A unique feature of REMEM is that it can be integrated into existing disk-based checkpointing systems seamlessly. A user can flexibly switch between REMEM and disk as checkpointing storage to balance the efficiency and reliability. The implementation of REMEM on Open MPI is also introduced. The experimental results confirm that REMEM and the proposed adaptive checkpointing storage selection are promising in both performance, reliability and scalability.

  11. Internal V-Band Clamp

    DOEpatents

    Vaughn, Mark R.; Hafenrichter, Everett S.; Chapa, Agapito C.; Harris, Steven M.; Martinez, Marcus J.; Baty, Roy S.

    2006-02-28

    A system for clamping two tubular members together in an end-to-end relationship uses a split ring with a V-shaped outer rim that can engage a clamping surface on each member. The split ring has a relaxed closed state where the ends of the ring are adjacent and the outside diameter of the split ring is less than the minimum inside diameter of the members at their ends. The members are clamped when the split ring is spread into an elastically stretched position where the ring rim is pressed tightly against the interior surfaces of the members. Mechanisms are provided for removing the spreader so the split ring will return to the relaxed state, releasing the clamped members.

  12. DNA damage checkpoint recovery and cancer development

    SciTech Connect

    Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong; Legerski, Randy J.

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  13. Reducing space overhead for independendent checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Chung, Pi-Yu; Lin, In-Jen; Fuchs, W. Kent

    1992-01-01

    The main disadvantages of independent checkpointing are the possible domino effect and the associated storage space overhead for maintaining multiple checkpoints. In most previous work, it has been assumed that only the checkpoints older than the current global recovery line can be discarded. Here, we generalize a notion of recovery line to potential recovery line. Only the checkpoints belonging to at least one of the potential recovery lines cannot be discarded. By using the model of maximum-sized antichains on a partially ordered set, an efficient algorithm is developed for finding all non-discardable checkpoints, and we show that the number of non-discardable checkpoints cannot exceed N(N+1)/2, where N is the number of processors. Communication trace driven simulation for several hypercube programs is performed to show the benefit of the proposed algorithm for real applications.

  14. Lifting clamp positively grips structural shapes

    NASA Technical Reports Server (NTRS)

    Reinhardt, E. C.

    1966-01-01

    Welded steel clamps securely grip structural shapes of various sizes for crane operations. The clamp has adjustable clamping jaws and screw-operated internal v-jaws and provides greater safety than hoisting slings presently used. The structural member can be rotated in any manner, angle, or direction without being released by the clamp.

  15. Immune Checkpoint Modulators: An Emerging Antiglioma Armamentarium

    PubMed Central

    Kim, Eileen S.; Kim, Jennifer E.; Patel, Mira A.; Mangraviti, Antonella; Ruzevick, Jacob; Lim, Michael

    2016-01-01

    Immune checkpoints have come to the forefront of cancer therapies as a powerful and promising strategy to stimulate antitumor T cell activity. Results from recent preclinical and clinical studies demonstrate how checkpoint inhibition can be utilized to prevent tumor immune evasion and both local and systemic immune suppression. This review encompasses the key immune checkpoints that have been found to play a role in tumorigenesis and, more specifically, gliomagenesis. The review will provide an overview of the existing preclinical and clinical data, antitumor efficacy, and clinical applications for each checkpoint with respect to GBM, as well as a summary of combination therapies with chemotherapy and radiation. PMID:26881264

  16. Targeting the Checkpoint to Kill Cancer Cells

    PubMed Central

    Benada, Jan; Macurek, Libor

    2015-01-01

    Cancer treatments such as radiotherapy and most of the chemotherapies act by damaging DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Inhibiting the checkpoint allows entry to mitosis despite the presence of DNA damage and can lead to cell death. Importantly, as cancer cells exhibit increased levels of endogenous DNA damage due to an excessive replication stress, inhibiting the checkpoint kinases alone could act as a directed anti-cancer therapy. Here, we review the current status of inhibitors targeted towards the checkpoint effectors and discuss mechanisms of their actions in killing of cancer cells. PMID:26295265

  17. Chromosome breakage after G2 checkpoint release

    PubMed Central

    Deckbar, Dorothee; Birraux, Julie; Krempler, Andrea; Tchouandong, Leopoldine; Beucher, Andrea; Walker, Sarah; Stiff, Tom; Jeggo, Penny; Löbrich, Markus

    2007-01-01

    DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and ∼20 γH2AX foci remain. PMID:17353355

  18. Split-tapered joint clamping device

    DOEpatents

    Olsen, Max J.; Schwartz, Jr., John F.

    1988-01-01

    This invention relates to a clamping device for removably attaching a tool element to a bracket element wherein a bracket element is disposed in a groove in the tool and a clamping member is disposed in said groove and in engagement with a clamping face of the bracket and a wall of the groove and with the clamping member having pivot means engaging the bracket and about which the clamping member rotates.

  19. How to Assess the Quality of Glucose Clamps? Evaluation of Clamps Performed With ClampArt, a Novel Automated Clamp Device

    PubMed Central

    Benesch, Carsten; Heise, Tim; Klein, Oliver; Heinemann, Lutz; Arnolds, Sabine

    2015-01-01

    Background: There are no widely accepted parameters to assess the quality of glucose clamps. Thus, we selected different parameters describing clamp quality. These parameters were then evaluated in glucose clamps carried out with ClampArt, a novel CE-marked, state-of-the-art fully automated glucose clamp device employing continuous blood glucose (BG) measurements and minute-by-minute adaptations of glucose infusion rate (GIR). Methods: Thirty-nine glucose clamps were performed in 10 healthy and 29 subjects with type 1 diabetes (T1DM) (total duration 583 h). ClampArt-based BG measurements were compared with those obtained with a laboratory reference method. Clamp quality was assessed by 5 parameters: (1) difference (mg/dl) of all paired BG measurements of ClampArt versus reference method (“trueness”), (2) coefficient of variation (CV, %) of ClampArt’s BG measurements at target clamp level (“precision”), (3) mean absolute relative difference (MARD, %) at target clamp level (“accuracy”), (4) difference (mg/dl) between ClampArt and target BG (“control deviation”), and (5) percentage operational time (“utility”). Results: ClampArt-based BG measurements showed a trueness of 1.2 ± 2.5 mg/dl. CV and MARD at target BG were 5.5 ± 2.1% and 5.3 ± 2.3%, respectively. There were only small deviations from target level (1.2 ± 1.6 mg/dl). Operational time was as high as 95.4% ± 4.1% (means ± SD). Conclusions: The selected parameters seem to be adequate to characterize clamp quality. The novel, fully automated clamp device ClampArt achieves high clamp quality, which in future trials should be compared with other (automated and manual) clamp methods. PMID:25852075

  20. Biomarkers associated with checkpoint inhibitors.

    PubMed

    Manson, G; Norwood, J; Marabelle, A; Kohrt, H; Houot, R

    2016-07-01

    Checkpoint inhibitors (CPI), namely anti-CTLA4 and anti-PD1/PD-L1 antibodies, demonstrated efficacy across multiple types of cancer. However, only subgroups of patients respond to these therapies. Additionally, CPI can induce severe immune-related adverse events (irAE). Biomarkers that predict efficacy and toxicity may help define the patients who may benefit the most from these costly and potentially toxic therapies. In this study, we review the main biomarkers that have been associated with the efficacy (pharmacodynamics and clinical benefit) and the toxicity (irAE) of CPIs in patients. PMID:27122549

  1. A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9-Rad1-Hus1 Checkpoint Complex Interactions

    SciTech Connect

    P Luncsford; D Chang; G Shi; J Bernstein; A Madabushi; D Patterson; A Lu; E Toth

    2011-12-31

    The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

  2. Intellectual property issues of immune checkpoint inhibitors.

    PubMed

    Storz, Ulrich

    2016-01-01

    Immune checkpoint inhibitors are drugs that interfere with tumor escape responses. Some members of this class are already approved, and expected to be blockbusters in the future. Many companies have developed patent activities in this field. This article focuses on the patent landscape, and discusses key players and cases related to immune checkpoint inhibitors. PMID:26466763

  3. Limit analysis of pipe clamps

    SciTech Connect

    Flanders, H.E. Jr.

    1990-01-01

    The Service Level D (faulted) load capacity of a conventional three-bolt pipe-clamp based upon the limit analysis method is presented. The load distribution, plastic hinge locations, and collapse load are developed for the lower bound limit load method. The results of the limit analysis are compared with the manufacturer's rated loads. 3 refs.

  4. Overlapped checkpointing with hardware assist

    SciTech Connect

    Mitchell, Christopher J; Nunez, James A; Wang, Jun

    2009-01-01

    We present a new approach to handling the demanding I/O workload incurred during checkpoint writes encountered in High Performance Computing. Prior efforts to improve performance have been primarily bound by mechanical limitations of the hard drive. Our research surpasses this limitation by providing a method to: (1) write checkpoint data to a high-speed, non-volatile buffer, and (2) asynchronously write this data to permanent storage while resuming computation. This removes the hard drive from the critical data path because our I/O node based buffers isolate the compute nodes from the storage servers. This solution is feasible because of industry declines in cost for high-capacity, non-volatile storage technologies. Testing was conducted on a small-scale cluster to prove the design, and then scaled at Los Alamos National Laboratory. Results show a definitive speedup factor for select workloads over writing directly to a typical global parallel file system; the Panasas ActiveScale File System.

  5. Kinase signaling in the spindle checkpoint.

    PubMed

    Kang, Jungseog; Yu, Hongtao

    2009-06-01

    The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the "wait anaphase" signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects. PMID:19228686

  6. A clamped rectangular plate containing a crack

    NASA Technical Reports Server (NTRS)

    Tang, R.; Erdogan, F.

    1985-01-01

    The general problem of a rectangular plate clamped along two parallel sides and containing a crack parallel to the clamps is considered. The problem is formulated in terms of a system of singular integral equations and the asymptotic behavior of the stress state near the corners is investigated. Numerical examples are considered for a clamped plate without a crack and with a centrally located crack, and the stress intensity factors and the stresses along the clamps are calculated.

  7. Visualizing the spindle checkpoint in Drosophila spermatocytes

    PubMed Central

    Rebollo, Elena; González, Cayetano

    2000-01-01

    The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila. PMID:11256627

  8. Clinical Development of Immune Checkpoint Inhibitors.

    PubMed

    Ito, Ayumu; Kondo, Shunsuke; Tada, Kohei; Kitano, Shigehisa

    2015-01-01

    Recent progress in cancer immunotherapy has been remarkable. Most striking are the clinical development and approval of immunomodulators, also known as immune checkpoint inhibitors. These monoclonal antibodies (mAb) are directed to immune checkpoint molecules, which are expressed on immune cells and mediate signals to attenuate excessive immune reactions. Although mAbs targeting tumor associated antigens, such as anti-CD20 mAb and anti-Her2 mAb, directly recognize tumor cells and induce cell death, immune checkpoint inhibitors restore and augment the antitumor immune activities of cytotoxic T cells by blocking immune checkpoint molecules on T cells or their ligands on antigen presenting and tumor cells. Based on preclinical data, many clinical trials have demonstrated the acceptable safety profiles and efficacies of immune checkpoint inhibitors in a variety of cancers. The first in class approved immune checkpoint inhibitor is ipilimumab, an anti-CTLA-4 (cytotoxic T lymphocyte antigen-4) mAb. Two pivotal phase III randomized controlled trials demonstrated a survival benefit in patients with metastatic melanoma. In 2011, the US Food and Drug Administration (FDA) approved ipilimumab for metastatic melanoma. Several clinical trials have since investigated new agents, alone and in combination, for various cancers. In this review, we discuss the current development status of and future challenges in utilizing immune checkpoint inhibitors. PMID:26161407

  9. Clinical Development of Immune Checkpoint Inhibitors

    PubMed Central

    Ito, Ayumu; Kondo, Shunsuke; Tada, Kohei; Kitano, Shigehisa

    2015-01-01

    Recent progress in cancer immunotherapy has been remarkable. Most striking are the clinical development and approval of immunomodulators, also known as immune checkpoint inhibitors. These monoclonal antibodies (mAb) are directed to immune checkpoint molecules, which are expressed on immune cells and mediate signals to attenuate excessive immune reactions. Although mAbs targeting tumor associated antigens, such as anti-CD20 mAb and anti-Her2 mAb, directly recognize tumor cells and induce cell death, immune checkpoint inhibitors restore and augment the antitumor immune activities of cytotoxic T cells by blocking immune checkpoint molecules on T cells or their ligands on antigen presenting and tumor cells. Based on preclinical data, many clinical trials have demonstrated the acceptable safety profiles and efficacies of immune checkpoint inhibitors in a variety of cancers. The first in class approved immune checkpoint inhibitor is ipilimumab, an anti-CTLA-4 (cytotoxic T lymphocyte antigen-4) mAb. Two pivotal phase III randomized controlled trials demonstrated a survival benefit in patients with metastatic melanoma. In 2011, the US Food and Drug Administration (FDA) approved ipilimumab for metastatic melanoma. Several clinical trials have since investigated new agents, alone and in combination, for various cancers. In this review, we discuss the current development status of and future challenges in utilizing immune checkpoint inhibitors. PMID:26161407

  10. Berkeley Lab Checkpoint/Restart for Linux

    2003-11-15

    This package implements system-level checkpointing of scientific applications mnning on Linux clusters in a manner suitable for implementing preemption, migration and fault recovery by a batch scheduler The design includes documented interfaces for a cooperating application or library to implement extensions to the checkpoint system, such as consistent checkpointing of distnbuted MPI applications Using this package with an appropnate MPI implementation, the vast majority of scientific applications which use MPI for communucation are checkpointable withoutmore » any modifications to the application source code. Extending VMAdump code used in the bproc system, the BLCR kemel modules provide three additional features necessary for useful system-level checkpointing of scientific applications(installation of bproc is not required to use BLCR) First, this package provides the bookkeeping and coordination required for checkpointing and restoring multi-threaded and multi-process applications mnning on a single node Secondly, this package provides a system call interface allowing checkpoints to be requested by any aufhonzed process, such as a batch scheduler. Thirdly, this package provides a system call interface allowing applications and/or application libraries to extend the checkpoint capabilities in user space, for instance to proide coordination of checkpoints of distritsuted MPI applications. The "Iibcr" library in this package implements a wrapper around the system call interface exported by the kemel modules, and mantains bookkeeping to allow registration of callbacks by runtime libraries This library also provides the necesary thread-saftety and signal-safety mechanisms Thus, this library provides the means for applications and run-time libranes, such as MPI, to register callback functions to be run when a checkpoint is taken or when restarting from one. This library may also be used as a LD_PRELOAD to enable checkpointing of applications with development

  11. Solution structure of an "open" E. coli Pol III clamp loader sliding clamp complex.

    PubMed

    Tondnevis, Farzaneh; Weiss, Thomas M; Matsui, Tsutomu; Bloom, Linda B; McKenna, Robert

    2016-06-01

    Sliding clamps are opened and loaded onto primer template junctions by clamp loaders, and once loaded on DNA, confer processivity to replicative polymerases. Previously determined crystal structures of eukaryotic and T4 clamp loader-clamp complexes have captured the sliding clamps in either closed or only partially open interface conformations. In these solution structure studies, we have captured for the first time the clamp loader-sliding clamp complex from Escherichia coli using size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). The data suggests the sliding clamp is in an open conformation which is wide enough to permit duplex DNA binding. The data also provides information about spatial arrangement of the sliding clamp with respect to the clamp loader subunits and is compared to complex crystal structures determined from other organisms. PMID:26968362

  12. A survey of factors associated with the successful recognition of agonal breathing and cardiac arrest by 9-1-1 call takers: design and methodology

    PubMed Central

    Vaillancourt, Christian; Jensen, Jan L; Grimshaw, Jeremy; Brehaut, Jamie C; Charette, Manya; Kasaboski, Ann; Osmond, Martin; Wells, George A; Stiell, Ian G

    2009-01-01

    Background Cardiac arrest victims most often collapse at home, where only a modest proportion receives life-saving bystander cardiopulmonary resuscitation. As many as 40% of all sudden cardiac arrest victims have agonal or abnormal breathing in the first minutes following cardiac arrest. 9-1-1 call takers may wrongly interpret agonal breathing as a sign of life, and not initiate telephone cardiopulmonary resuscitation instructions. Improving 9-1-1 call takers' ability to recognize agonal breathing as a sign of cardiac arrest could result in improved bystander cardiopulmonary resuscitation and survival rates for out-of-hospital cardiac arrest victims. Methods/Design The overall goal of this study is to design and conduct a survey of 9-1-1 call takers in the province of Ontario to better understand the factors associated with the successful identification of cardiac arrest (including patients with agonal breathing) over the phone, and subsequent administration of cardiopulmonary resuscitation instructions to callers. This study will be conducted in three phases using the Theory of Planned Behaviour. In Phase One, we will conduct semi-structured qualitative interviews with a purposeful selection of 9-1-1 call takers from Ontario, and identify common themes and belief categories. In Phase Two, we will use the qualitative interview results to design and pilot a quantitative survey. In Phase Three, a final version of the quantitative survey will be administered via an electronic medium to all registered call takers in the province of Ontario. We will perform qualitative thematic analysis (Phase One) and regression modelling (Phases Two and Three), to determine direct and indirect relationship of behavioural constructs with intentions to provide cardiopulmonary resuscitation instructions. Discussion The results of this study will provide valuable insight into the factors associated with the successful recognition of agonal breathing and cardiac arrest by 9-1-1 call takers

  13. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies.

    PubMed

    Zabludoff, Sonya D; Deng, Chun; Grondine, Michael R; Sheehy, Adam M; Ashwell, Susan; Caleb, Benjamin L; Green, Stephen; Haye, Heather R; Horn, Candice L; Janetka, James W; Liu, Dongfang; Mouchet, Elizabeth; Ready, Shannon; Rosenthal, Judith L; Queva, Christophe; Schwartz, Gary K; Taylor, Karen J; Tse, Archie N; Walker, Graeme E; White, Anne M

    2008-09-01

    Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. PMID:18790776

  14. Dynamics of Open DNA Sliding Clamps.

    PubMed

    Oakley, Aaron J

    2016-01-01

    A range of enzymes in DNA replication and repair bind to DNA-clamps: torus-shaped proteins that encircle double-stranded DNA and act as mobile tethers. Clamps from viruses (such as gp45 from the T4 bacteriophage) and eukaryotes (PCNAs) are homotrimers, each protomer containing two repeats of the DNA-clamp motif, while bacterial clamps (pol III β) are homodimers, each protomer containing three DNA-clamp motifs. Clamps need to be flexible enough to allow opening and loading onto primed DNA by clamp loader complexes. Equilibrium and steered molecular dynamics simulations have been used to study DNA-clamp conformation in open and closed forms. The E. coli and PCNA clamps appear to prefer closed, planar conformations. Remarkably, gp45 appears to prefer an open right-handed spiral conformation in solution, in agreement with previously reported biophysical data. The structural preferences of DNA clamps in solution have implications for understanding the duty cycle of clamp-loaders. PMID:27148748

  15. Dynamics of Open DNA Sliding Clamps

    PubMed Central

    Oakley, Aaron J.

    2016-01-01

    A range of enzymes in DNA replication and repair bind to DNA-clamps: torus-shaped proteins that encircle double-stranded DNA and act as mobile tethers. Clamps from viruses (such as gp45 from the T4 bacteriophage) and eukaryotes (PCNAs) are homotrimers, each protomer containing two repeats of the DNA-clamp motif, while bacterial clamps (pol III β) are homodimers, each protomer containing three DNA-clamp motifs. Clamps need to be flexible enough to allow opening and loading onto primed DNA by clamp loader complexes. Equilibrium and steered molecular dynamics simulations have been used to study DNA-clamp conformation in open and closed forms. The E. coli and PCNA clamps appear to prefer closed, planar conformations. Remarkably, gp45 appears to prefer an open right-handed spiral conformation in solution, in agreement with previously reported biophysical data. The structural preferences of DNA clamps in solution have implications for understanding the duty cycle of clamp-loaders. PMID:27148748

  16. The Scalable Checkpoint/Restart Library

    2009-02-23

    The Scalable Checkpoint/Restart (SCR) library provides an interface that codes may use to worite our and read in application-level checkpoints in a scalable fashion. In the current implementation, checkpoint files are cached in local storage (hard disk or RAM disk) on the compute nodes. This technique provides scalable aggregate bandwidth and uses storage resources that are fully dedicated to the job. This approach addresses the two common drawbacks of checkpointing a large-scale application to amore » shared parallel file system, namely, limited bandwidth and file system contention. In fact, on current platforms, SCR scales linearly with the number of compute nodes. It has been benchmarked as high as 720GB/s on 1094 nodes of Atlas, which is nearly two orders of magnitude faster thanthe parallel file system.« less

  17. The Scalable Checkpoint/Restart Library

    SciTech Connect

    Moody, A.

    2009-02-23

    The Scalable Checkpoint/Restart (SCR) library provides an interface that codes may use to worite our and read in application-level checkpoints in a scalable fashion. In the current implementation, checkpoint files are cached in local storage (hard disk or RAM disk) on the compute nodes. This technique provides scalable aggregate bandwidth and uses storage resources that are fully dedicated to the job. This approach addresses the two common drawbacks of checkpointing a large-scale application to a shared parallel file system, namely, limited bandwidth and file system contention. In fact, on current platforms, SCR scales linearly with the number of compute nodes. It has been benchmarked as high as 720GB/s on 1094 nodes of Atlas, which is nearly two orders of magnitude faster thanthe parallel file system.

  18. Optimal message log reclamation for uncoordinated checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. K.

    1994-01-01

    Uncoordinated checkpointing for message-passing systems allows maximum process autonomy and general nondeterministic execution, but suffers from potential domino effect and the large space overhead for maintaining checkpoints and message logs. Traditionally, it has been assumed that only obsolete checkpoints and message logs before the global recovery line can be garbage-collected. Recently, an approach to identifying all garbage checkpoints based on recovery line transformation and decomposition has been developed. We show in this paper that the same approach can be applied to the problem of identifying all garbage message logs for systems requiring message logging to record in-transit messages. Communication trace-driven simulation for several parallel programs is used to evaluate the proposed algorithm.

  19. High-speed pressure clamp.

    PubMed

    Besch, Stephen R; Suchyna, Thomas; Sachs, Frederick

    2002-10-01

    We built a high-speed, pneumatic pressure clamp to stimulate patch-clamped membranes mechanically. The key control element is a newly designed differential valve that uses a single, nickel-plated piezoelectric bending element to control both pressure and vacuum. To minimize response time, the valve body was designed with minimum dead volume. The result is improved response time and stability with a threefold decrease in actuation latency. Tight valve clearances minimize the steady-state air flow, permitting us to use small resonant-piston pumps to supply pressure and vacuum. To protect the valve from water contamination in the event of a broken pipette, an optical sensor detects water entering the valve and increases pressure rapidly to clear the system. The open-loop time constant for pressure is 2.5 ms for a 100-mmHg step, and the closed-loop settling time is 500-600 micros. Valve actuation latency is 120 micros. The system performance is illustrated for mechanically induced changes in patch capacitance. PMID:12397401

  20. Checkpoint kinase inhibitor synergizes with DNA-damaging agents in G1 checkpoint-defective neuroblastoma.

    PubMed

    Xu, Hong; Cheung, Irene Y; Wei, Xiao X; Tran, Hoa; Gao, Xiaoni; Cheung, Nai-Kong V

    2011-10-15

    Checkpoint kinase inhibitors can enhance the cancer killing action of DNA-damaging chemotherapeutic agents by disrupting the S/G(2) cell cycle checkpoints. The in vitro and in vivo effects of the Chk1/2 inhibitor AZD7762 when combined with these agents were examined using neuroblastoma cell lines with known p53/MDM2/p14(ARF) genomic status. Four of four p53 mutant lines and three of five MDM2/p14(ARF) abnormal lines were defective in G(1) checkpoint, correlating with failure to induce endogenous p21 after treatment with DNA-damaging agents. In cytotoxicity assays, these G(1) checkpoint-defective lines were more resistant to DNA-damaging agents when compared to G(1) checkpoint intact lines, yet becoming more sensitive when AZD7762 was added. Moreover, AZD7762 abrogated DNA damage-induced S/G(2) checkpoint arrest both in vitro and in vivo. In xenograft models, a significant delay in tumor growth accompanied by histological evidence of increased apoptosis was observed, when AZD7762 was added to the DNA-damaging drug gemcitabine. These results suggest a therapeutic potential of combination therapy using checkpoint kinase inhibitor and chemotherapy to reverse or prevent drug resistance in treating neuroblastomas with defective G(1) checkpoints. PMID:21154747

  1. A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

    PubMed

    García-Rodríguez, Luis J; De Piccoli, Giacomo; Marchesi, Vanessa; Jones, Richard C; Edmondson, Ricky D; Labib, Karim

    2015-10-15

    Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint. PMID:26250113

  2. Lazy Checkpointing : Exploiting Temporal Locality in Failures to Mitigate Checkpointing Overheads on Extreme-Scale Systems

    SciTech Connect

    Tiwari, Devesh; Gupta, Saurabh; Vazhkudai, Sudharshan S

    2014-01-01

    Continuing increase in the computational power of supercomputers has enabled large-scale scientific applications in the areas of astrophysics, fusion, climate and combustion to run larger and longer-running simulations, facilitating deeper scientific insights. However, these long-running simulations are often interrupted by multiple system failures. Therefore, these applications rely on ``checkpointing'' as a resilience mechanism to store application state to permanent storage and recover from failures. \\\\ \\indent Unfortunately, checkpointing incurs excessive I/O overhead on supercomputers due to large size of checkpoints, resulting in a sub-optimal performance and resource utilization. In this paper, we devise novel mechanisms to show how checkpointing overhead can be mitigated significantly by exploiting the temporal characteristics of system failures. We provide new insights and detailed quantitative understanding of the checkpointing overheads and trade-offs on large-scale machines. Our prototype implementation shows the viability of our approach on extreme-scale machines.

  3. Asynchronous Checkpoint Migration with MRNet in the Scalable Checkpoint / Restart Library

    SciTech Connect

    Mohror, K; Moody, A; de Supinski, B R

    2012-03-20

    Applications running on today's supercomputers tolerate failures by periodically saving their state in checkpoint files on stable storage, such as a parallel file system. Although this approach is simple, the overhead of writing the checkpoints can be prohibitive, especially for large-scale jobs. In this paper, we present initial results of an enhancement to our Scalable Checkpoint/Restart Library (SCR). We employ MRNet, a tree-based overlay network library, to transfer checkpoints from the compute nodes to the parallel file system asynchronously. This enhancement increases application efficiency by removing the need for an application to block while checkpoints are transferred to the parallel file system. We show that the integration of SCR with MRNet can reduce the time spent in I/O operations by as much as 15x. However, our experiments exposed new scalability issues with our initial implementation. We discuss the sources of the scalability problems and our plans to address them.

  4. Advanced motor driven clamped borehole seismic receiver

    DOEpatents

    Engler, Bruce P.; Sleefe, Gerard E.; Striker, Richard P.

    1993-01-01

    A borehole seismic tool including a borehole clamp which only moves perpendicular to the borehole. The clamp is driven by an electric motor, via a right angle drive. When used as a seismic receiver, the tool has a three part housing, two of which are hermetically sealed. Accelerometers or geophones are mounted in one hermetically sealed part, the electric meter in the other hermetically sealed part, and the clamp and right angle drive in the third part. Preferably the tool includes cable connectors at both ends. Optionally a shear plate can be added to the clamp to extend the range of the tool.

  5. Advanced motor driven clamped borehole seismic receiver

    DOEpatents

    Engler, B.P.; Sleefe, G.E.; Striker, R.P.

    1993-02-23

    A borehole seismic tool is described including a borehole clamp which only moves perpendicular to the borehole. The clamp is driven by an electric motor, via a right angle drive. When used as a seismic receiver, the tool has a three part housing, two of which are hermetically sealed. Accelerometers or geophones are mounted in one hermetically sealed part, the electric motor in the other hermetically sealed part, and the clamp and right angle drive in the third part. Preferably the tool includes cable connectors at both ends. Optionally a shear plate can be added to the clamp to extend the range of the tool.

  6. Diverless pipeline repair clamp: Phase 2

    SciTech Connect

    Miller, J.E.; Lane, B. )

    1992-04-01

    The objective of this project sponsored by the Pipeline Research Committee of the American Gas Association, is to develop a system suitable for repairing small leaks on deepwater pipelines. Phase I of the project, completed in 1990 by Stress Engineering Services, Inc. investigated the types of problems that would have to be overcome to effect a diverless clamp-type repair. Several repair systems were investigated and ten mechanisms were proposed that could be used to secure two clamp halves together. This current Phase 11 effort, is to take two most promising clamp concepts from Phase 1, further evaluate hardware and installation issues, develop conceptual designs, and determine which concept should be carried forward to detailed design. The two concepts evaluated were (1) a bolted split-sleeve clamp suited for ROV installation, and (2) a hydraulically self-actuating clamp requiring only placement on the pipe and actuation by ROV hydraulic hot stabs. Both concepts were evaluated for a 12-inch (324 mm) nominal pipe diameter with an ANSI 900 (15.3 mPa) pressure rating, presuming either system could be adapted to a wider range of pipe sizes and design pressures. Based on the results of this investigation a modified bolted split-sleeve clamp was recommended over the hydraulically self-actuating clamp. The main reasons are (1) the bolted split-sleeve clamp can be adapted to installation by a ROV, (2) sealing and clamping mechanisms borrow from available proven technology, (3) it would require less development effort than the hydraulically self-actuating clamp, and (4) the bolted split-sleeve clamp would probably result in a simpler, less costly design.

  7. Checkpoint triggering in a computer system

    DOEpatents

    Cher, Chen-Yong

    2016-09-06

    According to an aspect, a method for triggering creation of a checkpoint in a computer system includes executing a task in a processing node of the computer system and determining whether it is time to read a monitor associated with a metric of the task. The monitor is read to determine a value of the metric based on determining that it is time to read the monitor. A threshold for triggering creation of the checkpoint is determined based on the value of the metric. Based on determining that the value of the metric has crossed the threshold, the checkpoint including state data of the task is created to enable restarting execution of the task upon a restart operation.

  8. Cell cycle checkpoint regulators reach a zillion

    PubMed Central

    Yasutis, Kimberly M.; Kozminski, Keith G.

    2013-01-01

    Entry into mitosis is regulated by a checkpoint at the boundary between the G2 and M phases of the cell cycle (G2/M). In many organisms, this checkpoint surveys DNA damage and cell size and is controlled by both the activation of mitotic cyclin-dependent kinases (Cdks) and the inhibition of an opposing phosphatase, protein phosphatase 2A (PP2A). Misregulation of mitotic entry can often lead to oncogenesis or cell death. Recent research has focused on discovering the signaling pathways that feed into the core checkpoint control mechanisms dependent on Cdk and PP2A. Herein, we review the conserved mechanisms of the G2/M transition, including recently discovered upstream signaling pathways that link cell growth and DNA replication to cell cycle progression. Critical consideration of the human, frog and yeast models of mitotic entry frame unresolved and emerging questions in this field, providing a prediction of signaling molecules and pathways yet to be discovered. PMID:23598718

  9. Optimal message log reclamation for independent checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. Kent

    1993-01-01

    Independent (uncoordinated) check pointing for parallel and distributed systems allows maximum process autonomy but suffers from possible domino effects and the associated storage space overhead for maintaining multiple checkpoints and message logs. In most research on check pointing and recovery, it was assumed that only the checkpoints and message logs older than the global recovery line can be discarded. It is shown how recovery line transformation and decomposition can be applied to the problem of efficiently identifying all discardable message logs, thereby achieving optimal garbage collection. Communication trace-driven simulation for several parallel programs is used to show the benefits of the proposed algorithm for message log reclamation.

  10. Immune checkpoint blockade in lung cancer.

    PubMed

    Somasundaram, Aswin; Socinski, Mark A; Villaruz, Liza C

    2016-08-01

    Immunotherapy has revolutionized the therapeutic landscape of advanced lung cancer. The adaptive immune system has developed a sophisticated method of tumor growth control, but T-cell activation is regulated by various checkpoints. Blockade of the immune checkpoints with therapies targeting the PD-1 pathway, such as nivolumab and pembrolizumab, has been validated as a therapeutic approach in non-small cell lung cancer. Newer therapies and novel combinations are also being evaluated, and the use of biomarkers in conjunction with these drugs is an area of active investigation. This review summarizes the current evidence for the efficacy and safety of the above approaches in the treatment of lung cancer. PMID:27585231

  11. Protein folding in a force clamp

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Szymczak, P.

    2006-05-01

    Kinetics of folding of a protein held in a force clamp are compared to an unconstrained folding. The comparison is made within a simple topology-based dynamical model of ubiquitin. We demonstrate that the experimentally observed variations in the end-to-end distance reflect microscopic events during folding. However, the folding scenarios in and out of the force clamp are distinct.

  12. Checkpointing and Recovery in Distributed and Database Systems

    ERIC Educational Resources Information Center

    Wu, Jiang

    2011-01-01

    A transaction-consistent global checkpoint of a database records a state of the database which reflects the effect of only completed transactions and not the results of any partially executed transactions. This thesis establishes the necessary and sufficient conditions for a checkpoint of a data item (or the checkpoints of a set of data items) to…

  13. The Schizosaccharomyces pombe rad3 checkpoint gene.

    PubMed Central

    Bentley, N J; Holtzman, D A; Flaggs, G; Keegan, K S; DeMaggio, A; Ford, J C; Hoekstra, M; Carr, A M

    1996-01-01

    The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution. Images PMID:8978690

  14. A Wee1 checkpoint inhibits anaphase onset

    PubMed Central

    Lianga, Noel; Williams, Elizabeth C.; Kennedy, Erin K.; Doré, Carole; Pilon, Sophie; Girard, Stéphanie L.; Deneault, Jean-Sebastien

    2013-01-01

    Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APCCdc20). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25+) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2ACdc55 triggers anaphase onset. PMID:23751495

  15. The Monogenean Which Lost Its Clamps

    PubMed Central

    Justine, Jean-Lou; Rahmouni, Chahrazed; Gey, Delphine; Schoelinck, Charlotte; Hoberg, Eric P.

    2013-01-01

    Ectoparasites face a daily challenge: to remain attached to their hosts. Polyopisthocotylean monogeneans usually attach to the surface of fish gills using highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 60 years ago, the clamps were considered to be absent but few specimens were available and this observation was later questioned. In addition, genera within the family Protomicrocotylidae have either clamps of the “gastrocotylid” or the “microcotylid” types; this puzzled systematists because these clamp types are characteristic of distinct, major groups. Discovery of another, new, species of the genus Lethacotyle, has allowed us to explore the nature of the attachment structures in protomicrocotylids. Lethacotyle vera n. sp. is described from the gills of the carangid Caranx papuensis off New Caledonia. It is distinguished from Lethacotyle fijiensis, the only other species of the genus, by the length of the male copulatory spines. Sequences of 28S rDNA were used to build a tree, in which Lethacotyle vera grouped with other protomicrocotylids. The identity of the host fish was confirmed with COI barcodes. We observed that protomicrocotylids have specialized structures associated with their attachment organ, such as lateral flaps and transverse striations, which are not known in other monogeneans. We thus hypothesized that the clamps in protomicrocotylids were sequentially lost during evolution, coinciding with the development of other attachment structures. To test the hypothesis, we calculated the surfaces of clamps and body in 120 species of gastrocotylinean monogeneans, based on published descriptions. The ratio of clamp surface: body surface was the lowest in protomicrocotylids. We conclude that clamps in protomicrocotylids are vestigial organs, and that occurrence of “gastrocotylid” and simpler “microcotylid” clamps within the same family are

  16. 33 CFR 183.560 - Hose clamps: Installation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Hose clamps: Installation. Each hose clamp on a hose from the fuel tank to the fuel inlet connection on..., pipe, or hose fitting; and (d) Not depend solely on the spring tension of the clamp for...

  17. Surface characterization of selected LDEF tray clamps

    NASA Technical Reports Server (NTRS)

    Cromer, T. F.; Grammer, H. L.; Wightman, J. P.; Young, Philip R.; Slemp, Wayne S.

    1993-01-01

    The surface characterization of chromic acid anodized 6061-T6 aluminum alloy tray clamps has shown differences in surface chemistry depending upon the position on the Long Duration Exposure Facility (LDEF). Water contact angle results showed no changes in wettability of the tray clamps. The overall surface topography of the control, trailing edge(E3) and leading edge(D9) samples was similar. The thickness of the aluminum oxide layer for all samples determined by Auger depth profiling was less than one micron. X-ray photoelectron spectroscopy (XPS) analysis of the tray clamps showed significant differences in the surface composition. Carbon and silicon containing compounds were the primary contaminants detected.

  18. Compiler-Enhanced Incremental Checkpointing for OpenMP Applications

    SciTech Connect

    Bronevetsky, G; Marques, D; Pingali, K; Rugina, R; McKee, S A

    2008-01-21

    As modern supercomputing systems reach the peta-flop performance range, they grow in both size and complexity. This makes them increasingly vulnerable to failures from a variety of causes. Checkpointing is a popular technique for tolerating such failures, enabling applications to periodically save their state and restart computation after a failure. Although a variety of automated system-level checkpointing solutions are currently available to HPC users, manual application-level checkpointing remains more popular due to its superior performance. This paper improves performance of automated checkpointing via a compiler analysis for incremental checkpointing. This analysis, which works with both sequential and OpenMP applications, reduces checkpoint sizes by as much as 80% and enables asynchronous checkpointing.

  19. Compiler-Enhanced Incremental Checkpointing for OpenMP Applications

    SciTech Connect

    Bronevetsky, G; Marques, D; Pingali, K; McKee, S; Rugina, R

    2009-02-18

    As modern supercomputing systems reach the peta-flop performance range, they grow in both size and complexity. This makes them increasingly vulnerable to failures from a variety of causes. Checkpointing is a popular technique for tolerating such failures, enabling applications to periodically save their state and restart computation after a failure. Although a variety of automated system-level checkpointing solutions are currently available to HPC users, manual application-level checkpointing remains more popular due to its superior performance. This paper improves performance of automated checkpointing via a compiler analysis for incremental checkpointing. This analysis, which works with both sequential and OpenMP applications, significantly reduces checkpoint sizes and enables asynchronous checkpointing.

  20. The spindle checkpoint and chromosome segregation in meiosis

    PubMed Central

    Gorbsky, Gary J.

    2014-01-01

    The spindle checkpoint is a key regulator of chromosome segregation in mitosis and meiosis. Its function is to prevent precocious anaphase onset before chromosomes have achieved bipolar attachment to the spindle. The spindle checkpoint comprises a complex set of signaling pathways that integrate microtubule dynamics, biomechanical forces at the kinetochores, and intricate regulation of protein interactions and post-translational modifications. Historically, many key observations that gave rise to the initial concepts of the spindle checkpoint were carried out in meiotic systems. In contrast with mitosis, the two distinct chromosome segregation events of meiosis present a special challenge for the regulation of checkpoint signaling. Preservation of fidelity in chromosome segregation in meiosis, controlled by the spindle checkpoint, also has significant impact in human health. This review highlights the contributions from meiotic systems in understanding the spindle checkpoint as well as the role of checkpoint signaling in controlling the complex divisions of meiosis. PMID:25470754

  1. Regulation of mitotic progression by the spindle assembly checkpoint

    PubMed Central

    Lischetti, Tiziana; Nilsson, Jakob

    2015-01-01

    Equal segregation of sister chromatids during mitosis requires that pairs of kinetochores establish proper attachment to microtubules emanating from opposite poles of the mitotic spindle. The spindle assembly checkpoint (SAC) protects against errors in segregation by delaying sister separation in response to improper kinetochore–microtubule interactions, and certain checkpoint proteins help to establish proper attachments. Anaphase entry is inhibited by the checkpoint through assembly of the mitotic checkpoint complex (MCC) composed of the 2 checkpoint proteins, Mad2 and BubR1, bound to Cdc20. The outer kinetochore acts as a catalyst for MCC production through the recruitment and proper positioning of checkpoint proteins and recently there has been remarkable progress in understanding how this is achieved. Here, we highlight recent advances in our understanding of kinetochore–checkpoint protein interactions and inhibition of the anaphase promoting complex by the MCC. PMID:27308407

  2. Dual Functions, Clamp Opening and Primer-Template Recognition, Define a Key Clamp Loader Subunit

    PubMed Central

    Coman, Maria Magdalena; Jin, Mi; Ceapa, Razvan; Finkelstein, Jeff; O'Donnell, Michael; Chait, Brian T.; Hingorani, Manju M.

    2010-01-01

    Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multi-protein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E. coli γ complex clamp loader and DNA using UV-induced protein–DNA cross-linking and mass spectrometry. The results show that the δ subunit in the γ complex makes close contact with the primer-template junction. Tryptophan 279 in the δ C-terminal domain lies near the 3′-OH primer end and may play a key role in primer-template recognition. Previous studies have shown that δ also binds and opens the β clamp (hydrophobic residues in the N-terminal domain of δ contact β. The clamp-binding and DNA-binding sites on δ appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S. cerevisiae RFC complex suggests that the dual functionality observed for δ in the γ complex may be true also for clamp loaders from other organisms. PMID:15364574

  3. Dynamic clamp with StdpC software

    PubMed Central

    2011-01-01

    Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here, we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, Spike timing dependent plasticity Clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments via an intuitive and powerful interface with a minimal initial lead-time of a few hours. After initial configuration, experimental results can be generated within minutes of cell impalement. PMID:21372819

  4. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  5. Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

    PubMed Central

    Kusch, Jana; Zifarelli, Giovanni

    2014-01-01

    Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research. PMID:24655500

  6. Structural analysis of a eukaryotic sliding DNA clamp-clamp loadercomplex.

    SciTech Connect

    Bowman, Gregory D.; O'Donnell, Mike; Kuriyan, John

    2006-06-17

    Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-?-S by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.

  7. Protein folding in a force-clamp

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Szymczak, Piotr

    2006-03-01

    Kinetics of folding of a protein held in a force-clamp are compared to an unconstrained folding. The comparison is made within a simple topology-based dynamical model of ubiquitin. We demonstrate that the experimentally observed rapid changes in the end-to-end distance mirror microscopic events during folding. However, the folding scenarios in and out of the force-clamp are distinct.

  8. Rfc4 Interacts with Rpa1 and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae

    PubMed Central

    Kim, Hee-Sook; Brill, Steven J.

    2001-01-01

    The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G1/S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G2/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles. PMID:11340166

  9. [Cancer immunotherapy by immuno-checkpoint blockade].

    PubMed

    Kawakami, Yutaka

    2015-10-01

    As cancer immunotherapies utilizing anti-tumor T-cell responses, immuno-checkpoint blockade and adoptive T-cell immunotherapy have recently achieved durable responses even in advanced cancer patients with metastases. Administration of antibodies on the T-cell surface, CTLA-4 and PD-1 (or PD-1 ligand PD-L1), resulted in tumor regression of not only melanoma and renal cell cancer which were known to be relatively sensitive to immunotherapy, but also various malignancies including lung, bladder, ovarian, gastric, and head and neck cancers, as well as hematological malignancies such as Hodgkin and B-cell malignant lymphomas. These findings have changed the status of immunotherapy in the development of cancer treatments. Currently, development of combinations employing cancer immunotherapy with immuno-checkpoint blockade, as well as personalized cancer immunotherapy based on the evaluation of pretreatment immune status, are in progress. PMID:26458459

  10. Immune checkpoints and immunotherapy for colorectal cancer

    PubMed Central

    Singh, Preet Paul; Sharma, Piyush K.; Krishnan, Gayathri; Lockhart, A. Craig

    2015-01-01

    Colorectal cancer (CRC) remains one of the major causes of death worldwide, despite steady improvement in early detection and overall survival over the past decade. Current treatment paradigms, with chemotherapy and biologics, appear to have reached their maximum benefit. Immunotherapy, especially with checkpoint inhibitors, has shown considerable clinical benefit in various cancers, including mismatch-repair-deficient CRC. This has led to the planning and initiation of several clinical trials evaluating novel immunotherapy agents—as single agents, combinations and in conjunction with chemotherapy—in patients with CRC. This article reviews biological and preclinical data for checkpoint inhibitors and discusses various immunotherapy trials in CRC, as well as current efforts in CRC immunotherapy. PMID:26510455

  11. MRN- and 9-1-1-Independent Activation of the ATR-Chk1 Pathway during the Induction of the Virulence Program in the Phytopathogen Ustilago maydis

    PubMed Central

    Tenorio-Gómez, María; de Sena-Tomás, Carmen; Pérez-Martín, Jose

    2015-01-01

    DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation. PMID:26367864

  12. MRN- and 9-1-1-Independent Activation of the ATR-Chk1 Pathway during the Induction of the Virulence Program in the Phytopathogen Ustilago maydis.

    PubMed

    Tenorio-Gómez, María; de Sena-Tomás, Carmen; Pérez-Martín, Jose

    2015-01-01

    DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation. PMID:26367864

  13. Immune checkpoint inhibitors: therapeutic advances in melanoma

    PubMed Central

    Márquez-Rodas, Ivan; Cerezuela, Pablo; Soria, Ainara; Berrocal, Alfonso; Riso, Aldo; Martín-Algarra, Salvador

    2015-01-01

    In recent years, new strategies for treating melanoma have been introduced, improving the outlook for this challenging disease. One of the most important advances has been the development of immunotherapy. The better understanding of the role of the immunological system in tumor control has paved the way for strategies to enhance the immune response against cancer cells. Monoclonal antibodies (mAbs) against the immune checkpoints cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have demonstrated high activity in melanoma and other tumors. Ipilimumab, an anti CTLA-4 antibody, was the first drug of this class that was approved. Although the response rate with ipilimumab is low (less than 20% of patients have objective responses), 20% of patients have long survival, with similar results in the first and second line settings. Nivolumab and pembrolizumab, both anti PD-1 inhibitors, have been approved for the treatment of melanoma, with response rates of 40% and a demonstrated survival advantage in phase III trials. This has marked a new era in the treatment of metastatic melanoma and much research is now ongoing with other drugs targeting checkpoint inhibitors. In addition, the agonist of activating molecules on T cells and their combinations are being investigated. Herein we review the clinical development of checkpoint inhibitors and their approval for treatment of metastatic melanoma. PMID:26605313

  14. Immune Checkpoint Inhibitors in Older Adults.

    PubMed

    Elias, Rawad; Morales, Joshua; Rehman, Yasser; Khurshid, Humera

    2016-08-01

    Cancer is primarily a disease of older adults. The treatment of advanced stage tumors usually involves the use of systemic agents that may be associated with significant risk of toxicity, especially in older patients. Immune checkpoint inhibitors are newcomers to the oncology world with improved efficacy and better safety profiles when compared to traditional cytotoxic drugs. This makes them an attractive treatment option. While there are no elderly specific trials, this review attempts to look at the current available data from a geriatric oncology perspective. We reviewed data from phase III studies that led to newly approved indications of checkpoint inhibitors in non-small cell lung cancer, melanoma, and renal cell cancer. Data were reviewed with respect to response, survival, and toxicity according to three groups: <65 years, 65-75 years, and >75 years. Current literature does not allow one to draw definitive conclusions regarding the role of immune checkpoint inhibitors in older adults. However, they may offer a potentially less toxic but equally efficacious treatment option for the senior adult oncology patient. PMID:27287329

  15. Diverless pipeline repair clamp, Phase 3

    SciTech Connect

    Miller, J.E.

    1993-08-01

    The objective of this project is to develop a system suitable for repairing small leaks in deep water pipelines. It is assumed that leak repair operations at the water depths in question will be performed by Remotely Operated Vehicles (ROV`s). This report summarizes the results of the third and final phase of this project. Phase 3 work included design, manufacture, and dry testing of (1) a one-half scale model of a 12 inch repair clamp, (2) a full-scale bolt test fixture to demonstrate bolt containment and startup under realistic misalignment of the clamp halves, and (3) a full-scale one-way cylinder for end seal activation. Phase 3 also included a study commissioned from Oceaneering directed at defining the interfaces of the clamp package and the ROV, including suggested procedures for deployment and positioning of the clamp package on the pipeline. Issues regarding bolt make-up by the ROV were also studied in detail and limitations in bolting capability were outlined. The conclusion of this work is that the clamping system described herein may be implemented in a direct manner. The design issues causing the most concern have been resolved through laboratory tests. Note however that all testing performed was mechanical in nature and performed in a dry environment. The recommended next development step, prior to declaring the system operational, is to manufacture a fully outfitted clamp package and to perform installation tests in a controlled underwater environment using a typical deepwater ROV. Wet tests are required in order to demonstrate ROV interfaces and installation procedures, however, the major mechanical features represented by the clamp design as well as its operation have been proven.

  16. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  17. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  18. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  19. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  20. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  1. Template based parallel checkpointing in a massively parallel computer system

    DOEpatents

    Archer, Charles Jens; Inglett, Todd Alan

    2009-01-13

    A method and apparatus for a template based parallel checkpoint save for a massively parallel super computer system using a parallel variation of the rsync protocol, and network broadcast. In preferred embodiments, the checkpoint data for each node is compared to a template checkpoint file that resides in the storage and that was previously produced. Embodiments herein greatly decrease the amount of data that must be transmitted and stored for faster checkpointing and increased efficiency of the computer system. Embodiments are directed to a parallel computer system with nodes arranged in a cluster with a high speed interconnect that can perform broadcast communication. The checkpoint contains a set of actual small data blocks with their corresponding checksums from all nodes in the system. The data blocks may be compressed using conventional non-lossy data compression algorithms to further reduce the overall checkpoint size.

  2. Piezoresistive cantilever force-clamp system

    SciTech Connect

    Park, Sung-Jin; Petzold, Bryan C.; Pruitt, Beth L.; Goodman, Miriam B.

    2011-04-15

    We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or ''clamps'' the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to a sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of {mu}N force and nm up to tens of {mu}m displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode.

  3. An Ultrasonic Clamp for Bloodless Partial Nephrectomy

    NASA Astrophysics Data System (ADS)

    Lafon, Cyril; Bouchoux, Guillaume; Murat, François Joseph; Birer, Alain; Theillère, Yves; Chapelon, Jean Yves; Cathignol, Dominique

    2007-05-01

    Maximum conservation of the kidney is preferable through partial nephrectomy for patients at risk of disease recurrence of renal cancers. Haemostatic tools are needed in order to achieve bloodless surgery and reduce post surgery morbidity. Two piezo-ceramic transducers operating at a frequency of 4 MHz were mounted on each arm of a clamp. When used for coagulation purposes, two transducers situated on opposite arms of the clamp were driven simultaneously. Heat delivery was optimized as each transducers mirrored back to targeted tissues the wave generated by the opposite transducer. Real-time treatment monitoring with an echo-based technique was also envisaged with this clamp. Therapy was periodically interrupted so one transducer could generate a pulse. The echo returning from the opposite transducer was treated. Coagulation necroses were obtained in vitro on substantial thicknesses (23-38mm) of pig liver over exposure durations ranging from 30s to 130s, and with acoustic intensities of less than 15W/cm2 per transducer. Both kidneys of two pigs were treated in vivo with the clamp (14.5W/cm2 for 90s), and the partial nephrectomies performed proved to be bloodless. In vitro and in vivo, wide transfixing lesions corresponded to an echo energy decrease superior to -10dB and parabolic form of the time of flight versus treatment time. In conclusion, this ultrasound clamp has proven to be an excellent mean for achieving monitored haemostasis in kidney.

  4. Compact, Stiff, Remotely-Actuable Quick-Release Clamp

    NASA Technical Reports Server (NTRS)

    Tsai, Ted W. (Inventor)

    2000-01-01

    The present invention provides a clamp that is compact and lightweight, yet provides high holding strength and stiffness or rigidity. The clamp uses a unique double slant interface design which provides mechanical advantages to resist forces applied to the clamp member as the load increases. The clamp allows for rapid and remote-activated release of the clamp jaws by applying only a small operating force to an over-center lock/release mechanism, such as by pulling a manual tether.

  5. Efficient Checkpointing of Virtual Machines using Virtual Machine Introspection

    SciTech Connect

    Aderholdt, Ferrol; Han, Fang; Scott, Stephen L; Naughton, III, Thomas J

    2014-01-01

    Cloud Computing environments rely heavily on system-level virtualization. This is due to the inherent benefits of virtualization including fault tolerance through checkpoint/restart (C/R) mechanisms. Because clouds are the abstraction of large data centers and large data centers have a higher potential for failure, it is imperative that a C/R mechanism for such an environment provide minimal latency as well as a small checkpoint file size. Recently, there has been much research into C/R with respect to virtual machines (VM) providing excellent solutions to reduce either checkpoint latency or checkpoint file size. However, these approaches do not provide both. This paper presents a method of checkpointing VMs by utilizing virtual machine introspection (VMI). Through the usage of VMI, we are able to determine which pages of memory within the guest are used or free and are better able to reduce the amount of pages written to disk during a checkpoint. We have validated this work by using various benchmarks to measure the latency along with the checkpoint size. With respect to checkpoint file size, our approach results in file sizes within 24% or less of the actual used memory within the guest. Additionally, the checkpoint latency of our approach is up to 52% faster than KVM s default method.

  6. Targeted Therapies Combined With Immune Checkpoint Therapy.

    PubMed

    Prieto, Peter A; Reuben, Alexandre; Cooper, Zachary A; Wargo, Jennifer A

    2016-01-01

    The age of personalized medicine continues to evolve within clinical oncology with the arsenal available to clinicians in a variety of malignancies expanding at an exponential rate. The development and advancement of molecular treatment modalities, including targeted therapy and immune checkpoint blockade, continue to flourish. Treatment with targeted therapy (BRAF, MEK, and other small molecule inhibitors) can be associated with swift disease control and high response rates, but limited durability when used as monotherapy. Conversely, treatment with immune checkpoint blockade monotherapy regimens (anti-cytotoxic T-lymphocyte antigen 4 and anti-programmed cell death protein 1/programmed cell death protein 1 ligand) tends to have lower response rates than that observed with BRAF-targeted therapy, although these treatments may offer long-term durable disease control. With the advent of these forms of therapy, there was interest early on in empirically combining targeted therapy with immune checkpoint blockade with the hopes of preserving high response rates and adding durability; however, there is now strong scientific rationale for combining these forms of therapy-and early evidence of synergy in preclinical models of melanoma. Clinical trials combining these strategies are ongoing, and mature data regarding response rates and durability are not yet available. Synergy may ultimately be apparent; however, it has also become clear that complexities exist regarding toxicity when combining these therapies. Nonetheless, this increased appreciation of the complex interplay between oncogenic mutations and antitumor immunity has opened up tremendous opportunities for studying targeted agents and immunotherapy in combination, which extends far beyond melanoma to other solid tumors and also to hematologic malignancies. PMID:27111910

  7. Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

    PubMed Central

    Marzahn, Melissa R.; Hayner, Jaclyn N.; Meyer, Jennifer A.; Bloom, Linda B.

    2014-01-01

    DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+ family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the S. cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway. PMID:25450506

  8. Limit analysis of pipe clamps. Revision 1

    SciTech Connect

    Flanders, H.E. Jr.

    1990-12-31

    The Service Level D (faulted) load capacity of a conventional three-bolt pipe-clamp based upon the limit analysis method is presented. The load distribution, plastic hinge locations, and collapse load are developed for the lower bound limit load method. The results of the limit analysis are compared with the manufacturer`s rated loads. 3 refs.

  9. Clamp and Gas Nozzle for TIG Welding

    NASA Technical Reports Server (NTRS)

    Gue, G. B.; Goller, H. L.

    1982-01-01

    Tool that combines clamp with gas nozzle is aid to tungsten/inert-gas (TIG) welding in hard-to-reach spots. Tool holds work to be welded while directing a stream of argon gas at weld joint, providing an oxygen-free environment for tungsten-arc welding.

  10. Piezoresistive cantilever force-clamp system

    PubMed Central

    Park, Sung-Jin; Petzold, Bryan C.; Goodman, Miriam B.; Pruitt, Beth L.

    2011-01-01

    We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or “clamps” the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to a sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of μN force and nm up to tens of μm displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode. PMID:21529009

  11. Toward a systems-level view of mitotic checkpoints.

    PubMed

    Ibrahim, Bashar

    2015-03-01

    Reproduction and natural selection are the key elements of life. In order to reproduce, the genetic material must be doubled, separated and placed into two new daughter cells, each containing a complete set of chromosomes and organelles. In mitosis, transition from one process to the next is guided by intricate surveillance mechanisms, known as the mitotic checkpoints. Dis-regulation of cell division through checkpoint malfunction can lead to developmental defects and contribute to the development or progression of tumors. This review approaches two important mitotic checkpoints, the spindle assembly checkpoint (SAC) and the spindle position checkpoint (SPOC). The highly conserved spindle assembly checkpoint (SAC) controls the onset of anaphase by preventing premature segregation of the sister chromatids of the duplicated genome, to the spindle poles. In contrast, the spindle position checkpoint (SPOC), in the budding yeast Saccharomyces cerevisiae, ensures that during asymmetric cell division mitotic exit does not occur until the spindle is properly aligned with the cell polarity axis. Although there are no known homologs, there is indication that functionally similar checkpoints exist also in animal cells. This review can be regarded as an "executable model", which could be easily translated into various quantitative concrete models like Petri nets, ODEs, PDEs, or stochastic particle simulations. It can also function as a base for developing quantitative models explaining the interplay of the various components and proteins controlling mitosis. PMID:25722206

  12. Developmental checkpoints guarded by regulated necrosis.

    PubMed

    Dillon, Christopher P; Tummers, Bart; Baran, Katherine; Green, Douglas R

    2016-06-01

    The process of embryonic development is highly regulated through the symbiotic control of differentiation and programmed cell death pathways, which together sculpt tissues and organs. The importance of programmed necrotic (RIPK-dependent necroptosis) cell death during development has recently been recognized as important and has largely been characterized using genetically engineered animals. Suppression of necroptosis appears to be essential for murine development and occurs at three distinct checkpoints, E10.5, E16.5, and P1. These distinct time points have helped delineate the molecular pathways and regulation of necroptosis. The embryonic lethality at E10.5 seen in knockouts of caspase-8, FADD, or FLIP (cflar), components of the extrinsic apoptosis pathway, resulted in pallid embryos that did not exhibit the expected cellular expansions. This was the first suggestion that these factors play an important role in the inhibition of necroptotic cell death. The embryonic lethality at E16.5 highlighted the importance of TNF engaging necroptosis in vivo, since elimination of TNFR1 from casp8 (-/-), fadd (-/-), or cflar (-/-), ripk3 (-/-) embryos delayed embryonic lethality from E10.5 until E16.5. The P1 checkpoint demonstrates the dual role of RIPK1 in both the induction and inhibition of necroptosis, depending on the upstream signal. This review summarizes the role of necroptosis in development and the genetic evidence that helped detail the molecular mechanisms of this novel pathway of programmed cell death. PMID:27056574

  13. Immune checkpoint inhibition in lymphoid disease.

    PubMed

    Eyre, Toby A; Collins, Graham P

    2015-08-01

    It has long been understood that the immune system has intrinsic anti-tumour activity in humans, and that a key mechanism of tumour progression is the ability of a tumour to escape this immune surveillance. A number of attempts have been made to harness this anti-tumour immunity in both solid tumour oncology and haematological malignancies with variable success. Examples include the use of allogeneic stem cell transplantation and donor lymphocyte infusion in haematological cancer and vaccine studies in solid tumours. Enhanced signalling of the Programmed cell death-1 (PDCD1, PD-1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA4) 'immune checkpoint' pathway has emerged recently as a critical mechanism by which tumours can escape the natural anti-tumour immune response. As such, novel therapies have been developed to help enhance this natural immunity by switching off the PDCD1/CTLA4 immune checkpoint pathway. The following review will discuss the pathobiology of these pathways and the exciting new data now available in lymphoid malignancies. PMID:25824455

  14. Immune Checkpoint Blockade in Cancer Therapy

    PubMed Central

    Postow, Michael A.; Callahan, Margaret K.; Wolchok, Jedd D.

    2015-01-01

    Immunologic checkpoint blockade with antibodies that target cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) and the programmed cell death protein 1 pathway (PD-1/PD-L1) have demonstrated promise in a variety of malignancies. Ipilimumab (CTLA-4) and pembrolizumab (PD-1) are approved by the US Food and Drug Administration for the treatment of advanced melanoma, and additional regulatory approvals are expected across the oncologic spectrum for a variety of other agents that target these pathways. Treatment with both CTLA-4 and PD-1/PD-L1 blockade is associated with a unique pattern of adverse events called immune-related adverse events, and occasionally, unusual kinetics of tumor response are seen. Combination approaches involving CTLA-4 and PD-1/PD-L1 blockade are being investigated to determine whether they enhance the efficacy of either approach alone. Principles learned during the development of CTLA-4 and PD-1/PD-L1 approaches will likely be used as new immunologic checkpoint blocking antibodies begin clinical investigation. PMID:25605845

  15. Electrical cable connector-clamp has smooth exterior surface

    NASA Technical Reports Server (NTRS)

    1965-01-01

    Electrical cable connector-clamp fitted with a collet has a smooth exterior surface that can be easily gripped. The collet clamps a portion of the cable and provides for connecting it to a standard electrical connector.

  16. Mechanical and metallurgical properties of carotid artery clamps.

    PubMed

    Dujovny, M; Kossovsky, N; Kossowsky, R; Segal, R; Diaz, F G; Kaufman, H; Perlin, A; Cook, E E

    1985-11-01

    The mechanical and metallurgical properties of carotid artery clamps were evaluated. The pressure plate retreat propensity, metallurgical composition, surface morphology, magnetic properties, and corrosion resistance of the Crutchfield, Selverstone, Salibi, and Kindt clamps were tested. None of the clamps showed evidence of pressure plate retreat. The clamps differed significantly in their composition, surface cleanliness, magnetic properties, and corrosion resistance. The Crutchfield clamp was the only one manufactured from an ASTM-ANSI-approved implantable stainless steel (AISI 316) and the only clamp in which the surfaces were clean and free of debris. The Selverstone clamp was made principally from AISI 304 stainless steel, as was one Salibi clamp. The pressure plate on another Salibi clamp was made from a 1% chromium and 1% manganese steel. Machining and surface debris consisting principally of aluminum, silicon, and sulfur was abundant on the Selverstone and Salibi clamps. The Kindt clamp was manufactured from AISI 301 stainless steel with a silicate-aluminized outer coating. The Crutchfield and Selverstone clamps were essentially nonferromagnetic, whereas the Salibi and Kindt clamps were sensitive to magnetic flux. In the pitting potential corrosion test, the Crutchfield clamp demonstrated good corrosion resistance with a pitting potential of 310 mV and no surface corrosion or pitting by scanning electron microscopy examination. The Selverstone clamp had lower pitting potentials and showed various degrees of corrosion and surface pitting by scanning electron microscopy. The Salibi pressure plate had a very low pitting potential of -525 mV and showed severe corrosion. By metallurgical criteria, only the Crutchfield clamp is suitable for long term implantation. PMID:4069328

  17. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Requirements § 18.40 Cable clamps and grips. Insulated clamps shall be provided for all portable (trailing) cables to prevent strain on the cable terminals of a machine. Also insulated clamps shall be provided to... mounted component. Cable grips anchored to the cable may be used in lieu of insulated strain...

  18. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Requirements § 18.40 Cable clamps and grips. Insulated clamps shall be provided for all portable (trailing) cables to prevent strain on the cable terminals of a machine. Also insulated clamps shall be provided to... mounted component. Cable grips anchored to the cable may be used in lieu of insulated strain...

  19. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Requirements § 18.40 Cable clamps and grips. Insulated clamps shall be provided for all portable (trailing) cables to prevent strain on the cable terminals of a machine. Also insulated clamps shall be provided to... mounted component. Cable grips anchored to the cable may be used in lieu of insulated strain...

  20. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Requirements § 18.40 Cable clamps and grips. Insulated clamps shall be provided for all portable (trailing) cables to prevent strain on the cable terminals of a machine. Also insulated clamps shall be provided to... mounted component. Cable grips anchored to the cable may be used in lieu of insulated strain...

  1. Single molecule study of a processivity clamp sliding on DNA

    SciTech Connect

    Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A; Barsky, D

    2007-07-05

    Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.

  2. Using the Sirocco File System for high-bandwidth checkpoints.

    SciTech Connect

    Klundt, Ruth Ann; Curry, Matthew L.; Ward, H. Lee

    2012-02-01

    The Sirocco File System, a file system for exascale under active development, is designed to allow the storage software to maximize quality of service through increased flexibility and local decision-making. By allowing the storage system to manage a range of storage targets that have varying speeds and capacities, the system can increase the speed and surety of storage to the application. We instrument CTH to use a group of RAM-based Sirocco storage servers allocated within the job as a high-performance storage tier to accept checkpoints, allowing computation to potentially continue asynchronously of checkpoint migration to slower, more permanent storage. The result is a 10-60x speedup in constructing and moving checkpoint data from the compute nodes. This demonstration of early Sirocco functionality shows a significant benefit for a real I/O workload, checkpointing, in a real application, CTH. By running Sirocco storage servers within a job as RAM-only stores, CTH was able to store checkpoints 10-60x faster than storing to PanFS, allowing the job to continue computing sooner. While this prototype did not include automatic data migration, the checkpoint was available to be pushed or pulled to disk-based storage as needed after the compute nodes continued computing. Future developments include the ability to dynamically spawn Sirocco nodes to absorb checkpoints, expanding this mechanism to other fast tiers of storage like flash memory, and sharing of dynamic Sirocco nodes between multiple jobs as needed.

  3. Keeping checkpoint/restart viable for exascale systems.

    SciTech Connect

    Riesen, Rolf E.; Bridges, Patrick G.; Stearley, Jon R.; Laros, James H., III; Oldfield, Ron A.; Arnold, Dorian; Pedretti, Kevin Thomas Tauke; Ferreira, Kurt Brian; Brightwell, Ronald Brian

    2011-09-01

    Next-generation exascale systems, those capable of performing a quintillion (10{sup 18}) operations per second, are expected to be delivered in the next 8-10 years. These systems, which will be 1,000 times faster than current systems, will be of unprecedented scale. As these systems continue to grow in size, faults will become increasingly common, even over the course of small calculations. Therefore, issues such as fault tolerance and reliability will limit application scalability. Current techniques to ensure progress across faults like checkpoint/restart, the dominant fault tolerance mechanism for the last 25 years, are increasingly problematic at the scales of future systems due to their excessive overheads. In this work, we evaluate a number of techniques to decrease the overhead of checkpoint/restart and keep this method viable for future exascale systems. More specifically, this work evaluates state-machine replication to dramatically increase the checkpoint interval (the time between successive checkpoint) and hash-based, probabilistic incremental checkpointing using graphics processing units to decrease the checkpoint commit time (the time to save one checkpoint). Using a combination of empirical analysis, modeling, and simulation, we study the costs and benefits of these approaches on a wide range of parameters. These results, which cover of number of high-performance computing capability workloads, different failure distributions, hardware mean time to failures, and I/O bandwidths, show the potential benefits of these techniques for meeting the reliability demands of future exascale platforms.

  4. Managing Adverse Events With Immune Checkpoint Agents.

    PubMed

    Dadu, Ramona; Zobniw, Chrystia; Diab, Adi

    2016-01-01

    Immune checkpoint inhibitors (anti-cytotoxic T-lymphocyte antigen 4 and anti programmed cell death 1/programmed cell death 1 ligand antibodies) have shown impressive clinical activity in multiple cancer types. Despite achieving great clinical success, challenges and limitations of these drugs as monotherapy or various combinational strategies include the development of a unique set of immune-related adverse events (irAEs) that can be severe and even fatal. Therefore, identification of patients at risk, prevention, consistent communication between patients and medical team, rapid recognition, and treatment of irAEs are critical in optimizing treatment outcomes. This review focuses on the description of more common irAEs and provides a suggested approach for management of specific irAEs. PMID:27111908

  5. π-Clamp Mediated Cysteine Conjugation

    PubMed Central

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; Van Voorhis, Troy; Pentelute, Bradley L.

    2016-01-01

    Site-selective functionalization of complex molecules is a grand challenge in chemistry. Protecting groups or catalysts must be used to selectively modify one site among many that are similarly reactive. General strategies are rare such the local chemical environment around the target site is tuned for selective transformation. Here we show a four amino acid sequence (Phe-Cys-Pro-Phe), which we call the “π-clamp”, tunes the reactivity of its cysteine thiol for the site-selective conjugation with perfluoroaromatic reagents. We used the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues (e.g. antibodies and cysteine-based enzymes), which was impossible with prior cysteine modification methods. The modified π-clamp antibodies retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates (ADCs) for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach for site-selective chemistry and provides opportunities to modify biomolecules for research and therapeutics. PMID:26791894

  6. Temperature-Controlled Clamping and Releasing Mechanism

    NASA Technical Reports Server (NTRS)

    Rosing, David; Ford, Virginia

    2005-01-01

    A report describes the development of a mechanism that automatically clamps upon warming and releases upon cooling between temperature limits of approx. =180 K and approx. =293 K. The mechanism satisfied a need specific to a program that involved repeated excursions of a spectrometer between a room-temperature atmospheric environment and a cryogenic vacuum testing environment. The mechanism was also to be utilized in the intended application of the spectrometer, in which the spectrometer would be clamped for protection during launch of a spacecraft and released in the cold of outer space to allow it to assume its nominal configuration for scientific observations. The mechanism is passive in the sense that its operation does not depend on a control system and does not require any power other than that incidental to heating and cooling. The clamping and releasing action is effected by bolt-preloaded stacks of shape-memory-alloy (SMA) cylinders. In designing this mechanism, as in designing other, similar SMA mechanisms, it was necessary to account for the complex interplay among thermal expansion, elastic and inelastic deformation under load, and SMA thermomechanical properties.

  7. Carbon nanotube-clamped metal atomic chain

    PubMed Central

    Tang, Dai-Ming; Yin, Li-Chang; Li, Feng; Liu, Chang; Yu, Wan-Jing; Hou, Peng-Xiang; Wu, Bo; Lee, Young-Hee; Ma, Xiu-Liang; Cheng, Hui-Ming

    2010-01-01

    Metal atomic chain (MAC) is an ultimate one-dimensional structure with unique physical properties, such as quantized conductance, colossal magnetic anisotropy, and quantized magnetoresistance. Therefore, MACs show great potential as possible components of nanoscale electronic and spintronic devices. However, MACs are usually suspended between two macroscale metallic electrodes; hence obvious technical barriers exist in the interconnection and integration of MACs. Here we report a carbon nanotube (CNT)-clamped MAC, where CNTs play the roles of both nanoconnector and electrodes. This nanostructure is prepared by in situ machining a metal-filled CNT, including peeling off carbon shells by spatially and elementally selective electron beam irradiation and further elongating the exposed metal nanorod. The microstructure and formation process of this CNT-clamped MAC are explored by both transmission electron microscopy observations and theoretical simulations. First-principles calculations indicate that strong covalent bonds are formed between the CNT and MAC. The electrical transport property of the CNT-clamped MAC was experimentally measured, and quantized conductance was observed. PMID:20427743

  8. π-Clamp-mediated cysteine conjugation

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  9. Checkpoint Blockade for the Treatment of Advanced Melanoma.

    PubMed

    Callahan, Margaret K; Flaherty, Catherine R; Postow, Michael A

    2016-01-01

    Immunotherapy with immune checkpoint inhibition has been improving the outcomes of patients with many different types of malignancies. Immune checkpoint inhibition has been most extensively studied in patients with advanced melanoma and there are three FDA approved antibodies already widely used in clinical practice (ipilimumab, nivolumab, and pembrolizumab). In this chapter, we review the mechanistic basis behind the development of immune checkpoint blocking antibodies. We then discuss specifics regarding each agent, unique clinical considerations in treating patients with this approach, and future directions, including combination strategies. This chapter is focused on melanoma, but the principles related to this immunotherapy approach are applicable to patients with many types of malignancies. PMID:26601865

  10. Condition of chromic acid anodized aluminum clamps flown

    NASA Technical Reports Server (NTRS)

    Plagemann, W. L.

    1991-01-01

    A survey of the condition of the chromic acid anodized (CAA) coating on selected LDEF tray clamps was carried out. Measurements of solar absorptance and thermal emittance were carried out at multiple locations on both the space exposed and spacecraft facing sides of the clamps. Multiple clamps from each available angle relative to the ram direction were examined. The diffuse component of the reflectance spectrum was measured for a selected subset of the clamps. The thickness of the CAA was determined for a small set of clamps. Examples of variation in integrity of the coatings from leading to trailing edge will be shown.

  11. Analyzing the ATR-mediated checkpoint using Xenopus egg extracts

    PubMed Central

    Lupardus, Patrick J.; Van, Christopher; Cimprich, Karlene A.

    2009-01-01

    Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR. PMID:17189864

  12. Immune checkpoint inhibitor combinations in solid tumors: opportunities and challenges.

    PubMed

    Kyi, Chrisann; Postow, Michael A

    2016-06-01

    The emergence of immune 'checkpoint inhibitors' such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death receptor 1 (PD-1) has revolutionized treatment of solid tumors including melanoma, lung cancer, among many others. The goal of checkpoint inhibitor combination therapy is to improve clinical response and minimize toxicities. Rational design of checkpoint combinations considers immune-mediated mechanisms of antitumor activity: immunogenic cell death, antigen release and presentation, activation of T-cell responses, lymphocytic infiltration into tumors and depletion of immunosuppression. Potential synergistic combinations include checkpoint blockade with conventional (radiation, chemotherapy and targeted therapies) and newer immunotherapies (cancer vaccines, oncolytic viruses, among others). Reliable biomarkers are necessary to define patients who will achieve best clinical benefit with minimal toxicity in combination therapy. PMID:27349981

  13. Checkpoint/Restart of Virtual Machines Based on Xen

    SciTech Connect

    Vallee, Geoffroy R; Naughton, III, Thomas J; Ong, Hong Hoe; Scott, Stephen L

    2006-01-01

    System level virtualization provides several advantages: (i) customization is eased since virtual machines may be based on different systems; (ii) virtual machines are isolated from hardware, subsequently applications are isolated via the virtual machines; (iii) basic fault tolerance mechanisms -- pro-active fault tolerance through virtual machine migration and virtual machine snapshot/restore; and (iv) basic load balancing mechanisms -- the capability to move and stop virtual machines running in the system. However, the current Xen implementation does not natively provide mechanisms for virtual machine checkpoint/restart. This document presents the design of a reactive fault tolerant system, based on a checkpoint/restart mechanism for Xen virtual machines. We present the infrastructure for the management of virtual machines' checkpoint data as well as challenges for the implementation of a virtual machine checkpoint/restart mechanism based on Xen.

  14. Immune checkpoint blockade: a common denominator approach to cancer therapy

    PubMed Central

    Drake, Charles G.; Pardoll, Drew M.

    2015-01-01

    Summary The immune system recognizes and is poised to eliminate cancer, but is held in check by inhibitory receptors and ligands. These immune checkpoint pathways, which normally maintain self-tolerance and limit collateral tissue damage during anti-microbial immune responses, can be co-opted by cancer to evade immune destruction. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, and others in early development can unleash anti-tumor immunity and mediate durable cancer regressions. The complex biology of immune checkpoint pathways still contains many mysteries, and the full activity spectrum of checkpoint-blocking drugs, used alone or in combination, is currently the subject of intense study. PMID:25858804

  15. PD-1 Checkpoint Blockade in Acute Myeloid Leukemia

    PubMed Central

    Sehgal, Alison; Whiteside, Theresa L.; Boyiadzis, Michael

    2015-01-01

    Introduction Immune checkpoints are regulatory pathways induced in activated T lymphocytes that regulate antigen responsiveness. These immune checkpoints are hijacked by tumors to promote dysfunction of anti-tumor effector cells and consequently of tumor escape from the host immune system. Areas covered PD1/PDL-1, a checkpoint pathway, has been extensively investigated in leukemia mouse models. Expression of PD-1 on the surface of activated immune cells and of its ligands, PD-L1 and PD-L2, on leukemic blasts has been documented. Clinical trials with PD-1 inhibitors in patients with hematological malignancies are ongoing with promising clinical responses. Expert Opinion Therapy of hematological cancers with antibodies blocking inhibitory receptors is expected to be highly clinically effective. Checkpoint inhibitory receptors and their ligands are co-expressed on hematopoietic cells found in the leukemic milieu. Several distinct immunological mechanisms are likely to be engaged by antibody-based checkpoint blockade. Co-expression of multiple inhibitory receptors on hematopoietic cells offers an opportunity for combining blocking antibodies to achieve more effective therapy. Up-regulation of receptor/ligand expression in the leukemic milieu may provide a blood marker predictive of response. Finally, chemotherapy-induced up-regulation of PD-1 on T cells after conventional leukemia therapy creates a solid rationale for application of checkpoint blockade as a follow-up therapy. PMID:26036819

  16. Hypoxia-induced alterations of G2 checkpoint regulators.

    PubMed

    Hasvold, Grete; Lund-Andersen, Christin; Lando, Malin; Patzke, Sebastian; Hauge, Sissel; Suo, ZhenHe; Lyng, Heidi; Syljuåsen, Randi G

    2016-05-01

    Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage-induced cell cycle checkpoints. Here, we show that hypoxia (24 h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, the levels of several G2 checkpoint regulators, in particular Cyclin B, were reduced in G2 phase cells after hypoxic exposure, as shown by flow cytometric barcoding analysis of individual cells. These effects were accompanied by decreased phosphorylation of a Cyclin dependent kinase (CDK) target in G2 phase cells after hypoxia, suggesting decreased CDK activity. Furthermore, cells pre-exposed to hypoxia showed increased G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (∼0.03% O2 20 h and 0.2% O2 72 h). These results demonstrate that the DNA damage-induced G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors. PMID:26791779

  17. DMTCP: bringing interactive checkpoint-restart to Python

    NASA Astrophysics Data System (ADS)

    Arya, Kapil; Cooperman, Gene

    2015-01-01

    DMTCP (Distributed MultiThreaded CheckPointing) is a mature checkpoint-restart package. It operates in user space without kernel privilege, and adapts to application-specific requirements through plugins. While DMTCP has been able to checkpoint Python and IPython ‘from the outside’ for many years, a Python module has recently been created to support DMTCP. IPython support is included through a new DMTCP plugin. A checkpoint can be requested interactively within a Python session or under the control of a specific Python program. Further, the Python program can execute specific Python code prior to checkpoint, upon resuming (within the original process) and upon restarting (from a checkpoint image). Applications of DMTCP are demonstrated for: (i) Python-based graphics using virtual network client, (ii) a fast/slow technique to use multiple hosts or cores to check one (Cython Behnel S et al 2011 Comput. Sci. Eng. 13 31-39) computation in parallel, and (iii) a reversible debugger, FReD, with a novel reverse-expression watchpoint feature for locating the cause of a bug.

  18. Biological cell controllable patch-clamp microchip

    NASA Astrophysics Data System (ADS)

    Penmetsa, Siva; Nagrajan, Krithika; Gong, Zhongcheng; Mills, David; Que, Long

    2010-12-01

    A patch-clamp (PC) microchip with cell sorting and positioning functions is reported, which can avoid drawbacks of random cell selection or positioning for a PC microchip. The cell sorting and positioning are enabled by air bubble (AB) actuators. AB actuators are pneumatic actuators, in which air pressure is generated by microheaters within sealed microchambers. The sorting, positioning, and capturing of 3T3 cells by this type of microchip have been demonstrated. Using human breast cancer cells MDA-MB-231 as the model, experiments have been demonstrated by this microchip as a label-free technical platform for real-time monitoring of the cell viability.

  19. Checkpoint kinase 1 modulates sensitivity to cisplatin after spindle checkpoint activation in SW620 cells.

    PubMed

    Peralta-Sastre, A; Manguan-Garcia, C; de Luis, A; Belda-Iniesta, C; Moreno, S; Perona, R; Sanchez-Perez, I

    2010-02-01

    Aneuploidy is a common feature of tumours that arise by errors in chromosome segregation during mitosis. The aim of this study was to evaluate possible signaling pathways involved in sensitization to chemotherapy in cells with chromosomal instability. We designed a screen using the fission yeast Squizossaccharomyces pombe, to isolate strains showing a phenotype of chromosome mis-segregation and higher sensitivity to the antitumoral drug Bleomycin. We examined differences in gene expression using a comparative analysis of genome-wide expression of the wild type strain and one of the mutants. The results revealed a set of genes involved in cell cycle control, including Mad3/BubR1 and Chk1. We then studied the levels of these two proteins in colorectal cancer human cell lines with different genomic content. Among these, SW620 cells showed higher BubR1 and Chk1 mRNA levels than control cells under normal conditions. Since Chk1 is required for both S and G2/M checkpoints, and the microtubule-destabilizing agent, nocodazole induces mitotic arrest, we attempted to investigate the potential anticancer effects of nocodazole in combination with cisplatin. These studies showed that SW620 cells undergo synergistic cell death after spindle checkpoint activation followed by cisplatin treatment, suggesting a role of Chk1 in this checkpoint, very likely dependent on BubR1 protein. Importantly, Chk1-depleted SW620 cells lost this synergistic effect. In summary, we propose that Chk1 could be a biomarker predictive of the efficacy of chemotherapy across different types of tumors with aneuploidy. These findings may be potentially very useful for the stratification of patients for treatment. PMID:19931410

  20. Checkpoint blockade in combination with cancer vaccines.

    PubMed

    Morse, Michael A; Lyerly, H Kim

    2015-12-16

    Checkpoint blockade, prevention of inhibitory signaling that limits activation or function of tumor antigen-specific T cells responses, is revolutionizing the treatment of many poor prognosis malignancies. Indeed monoclonal antibodies that modulate signaling through the inhibitory molecules CTLA-4 and PD-1 are now clinically available; however, many tumors, demonstrate minimal response suggesting the need for combinations with other therapeutic strategies. Because an inadequate frequency of activated tumor antigen-specific T cells in the tumor environment, the so-called non-inflamed phenotype, is observed in some malignancies, other rationale partners are modalities that lead to enhanced T cell activation (vaccines, cytokines, toll-like receptor agonists, and other anticancer therapies such as chemo-, radio- or targeted therapies that lead to release of antigen from tumors). This review will focus on preclinical and clinical data supporting the use of cancer vaccines with anti-CTLA-4 and anti-PD-1/PD-L1 antibodies. Preliminary preclinical data demonstrate enhanced antitumor activity although the results in human studies are less clear. Broader combinations of multiple immune modulators are now under study. PMID:26482147

  1. Laser-assisted patch clamping: a methodology

    NASA Technical Reports Server (NTRS)

    Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

  2. ClampOn acoustic solid fuel monitor

    SciTech Connect

    Vesterhus, T.

    1999-07-01

    The general idea of the project is to develop a ClampOn Solid Fuel Monitor, enabling optimization of the combustion process in pulverized coal fired boilers. The development will be based on adapting existing technology for measuring the content of sand particles in a flow of natural gas. The Norwegian firm ClampOn AS develops equipment for such measurements, and has already a proven track record as a result of its work with major oil companies throughout the world. The industry wants some sort of fuel indicator, e.g. a piece of equipment that enables the operator to measure and control the amounts of the fuel to each individual burner. The best techniques available today--as far as the author knows--can only offer samples of the fuel stream at discrete points of time. To truly optimize the combustion process, it is vital to continuously monitor the mass of fuel to each burner, and optimize the combustion process through continuous and infinitesimal adjustments of the fuel flow. This will minimize the NO{sub x} created by uneven temperature-distribution in the combustion chamber. In this way maximum power generation can be obtained at minimal emission of pollutants for a given amount of coal burned.

  3. The Replication Checkpoint Protects Fork Stability by Releasing Transcribed Genes from Nuclear Pores

    PubMed Central

    Bermejo, Rodrigo; Capra, Thelma; Jossen, Rachel; Colosio, Arianna; Frattini, Camilla; Carotenuto, Walter; Cocito, Andrea; Doksani, Ylli; Klein, Hannah; Gómez-González, Belén; Aguilera, Andrés; Katou, Yuki; Shirahige, Katsuhiko; Foiani, Marco

    2011-01-01

    Summary Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes. PMID:21784245

  4. Phosphorylation-dependent Assembly and Coordination of the DNA Damage Checkpoint Apparatus by Rad4TopBP1

    PubMed Central

    Garcia, Valerie; Ren, Jing-Yi; Day, Matthew; Carr, Antony M.; Oliver, Antony W.; Du, Li-Lin; Pearl, Laurence H.

    2016-01-01

    Summary The BRCT-domain protein, Rad4TopBP1, facilitates activation of the DNA damage checkpoint in S. pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3ATR-Rad26ATRIP kinase complex and Crb253BP1 mediator. We have now determined crystal structures of the BRCT repeats of Rad4TopBP1, revealing a distinctive domain architecture, and have characterized their phosphorylation-dependent interactions with Rad9 and Crb253BP1. We identify a cluster of phosphorylation sites in the N-terminal region of Crb253BP1 that mediate interaction with Rad4TopBP1, and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Thr215 and Thr235 promotes phosphorylation of the non-canonical Thr187 site by scaffolding CDK recruitment. Finally we show that simultaneous interaction of a single Rad4TopBP1 molecule with both Thr187 phosphorylation sites in a Crb253BP1 dimer, is essential for establishing the DNA damage checkpoint. PMID:24074952

  5. Cell cycle control, checkpoint mechanisms, and genotoxic stress.

    PubMed Central

    Shackelford, R E; Kaufmann, W K; Paules, R S

    1999-01-01

    The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle

  6. Characterization of the clamp pressure of electrostatic chucks

    NASA Astrophysics Data System (ADS)

    Ziemann, M.; Voss, S.; Baldus, O.; Schmidt, V.

    2010-04-01

    Berliner Glas KGaA is specialized on the manufacturing of high performance wafer and reticle chucks. Electrostatic chucks (ESC) are especially used in vacuum environments e.g. during lithographic processing, coating and etching. The main task of the chuck is to provide a well defined positioning and thermal stabilization of the wafer or reticle. Typical wafer materials are semiconductors like silicon and in some special cases dielectrics like magnesia, alumina or glass. For a functional characterization of the ESC clamps Berliner Glas has developed a measurement method to determine the clamp pressure with a Fizeau interferometer. The setup utilizes the local bending of clamped wafers to determine the effective clamp pressure. The clamp pressure is measured in the range of 20...500 mbar. This new method allows for a lateral resolution of the clamp pressure measurement. It can be calibrated by various methods. Direct computation of the clamp pressure based on the bending height or comparative measurements with vacuum chucking by the same chuck gives evidence for the quantitative results. Transient clamp pressure variation can be measured with a resolution of 2 mbar. The results can be used to qualify and optimize ESĆs and even for a local correction of the clamp force.

  7. An Optimal Cell Detection Technique for Automated Patch Clamping

    NASA Technical Reports Server (NTRS)

    McDowell, Mark; Gray, Elizabeth

    2004-01-01

    While there are several hardware techniques for the automated patch clamping of cells that describe the equipment apparatus used for patch clamping, very few explain the science behind the actual technique of locating the ideal cell for a patch clamping procedure. We present a machine vision approach to patch clamping cell selection by developing an intelligent algorithm technique that gives the user the ability to determine the good cell to patch clamp in an image within one second. This technique will aid the user in determining the best candidates for patch clamping and will ultimately save time, increase efficiency and reduce cost. The ultimate goal is to combine intelligent processing with instrumentation and controls in order to produce a complete turnkey automated patch clamping system capable of accurately and reliably patch clamping cells with a minimum amount of human intervention. We present a unique technique that identifies good patch clamping cell candidates based on feature metrics of a cell's (x, y) position, major axis length, minor axis length, area, elongation, roundness, smoothness, angle of orientation, thinness and whether or not the cell is only particularly in the field of view. A patent is pending for this research.

  8. Spectral infrared hemispherical reflectance measurements for LDEF tray clamps

    NASA Technical Reports Server (NTRS)

    Cromwell, B. K.; Shepherd, S. D.; Pender, C. W.; Wood, B. E.

    1993-01-01

    Infrared hemispherical reflectance measurements that were made on 58 chromic acid anodized tray clamps from LDEF are described. The measurements were made using a hemiellipsoidal mirror reflectometer with interferometer for wavelengths between 2-15 microns. The tray clamps investigated were from locations about the entire spacecraft and provided the opportunity for comparing the effects of atomic oxygen at each location. Results indicate there was essentially no dependence on atomic oxygen fluence for the surfaces studied, but there did appear to be a slight dependence on solar radiation exposure. The reflectances of the front sides of the tray clamps consistently were slightly higher than for the protected rear tray clamp surfaces.

  9. Re-visiting the trans insertion model for complexin clamping.

    PubMed

    Krishnakumar, Shyam S; Li, Feng; Coleman, Jeff; Schauder, Curtis M; Kümmel, Daniel; Pincet, Frederic; Rothman, James E; Reinisch, Karin M

    2015-01-01

    We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based. Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping. PMID:25831964

  10. Berkeley lab checkpoint/restart (BLCR) for Linux clusters

    NASA Astrophysics Data System (ADS)

    Hargrove, Paul H.; Duell, Jason C.

    2006-09-01

    This article describes the motivation, design and implementation of Berkeley Lab Checkpoint/Restart (BLCR), a system-level checkpoint/restart implementation for Linux clusters that targets the space of typical High Performance Computing applications, including MPI. Application-level solutions, including both checkpointing and fault-tolerant algorithms, are recognized as more time and space efficient than system-level checkpoints, which cannot make use of any application-specific knowledge. However, system-level checkpointing allows for preemption, making it suitable for responding to ''fault precursors'' (for instance, elevated error rates from ECC memory or network CRCs, or elevated temperature from sensors). Preemption can also increase the efficiency of batch scheduling; for instance reducing idle cycles (by allowing for shutdown without any queue draining period or reallocation of resources to eliminate idle nodes when better fitting jobs are queued), and reducing the average queued time (by limiting large jobs to running during off-peak hours, without the need to limit the length of such jobs). Each of these potential uses makes BLCR a valuable tool for efficient resource management in Linux clusters.

  11. Message Efficient Checkpointing and Rollback Recovery in Heterogeneous Mobile Networks

    NASA Astrophysics Data System (ADS)

    Jaggi, Parmeet Kaur; Singh, Awadhesh Kumar

    2016-06-01

    Heterogeneous networks provide an appealing way of expanding the computing capability of mobile networks by combining infrastructure-less mobile ad-hoc networks with the infrastructure-based cellular mobile networks. The nodes in such a network range from low-power nodes to macro base stations and thus, vary greatly in their capabilities such as computation power and battery power. The nodes are susceptible to different types of transient and permanent failures and therefore, the algorithms designed for such networks need to be fault-tolerant. The article presents a checkpointing algorithm for the rollback recovery of mobile hosts in a heterogeneous mobile network. Checkpointing is a well established approach to provide fault tolerance in static and cellular mobile distributed systems. However, the use of checkpointing for fault tolerance in a heterogeneous environment remains to be explored. The proposed protocol is based on the results of zigzag paths and zigzag cycles by Netzer-Xu. Considering the heterogeneity prevalent in the network, an uncoordinated checkpointing technique is employed. Yet, useless checkpoints are avoided without causing a high message overhead.

  12. Heterologous Vaccination and Checkpoint Blockade Synergize To Induce Antileukemia Immunity.

    PubMed

    Manlove, Luke S; Schenkel, Jason M; Manlove, Kezia R; Pauken, Kristen E; Williams, Richard T; Vezys, Vaiva; Farrar, Michael A

    2016-06-01

    Checkpoint blockade-based immunotherapies are effective in cancers with high numbers of nonsynonymous mutations. In contrast, current paradigms suggest that such approaches will be ineffective in cancers with few nonsynonymous mutations. To examine this issue, we made use of a murine model of BCR-ABL(+) B-lineage acute lymphoblastic leukemia. Using a principal component analysis, we found that robust MHC class II expression, coupled with appropriate costimulation, correlated with lower leukemic burden. We next assessed whether checkpoint blockade or therapeutic vaccination could improve survival in mice with pre-established leukemia. Consistent with the low mutation load in our leukemia model, we found that checkpoint blockade alone had only modest effects on survival. In contrast, robust heterologous vaccination with a peptide derived from the BCR-ABL fusion (BAp), a key driver mutation, generated a small population of mice that survived long-term. Checkpoint blockade strongly synergized with heterologous vaccination to enhance overall survival in mice with leukemia. Enhanced survival did not correlate with numbers of BAp:I-A(b)-specific T cells, but rather with increased expression of IL-10, IL-17, and granzyme B and decreased expression of programmed death 1 on these cells. Our findings demonstrate that vaccination to key driver mutations cooperates with checkpoint blockade and allows for immune control of cancers with low nonsynonymous mutation loads. PMID:27183622

  13. Immune Checkpoint Inhibitors and Prostate Cancer: A New Frontier?

    PubMed Central

    Modena, Alessandra; Ciccarese, Chiara; Iacovelli, Roberto; Brunelli, Matteo; Montironi, Rodolfo; Fiorentino, Michelangelo; Tortora, Giampaolo; Massari, Francesco

    2016-01-01

    Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (mCRPC), agents that provide durable disease control and long-term survival are still needed. It is a fact that a tumor-induced immunosuppressive status (mediated by aberrant activation of inhibitory immune checkpoint pathways as a mechanism to evade host immune surveillance) plays a crucial role in the pathogenesis of cancer, including prostate cancer (PC), making CRPC patients suitable candidates for immunotherapy. Therefore, growing interest of anticancer research aims at blocking immune checkpoints (mainly targeting CTLA-4 and PD1/PD-L1 pathways) to restore and enhance cellular-mediated antitumor immunity and achieve durable tumor regression. In this review, we describe the current knowledge regarding the role of immune checkpoints in mediating PC progression, focusing on CTLA-4 and PD1 pathways. We also provide current clinical data available, an update on ongoing trials of immune checkpoint inhibitors in PC. Finally, we discuss the necessity to identify prognostic and predictive biomarkers of immune activity, and we analyze new immune checkpoints with a role as promising targets for PC therapy. PMID:27471580

  14. Voltage clamp experiments on ventricular myocarial fibres.

    PubMed

    Beeler, G W; Reuter, H

    1970-03-01

    1. A voltage clamp method utilizing a sucrose gap and glass microelectrodes was developed and used to study dog ventricular myocardial fibre bundles. The limitations and the reliability of this method are demonstrated by a series of tests.2. A dynamic sodium current, excited at membrane potentials more positive than -65 mV, was measured. The equilibrium potential for this large, rapid inward current depends directly on [Na](o), shifting 29.0 +/- 2.3 mV (+/- S.E. of mean), as opposed to a theoretically expected value of 30.6 mV, when [Na](o) is reduced to 31% of normal.3. Sodium current is inactivated by conditioning depolarizations. Complete inactivation occurs with conditioning potentials more positive than -45 mV, and 50% inactivation occurs at about -55 mV. The location of the inactivation curve shifts along the voltage axis, when [Ca](o) is varied between 0.2 and 7.2 mM.4. A second, much smaller and slower net inward current, with a threshold around -30 mV, and an equilibrium potential above +40 mV was also observed.5. The ;steady-state' current-voltage relationship (after 300-600 msec) exhibits inward-going (anomalous) rectification with negative slope between -50 and -25 mV.6. A small, very slowly developing component of outward current was observed at inside positive potentials. The equilibrium potential for this current, although slightly dependent on [K](o), is neither identical with the potassium equilibrium potential nor with the resting potential in normal Tyrode solution.7. Anatomical limitations, primarily resistance in the extracellular space within the bundle, prevent complete characterization of the rapid, large sodium current, but do not limit the application of the clamp method to the study of other, smaller and slower currents. The evidence for this is discussed extensively in the Appendix. PMID:5503866

  15. Immune checkpoint blockade therapy for bladder cancer treatment.

    PubMed

    Kim, Jayoung

    2016-06-01

    Bladder cancer remains the most immunogenic and expensive malignant tumor in the United States today. As the 4th leading cause of death from cancer in United States, Immunotherapy blocking immune checkpoints have been recently been applied to many aggressive cancers and changed interventions of urological cancers including advanced bladder cancer. The applied inhibition of PD-1-PD-L1 interactions can restore antitumor T-cell activity and enhance the cellular immune attack on antigens. The overall goals of this short review article are to introduce current cancer immunotherapy and immune checkpoint inhibitors, and to provide new insight into the underlying mechanisms that block immune checkpoints in tumor microenvironment. Furthermore, this review will address the preclinical and clinical trials to determine whether bladder cancer patients could benefit from this new cancer therapy in near future. PMID:27326412

  16. Checkpoint Inhibitors for the Treatment of Hodgkin Lymphoma.

    PubMed

    Bennani-Baiti, Nabila; Thanarajasingam, Gita; Ansell, Stephen

    2016-06-01

    Hodgkin lymphoma's (HL) tumor composition is characterized by a paucity of malignant cells and a preponderance of immune and stromal cells. Despite the rich immune milieu within the tumor microenvironment, malignant cells are able to effectively evade the immune system and use immune support to promote lymphoma cell growth and proliferation. Recognizing this has led to the identification of checkpoint inhibitory signals that enable immune evasion and to opening the door to therapeutic strategies on how to exploit the immune system in targeting tumor cells. We discuss herein some of the tumor evasion mechanisms in HL with a particular focus on the immune checkpoint pathways and focus on recent clinical data of checkpoint blockade in HL treatment. PMID:26818843

  17. [Immune Checkpoint Therapy for Non-Small-Cell Lung Cancer].

    PubMed

    Miyauchi, Eisaku; Inoue, Akira

    2016-06-01

    Nivolumab is an anti-PD-1 antibody that has recently been approved in Japan, and has shown high response rates and more favorable safety profiles in 2 phase III clinical trials. Accordingly, immune checkpoint therapy has now been included as a new standard treatment for non-small-cell lung cancer. These immune checkpoints are receptors expressed on T cells that regulate the immune response. The PD-1/PD-L1 signal inhibits cytotoxic T lymphocyte proliferation and survival, induces apoptosis of infiltrative T cells, and increases the amount of regulatory T cells in the tumor microenvironment. Therefore, severe immune-related adverse event(irAE)have been observed, including enterocolitis, neuropathies, and endocrinopathies. There are different management approaches to irAEs with conventional cytotoxic drugs. This article reviews the available data regarding immune checkpoint therapy for patients with non-small-cell lung cancer. PMID:27306803

  18. CDK5RAP2 is required for spindle checkpoint function.

    PubMed

    Zhang, Xiaoying; Liu, Dongyun; Lv, Shuang; Wang, Haibo; Zhong, Xueyan; Liu, Bo; Wang, Bo; Liao, Ji; Li, Jing; Pfeifer, Gerd P; Xu, Xingzhi

    2009-04-15

    The combination of paclitaxel and doxorubicin is among the most successful chemotherapy regimens in cancer treatment. CDK5RAP2, when mutated, causes primary microcephaly. We show here that inhibition of CDK5RAP2 expression causes chromosome mis-segregation, fails to maintain the spindle checkpoint, and is associated with reduced expression of the spindle checkpoint proteins BUBR1 and MAD2 and an increase in chromatin-associated CDC20. CDK5RAP2 resides on the BUBR1 and MAD2 promoters and regulates their transcription. Furthermore, CDK5RAP2-knockdown cells have increased resistance to paclitaxel and doxorubicin, and this resistance is partially rescued upon restoration of CDK5RAP2 expression. Cancer cells cultured in the presence of paclitaxel or doxorubicin exhibit dramatically decreased CDK5RAP2 levels. These results suggest that CDK5RAP2 is required for spindle checkpoint function and is a common target in paclitaxel and doxorubicin resistance. PMID:19282672

  19. Inhibiting Immune Checkpoints for the Treatment of Bladder Cancer

    PubMed Central

    Bidnur, S.; Savdie, R.; Black, P.C.

    2016-01-01

    Background: Increasing evidence supporting the role of immune checkpoint blockade in cancer management has been bolstered by recent reports demonstrating significant and durable clinical responses across multiple tumour types, including metastatic urothelial carcinoma (mUC). The majority of these results are achieved via blockade of the programmed death (PD) axis, which like CTLA-4 blockade permits T-cell activation and immune-mediated anti-tumour activity- essentially harnessing the patient’s own immune system to mount an anti-neoplastic response. However, while clinical responses can be striking, our understanding of the biology of immune checkpoint blockade is only beginning to shed light on how to maximize and even improve patient outcomes with immune checkpoint blockade, especially in UC. Methods: We performed a literature review for immune checkpoint blockade with a focus on rationale for checkpoint therapy and outcomes in UC. We also highlight the advances made in other tumour types, with a focus on the recent 2015 meeting of the American Society for Clinical Oncology. Results: In heavily pre-treated UC, trials are suggesting objective response rates above 30% . These impressive results are seen across multiple different tumour types, especially those with high burden of DNA level mutations. Identification of prognostic biomarkers is currently under investigation, in order to improve patient selection. Interestingly, response to PD-1 directed therapy is seen even in patients with no evidence of PD-1 positivity on immunohistochemistry. This has led to the development of enhanced biomarkers including assessing DNA mutation rates and immune gene signatures, to improve patient selection. Conclusions: Immune checkpoint blockade is an exciting cancer treatment modality which is demonstrating impressive clinical results across multiple tumour types. For UC, anti-PD directed therapy represents a much needed treatment in the metastatic, post chemotherapy context

  20. Combination Space Station Handrail Clamp and Pointing Device

    NASA Technical Reports Server (NTRS)

    Hughes, Stephen J. (Inventor)

    1999-01-01

    A device for attaching an experiment carrier to a space station handrail is provided. The device has two major components, a clamping mechanism for attachment to a space station handrail, and a pointing carrier on which an experiment package can be mounted and oriented. The handrail clamp uses an overcenter mechanism and the carrier mechanism uses an adjustable preload ball and socket for carrier positioning. The handrail clamp uses a stack of disk springs to provide a spring loaded button. This configuration provides consistent clamping force over a range of possible handrail thicknesses. Three load points are incorporated in the clamping mechanism thereby spreading the clamping load onto three separate points on the handrail. A four bar linkage is used to provide for a single actuation lever for all three load points. For additional safety, a secondary lock consisting of a capture plate and push lock keeps the clamp attached to the handrail in the event of main clamp failure. For the carrier positioning mechanism, a ball in a spring loaded socket uses friction to provide locking torque; however. the ball and socket are torque limited so that the ball ran slip under kick loads (125 pounds or greater). A lead screw attached to disk spring stacks is used to provide an adjustable spring force on the socket. A locking knob is attached to the lead screw to allow for hand manipulation of the lead screw.

  1. OPTIMAL TIMING FOR CLAMPING THE UMBILICAL CORD AFTER BIRTH

    PubMed Central

    Raju, Tonse N. K.; Singal, Nalini

    2013-01-01

    Synopsis This paper provides a brief overview of pros and cons of clamping the cord too early (within seconds) after birth. It also highlights evolving data that suggests that delaying cord clamping for 30–60 seconds after birth is beneficial to the baby and the mother, with no measurable negative effects. PMID:23164185

  2. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for males. (a) Identification. A urological...

  3. Off-clamp robotic partial nephrectomy: Technique and outcome

    PubMed Central

    Lamoshi, Abdulraouf Y.; Salkini, Mohamad W.

    2015-01-01

    Introduction: Robotic partial nephrectomy (RPN) is a technically challenging procedure. Advanced skills are needed to accomplish tumor resection, hemostasis, and renorrhaphy within short ischemia time in RPN. Off-clamp RPN with zero ischemia may decrease the risk of ischemic reperfusion injury to the kidney. However, the off-clamp technique has been associated with an increased risk of blood loss. The purpose of this study was to evaluate the outcome of our modified off-clamp technique utilized in certain RPN cases. Patients and Methods: A total of 81 patients underwent RPN between September 2009 and July 2013 for renal masses. We studied a subgroup of patients who underwent off-clamp RPN with zero ischemia time. The off-clamp technique was utilized for exophytic, nonhilar tumors that have a base of 2 cm or less. We developed a novel technique to avoid ischemia reperfusion renal injury while minimizing blood loss in certain cases of RPN. Results: Of the 81 cases of RPN, we reviewed and adopted the off-clamp technique in 34 patients (41.98%). Utilizing off-clamp RPN resulted in an average blood loss of 96.29 ml and 1.56 days (range: 1-3 days) of hospital stay and minimal change in serum creatinine. Conclusions: Off-clamp RPN is safe and feasible approach to excise certain kidney tumors. It carries the benefits of RPN and prevents ischemia reperfusion renal injury. PMID:25835489

  4. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for...

  5. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for males. (a) Identification. A urological...

  6. The Dynamical Mechanisms of the Cell Cycle Size Checkpoint

    NASA Astrophysics Data System (ADS)

    Feng, Shi-Fu; Yan, Jie; Liu, Zeng-Rong; Yang, Ling

    2012-10-01

    Cell division must be tightly coupled to cell growth in order to maintain cell size, whereas the mechanisms of how initialization of mitosis is regulated by cell size remain to be elucidated. We develop a mathematical model of the cell cycle, which incorporates cell growth to investigate the dynamical properties of the size checkpoint in embryos of Xenopus laevis. We show that the size checkpoint is naturally raised from a saddle-node bifurcation, and in a mutant case, the cell loses its size control ability due to the loss of this saddle-node point.

  7. Circumferential hoof clamp method of lameness induction in the horse.

    PubMed

    Swaab, M E; Mendez-Angulo, J L; Groschen, D M; Ernst, N S; Brown, M P; Trumble, T N

    2015-07-01

    A circumferential hoof clamp method to induce controlled and reversible lameness in the forelimbs of eight horses was assessed. Peak vertical forces and vertical impulses were recorded using a force plate to verify induced lameness. Video recordings were used by blinded observers to determine subjective lameness using a 0-5 scale and any residual lameness following clamp loosening. Tightening of clamps resulted in consistent, visible lameness in the selected limbs in all horses. Lameness was confirmed by significant decreases from baseline in the peak vertical force (P <0.01). Lameness was also confirmed subjectively by elevated median scores (0 at baseline and 2 during lameness). Lameness was not immediately reversible after clamp loosening (median score 1.5), but horses were not obviously lame after clamp removal and were no different from initial baseline (median score 0.5) approximately 3 days later. PMID:26045357

  8. Cell-Detection Technique for Automated Patch Clamping

    NASA Technical Reports Server (NTRS)

    McDowell, Mark; Gray, Elizabeth

    2008-01-01

    A unique and customizable machinevision and image-data-processing technique has been developed for use in automated identification of cells that are optimal for patch clamping. [Patch clamping (in which patch electrodes are pressed against cell membranes) is an electrophysiological technique widely applied for the study of ion channels, and of membrane proteins that regulate the flow of ions across the membranes. Patch clamping is used in many biological research fields such as neurobiology, pharmacology, and molecular biology.] While there exist several hardware techniques for automated patch clamping of cells, very few of those techniques incorporate machine vision for locating cells that are ideal subjects for patch clamping. In contrast, the present technique is embodied in a machine-vision algorithm that, in practical application, enables the user to identify good and bad cells for patch clamping in an image captured by a charge-coupled-device (CCD) camera attached to a microscope, within a processing time of one second. Hence, the present technique can save time, thereby increasing efficiency and reducing cost. The present technique involves the utilization of cell-feature metrics to accurately make decisions on the degree to which individual cells are "good" or "bad" candidates for patch clamping. These metrics include position coordinates (x,y) in the image plane, major-axis length, minor-axis length, area, elongation, roundness, smoothness, angle of orientation, and degree of inclusion in the field of view. The present technique does not require any special hardware beyond commercially available, off-the-shelf patch-clamping hardware: A standard patchclamping microscope system with an attached CCD camera, a personal computer with an imagedata- processing board, and some experience in utilizing imagedata- processing software are all that are needed. A cell image is first captured by the microscope CCD camera and image-data-processing board, then the image

  9. [Recent Development of Therapies for Melanoma Using Immune Checkpoint Blockades].

    PubMed

    Okuyama, Ryuhei

    2016-06-01

    Melanoma is a highly immune tumor, and tumor-specific T lymphocytes are occasionally induced. Recent progress in tumor immunology has made it possible to clinically develop new medicines targeting immune checkpoint molecules, such as cytotoxic T lymphocyte antigen 4(CTLA-4), programmed cell death 1(PD-1), and programmed cell death 1 ligand 1(PD-L1). CTLA-4 is expressed on naïve T cells and regulatory T cells. Ipilimumab, an anti-CTLA-4 antibody, shows a distinct durable clinical benefit by inhibiting the immunosuppressive function of CTLA-4. PD-1, which is expressed on activated T cells, inhibits T cell responses against tumor cells. The antibodies against PD-1, nivolumab and pembrolizumab, produce anti-tumor responses in melanoma and other cancers due to T cell reactivation. Furthermore, clinical trials of combination therapies using immune checkpoint blockades with molecularly targeted therapies and other chemotherapeutic agents are being conducted. However, immune checkpoint blockades frequently cause immune-related adverse events. Targeted therapies to immune checkpoint molecules are expected to be promising strategies for treatment of melanoma and other cancers. PMID:27306802

  10. Control of Swe1p degradation by the morphogenesis checkpoint.

    PubMed Central

    Sia, R A; Bardes, E S; Lew, D J

    1998-01-01

    In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2. PMID:9822611

  11. Immune-Checkpoint Blockade and Active Immunotherapy for Glioma

    PubMed Central

    Ahn, Brian J.; Pollack, Ian F.; Okada, Hideho

    2013-01-01

    Cancer immunotherapy has made tremendous progress, including promising results in patients with malignant gliomas. Nonetheless, the immunological microenvironment of the brain and tumors arising therein is still believed to be suboptimal for sufficient antitumor immune responses for a variety of reasons, including the operation of “immune-checkpoint” mechanisms. While these mechanisms prevent autoimmunity in physiological conditions, malignant tumors, including brain tumors, actively employ these mechanisms to evade from immunological attacks. Development of agents designed to unblock these checkpoint steps is currently one of the most active areas of cancer research. In this review, we summarize recent progresses in the field of brain tumor immunology with particular foci in the area of immune-checkpoint mechanisms and development of active immunotherapy strategies. In the last decade, a number of specific monoclonal antibodies designed to block immune-checkpoint mechanisms have been developed and show efficacy in other cancers, such as melanoma. On the other hand, active immunotherapy approaches, such as vaccines, have shown encouraging outcomes. We believe that development of effective immunotherapy approaches should ultimately integrate those checkpoint-blockade agents to enhance the efficacy of therapeutic approaches. With these agents available, it is going to be quite an exciting time in the field. The eventual success of immunotherapies for brain tumors will be dependent upon not only an in-depth understanding of immunology behind the brain and brain tumors, but also collaboration and teamwork for the development of novel trials that address multiple layers of immunological challenges in gliomas. PMID:24202450

  12. Development of cell-cycle checkpoint therapy for solid tumors.

    PubMed

    Tamura, Kenji

    2015-12-01

    Cellular proliferation is tightly controlled by several cell-cycle checkpoint proteins. In cancer, the genes encoding these proteins are often disrupted and cause unrestrained cancer growth. The proteins are over-expressed in many malignancies; thus, they are potential targets for anti-cancer therapies. These proteins include cyclin-dependent kinase, checkpoint kinase, WEE1 kinase, aurora kinase and polo-like kinase. Cyclin-dependent kinase inhibitors are the most advanced cell-cycle checkpoint therapeutics available. For instance, palbociclib (PD0332991) is a first-in-class, oral, highly selective inhibitor of CDK4/6 and, in combination with letrozole (Phase II; PALOMA-1) or with fulvestrant (Phase III; PALOMA-3), it has significantly prolonged progression-free survival, in patients with metastatic estrogen receptor-positive, HER2-negative breast cancer, in comparison with that observed in patients using letrozole, or fulvestrant alone, respectively. In this review, we provide an overview of the current compounds available for cell-cycle checkpoint protein-directed therapy for solid tumors. PMID:26486823

  13. Immune Checkpoint Inhibitors in Brain Metastases: From Biology to Treatment.

    PubMed

    Berghoff, Anna S; Venur, Vyshak A; Preusser, Matthias; Ahluwalia, Manmeet S

    2016-01-01

    Cancer immunotherapy has been a subject of intense research over the last several years, leading to new approaches for modulation of the immune system to treat malignancies. Immune checkpoint inhibitors (anti-CLTA-4 antibodies and anti-PD-1/PD-L1 antibodies) potentiate the host's own antitumor immune response. These immune checkpoint inhibitors have shown impressive clinical efficacy in advanced melanoma, metastatic kidney cancer, and metastatic non-small cell lung cancer (NSCLC)-all malignancies that frequently cause brain metastases. The immune response in the brain is highly regulated, challenging the treatment of brain metastases with immune-modulatory therapies. The immune microenvironment in brain metastases is active with a high density of tumor-infiltrating lymphocytes in certain patients and, therefore, may serve as a potential treatment target. However, clinical data of the efficacy of immune checkpoint inhibitors in brain metastases compared with extracranial metastases are limited, as most clinical trials with these new agents excluded patients with active brain metastases. In this article, we review the current scientific evidence of brain metastases biology with specific emphasis on inflammatory tumor microenvironment and the evolving state of clinical application of immune checkpoint inhibitors for patients with brain metastases. PMID:27249713

  14. SHP-1: the next checkpoint target for cancer immunotherapy?

    PubMed

    Watson, H Angharad; Wehenkel, Sophie; Matthews, James; Ager, Ann

    2016-04-15

    The immense power of the immune system is harnessed in healthy individuals by a range of negative regulatory signals and checkpoints. Manipulating these checkpoints through inhibition has resulted in striking immune-mediated clearance of otherwise untreatable tumours and metastases; unfortunately, not all patients respond to treatment with the currently available inhibitors of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Combinatorial studies using both anti-CTLA-4 and anti-PD-1 demonstrate synergistic effects of targeting multiple checkpoints, paving the way for other immune checkpoints to be targeted. Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T-cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein, and therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T-cells. This review will discuss the potential value of SHP-1 inhibition in future tumour immunotherapy. PMID:27068940

  15. Harnessing the Power of Onco-Immunotherapy with Checkpoint Inhibitors

    PubMed Central

    Rajani, Karishma R.; Vile, Richard G.

    2015-01-01

    Oncolytic viruses represent a diverse class of replication competent viruses that curtail tumor growth. These viruses, through their natural ability or through genetic modifications, can selectively replicate within tumor cells and induce cell death while leaving normal cells intact. Apart from the direct oncolytic activity, these viruses mediate tumor cell death via the induction of innate and adaptive immune responses. The field of oncolytic viruses has seen substantial advancement with the progression of numerous oncolytic viruses in various phases of clinical trials. Tumors employ a plethora of mechanisms to establish growth and subsequently metastasize. These include evasion of immune surveillance by inducing up-regulation of checkpoint proteins which function to abrogate T cell effector functions. Currently, antibodies blocking checkpoint proteins such as anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) and anti-programmed cell death-1 (PD-1) have been approved to treat cancer and shown to impart durable clinical responses. These antibodies typically need pre-existing active immune tumor microenvironment to establish durable clinical outcomes and not every patient responds to these therapies. This review provides an overview of published pre-clinical studies demonstrating superior therapeutic efficacy of combining oncolytic viruses with checkpoint blockade compared to monotherapies. These studies provide compelling evidence that oncolytic therapy can be potentiated by coupling it with checkpoint therapies. PMID:26580645

  16. DNA damage checkpoint, damage repair, and genome stability.

    PubMed

    Liu, Wei-Feng; Yu, Shan-Shan; Chen, Guan-Jun; Li, Yue-Zhong

    2006-05-01

    Genomic DNA is under constant attack from both endogenous and exogenous sources of DNA damaging agents. Without proper care, the ensuing DNA damages would lead to alteration of genomic structure thus affecting the faithful transmission of genetic information. During the process of evolution, organisms have acquired a series of mechanisms responding to and repairing DNA damage, thus assuring the maintenance of genome stability and faithful transmission of genetic information. DNA damage checkpoint is one such important mechanism by which, in the face of DNA damage, a cell can respond to amplified damage signals, either by actively halting the cell cycle until it ensures that critical processes such as DNA replication or mitosis are complete or by initiating apoptosis as a last resort. Over the last decade, complex hierarchical interactions between the key components like ATM/ATR in the checkpoint pathway and various other mediators, effectors including DNA damage repair proteins have begun to emerge. In the meantime, an intimate relationship between mechanisms of damage checkpoint pathway, DNA damage repair, and genome stability was also uncovered. Reviewed herein are the recent findings on both the mechanisms of activation of checkpoint pathways and their coordination with DNA damage repair machinery as well as their effect on genomic integrity. PMID:16722332

  17. Dynamic clamp: a powerful tool in cardiac electrophysiology.

    PubMed

    Wilders, Ronald

    2006-10-15

    Dynamic clamp is a collection of closely related techniques that have been employed in cardiac electrophysiology to provide direct answers to numerous research questions regarding basic cellular mechanisms of action potential formation, action potential transfer and action potential synchronization in health and disease. Building on traditional current clamp, dynamic clamp was initially used to create virtual gap junctions between isolated myocytes. More recent applications include the embedding of a real pacemaking myocyte in a simulated network of atrial or ventricular cells and the insertion of virtual ion channels, either simulated in real time or simultaneously recorded from an expression system, into the membrane of an isolated myocyte. These applications have proven that dynamic clamp, which is characterized by the real-time evaluation and injection of simulated membrane current, is a powerful tool in cardiac electrophysiology. Here, each of the three different experimental configurations used in cardiac electrophysiology is reviewed. Also, directions are given for the implementation of dynamic clamp in the cardiac electrophysiology laboratory. With the growing interest in the application of dynamic clamp in cardiac electrophysiology, it is anticipated that dynamic clamp will also prove to be a powerful tool in basic research on biological pacemakers and in identification of specific ion channels as targets for drug development. PMID:16873403

  18. Checkpointing for graceful degradation in distributed embedded systems

    NASA Astrophysics Data System (ADS)

    Sababha, Belal Hussein

    Graceful degradation is an approach to developing dependable safety-critical embedded applications, where redundant active or standby resources are used to cope with faults through a system reconfiguration at run-time. Compared to traditional hardware and software redundancy, it is a promising technique that may achieve dependability with a significant reduction in cost, size, weight, and power requirements. Reconfiguration at run-time necessitates using proper checkpointing protocols to support state reservation to ensure correct task restarts after a system reconfiguration. One of the most common checkpointing protocols are communication induced checkpointing (CIC) protocols, which are well developed and understood for large parallel and information systems, but not much has been done for resource limited embedded systems. This work implements and evaluates some of the most common CIC protocols in a periodic resource constrained distributed embedded system for graceful degradation purposes. A test-bed has been developed and used for the evaluation of the various protocols. The implemented protocols are thoroughly studied and performances are contrasted. Specifically the periodicity property and how it benefits checkpointing in embedded systems is investigated. This work introduces a unique effort of CIC protocol implementation and evaluation in the field of distributed embedded systems. Other than providing a test-bed for graceful degradation support, this work shows that some checkpointing protocols that are not efficient in large information systems and supercomputers perform well in embedded systems. We show that a simple index-based CIC protocol, such as the BCS protocol, is more appropriate in embedded system applications compared to other protocols that piggyback a significant amount of information to reduce the number of forced checkpoints. Finally, this work proposes a whole graceful degradation approach to achieve fault tolerance in resource constrained

  19. Fueling the engine and releasing the break: combinational therapy of cancer vaccines and immune checkpoint inhibitors

    PubMed Central

    Kleponis, Jennifer; Skelton, Richard; Zheng, Lei

    2015-01-01

    Immune checkpoint inhibitors are increasingly drawing much attention in the therapeutic development for cancer treatment. However, many cancer patients do not respond to treatments with immune checkpoint inhibitors, partly because of the lack of tumor-infiltrating effector T cells. Cancer vaccines may prime patients for treatments with immune checkpoint inhibitors by inducing effector T-cell infiltration into the tumors and immune checkpoint signals. The combination of cancer vaccine and an immune checkpoint inhibitor may function synergistically to induce more effective antitumor immune responses, and clinical trials to test the combination are currently ongoing. PMID:26487965

  20. Cell Size Checkpoint Control by the Retinoblastoma Tumor Suppressor Pathway

    PubMed Central

    Fang, Su-Chiung; de los Reyes, Chris; Umen, James G

    2006-01-01

    Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription. PMID:17040130

  1. Patch voltage clamp of squid axon membrane.

    PubMed

    Fishman, H M

    1975-12-01

    A small area (patch) of the external surface of a squid axon can be "isolated" electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude greater than 90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the exolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 komega (7 omega cm2). Patch capacitance ranges from 10-100 pF, which suggests areas of 10(-4) to 10(-5) cm2 and resting patch resistances of 10-100 Momega. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1-2 Momega. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an exoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise. PMID:1214276

  2. Insulin Tolerance Test and Hyperinsulinemic-Euglycemic Clamp

    PubMed Central

    Paschos, Georgios K.; FitzGerald, Garret A.

    2016-01-01

    The two tests are used to evaluate in vivo sensitivity to insulin in mouse. The hypoerinsulinemic-euglycemic clamp provides information about the sensitivity to insulin in liver and other metabolically relevant tissues.

  3. A clamp-like biohybrid catalyst for DNA oxidation

    NASA Astrophysics Data System (ADS)

    van Dongen, Stijn F. M.; Clerx, Joost; Nørgaard, Kasper; Bloemberg, Tom G.; Cornelissen, Jeroen J. L. M.; Trakselis, Michael A.; Nelson, Scott W.; Benkovic, Stephen J.; Rowan, Alan E.; Nolte, Roeland J. M.

    2013-11-01

    In processive catalysis, a catalyst binds to a substrate and remains bound as it performs several consecutive reactions, as exemplified by DNA polymerases. Processivity is essential in nature and is often mediated by a clamp-like structure that physically tethers the catalyst to its (polymeric) template. In the case of the bacteriophage T4 replisome, a dedicated clamp protein acts as a processivity mediator by encircling DNA and subsequently recruiting its polymerase. Here we use this DNA-binding protein to construct a biohybrid catalyst. Conjugation of the clamp protein to a chemical catalyst with sequence-specific oxidation behaviour formed a catalytic clamp that can be loaded onto a DNA plasmid. The catalytic activity of the biohybrid catalyst was visualized using a procedure based on an atomic force microscopy method that detects and spatially locates oxidized sites in DNA. Varying the experimental conditions enabled switching between processive and distributive catalysis and influencing the sliding direction of this rotaxane-like catalyst.

  4. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions

    PubMed Central

    Ólafsson, Guðjón; Thorpe, Peter H.

    2016-01-01

    The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2. PMID:27280788

  5. Injury response checkpoint and developmental timing in insects

    PubMed Central

    Hackney, Jennifer F; Cherbas, Peter

    2014-01-01

    In insects, localized tissue injury often leads to global (organism-wide) delays in development and retarded metamorphosis. In Drosophila, for example, injuries to the larval imaginal discs can retard pupariation and prolong metamorphosis. Injuries induced by treatments such as radiation, mechanical damage and induction of localized cell death can trigger similar delays. In most cases, the duration of the developmental delay appears to be correlated with the extent of damage, but the effect is also sensitive to the developmental stage of the treated animal. The proximate cause of the delays is likely a disruption of the ecdysone signaling pathway, but the intermediate steps leading from tissue injury and/or regeneration to that disruption remain unknown. Here, we review the evidence for injury-induced developmental delays, and for a checkpoint or checkpoints associated with the temporal progression of development and the on-going efforts to define the mechanisms involved. PMID:25833067

  6. Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling

    PubMed Central

    Kemmler, Stefan; Stach, Manuel; Knapp, Maria; Ortiz, Jennifer; Pfannstiel, Jens; Ruppert, Thomas; Lechner, Johannes

    2009-01-01

    The protein kinase Mps1 is, among others, essential for the spindle assembly checkpoint (SAC). We found that Saccharomyces cerevisiae Mps1 interacts physically with the N-terminal domain of Ndc80 (Ndc801−257), a constituent of the Ndc80 kinetochore complex. Furthermore, Mps1 effectively phosphorylates Ndc801−257 in vitro and facilitates Ndc80 phosphorylation in vivo. Mutating 14 of the phosphorylation sites to alanine results in compromised checkpoint signalling upon nocodazole treatment of mutants. Mutating the identical sites to aspartate (to simulate constitutive phosphorylation) causes a metaphase arrest with wild-type-like bipolar kinetochore–microtubule attachment. This arrest is due to a constitutively active SAC and consequently the inviable aspartate mutant can be rescued by disrupting SAC signalling. Therefore, we conclude that a putative Mps1-dependent phosphorylation of Ndc80 is important for SAC activation at kinetochores. PMID:19300438

  7. Immune Checkpoint Therapy and the Search for Predictive Biomarkers.

    PubMed

    Sharma, Padmanee

    2016-01-01

    Immune checkpoint therapy has started a revolution in the field of oncology. The concept that the immune system plays a critical role in antitumor responses, which has been around for decades, has finally been proven and firmly established with elegant preclinical studies and dramatic clinical responses in patients as a result of antibodies that block inhibitory T-cell pathways. However, the clinical responses being achieved are only in a subset of patients, and more work is needed to provide a better understanding of the mechanisms that elicit tumor rejection, which will enable identification of appropriate biomarkers, reveal new targets, provide data to guide combination studies, and eventually dictate a platform that allows more patients to derive clinical benefit, including cures, with immune checkpoint therapy. PMID:27111900

  8. A review of adverse events caused by immune checkpoint inhibitors.

    PubMed

    Fukushima, Satoshi

    2016-01-01

      There has been no effective therapy in the unresectable melanoma for more than 40 years. Anti-PD-1 antibody and anti-CTLA-4 antibody have totally changed the situation. They have clearly shown the survival benefits of the patients with metastatic melanoma. However, immune checkpoint inhibitors sometimes induce various kinds of immune-related adverse events (irAEs). It is very important for the clinicians to know the reported cases of irAEs and to keep in mind the symptoms of irAEs for the early detection. This review describes the previously reported irAEs and adequate managements for irAEs induced by immune checkpoint inhibitors. PMID:27181232

  9. Immune-Related Adverse Events From Immune Checkpoint Inhibitors.

    PubMed

    Marrone, K A; Ying, W; Naidoo, J

    2016-09-01

    Immunotherapy for cancer treatment has come of age, specifically with the use of immune checkpoint antibodies directed against molecules such as CTLA-4, PD-1, and PD-L1. Single-agent and combinatorial approaches utilizing these agents and other immunotherapies that may enhance antitumor effects are under investigation. With increasing clinical use of these agents, an appreciation for their toxicities comes to the fore. Adverse events that occur as a result of the immunologic effects of these therapies are termed "immune-related adverse events" (irAEs), and range in both frequency and severity in reported single-agent and combination studies. Improvements in our understanding of how and why irAEs develop and how to effectively manage them are needed. Herein we provide a state-of-the-art synopsis of the incidence, clinical features, mechanisms, and management of selected irAEs with immune checkpoint inhibitors currently in use. PMID:27170616

  10. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions.

    PubMed

    Ólafsson, Guðjón; Thorpe, Peter H

    2016-01-01

    The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2. PMID:27280788

  11. Epigenetic modifiers in immunotherapy: a focus on checkpoint inhibitors.

    PubMed

    Terranova-Barberio, Manuela; Thomas, Scott; Munster, Pamela N

    2016-06-01

    Immune surveillance should be directed to suppress tumor development and progression, involving a balance of coinhibitory and costimulatory signals that amplify immune response without overwhelming the host. Immunotherapy confers durable clinical benefit in 'immunogenic tumors', whereas in other tumors the responses are modest. Thus, immune checkpoint inhibitors may need to be combined with strategies to boost immune response or increase the tumor immune profile. Epigenetic aberrations contribute significantly to carcinogenesis. Recent findings suggest that epigenetic drugs prime the immune response by increasing expression of tumor-associated antigens and immune-related genes, as well as modulating chemokines and cytokines involved in immune system activation. This review describes our current understanding regarding epigenetic and immunotherapy combination, focusing on immune response priming to checkpoint blockade. PMID:27197539

  12. Checkpointing Shared Memory Programs at the Application-level

    SciTech Connect

    Bronevetsky, G; Schulz, M; Szwed, P; Marques, D; Pingali, K

    2004-09-08

    Trends in high-performance computing are making it necessary for long-running applications to tolerate hardware faults. The most commonly used approach is checkpoint and restart(CPR)-the state of the computation is saved periodically on disk, and when a failure occurs, the computation is restarted from the last saved state. At present, it is the responsibility of the programmer to instrument applications for CPR. Our group is investigating the use of compiler technology to instrument codes to make them self-checkpointing and self-restarting, thereby providing an automatic solution to the problem of making long-running scientific applications resilient to hardware faults. Our previous work focused on message-passing programs. In this paper, we describe such a system for shared-memory programs running on symmetric multiprocessors. The system has two components: (i)a pre-compiler for source-to-source modification of applications, and (ii) a runtime system that implements a protocol for coordinating CPR among the threads of the parallel application. For the sake of concreteness, we focus on a non-trivial subset of OpenMP that includes barriers and locks. One of the advantages of this approach is that the ability to tolerate faults becomes embedded within the application itself, so applications become self-checkpointing and self-restarting on any platform. We demonstrate this by showing that our transformed benchmarks can checkpoint and restart on three different platforms (Windows/x86, Linux/x86, and Tru64/Alpha). Our experiments show that the overhead introduced by this approach is usually quite small; they also suggest ways in which the current implementation can be tuned to reduced overheads further.

  13. Fanconi anemia proteins and the s phase checkpoint.

    PubMed

    Pichierri, Pietro; Rosselli, Filippo

    2004-06-01

    DNA interstrand crosslinks (ICLs) repair represents a formidable task for mammalian cells. Indeed, such DNA lesions, bridging both opposite DNA helices, function as a road-block for every DNA transaction, in particular DNA replication. The eight Fanconi anemia (FA) proteins interact in a common pathway that is thought to be central in ICLs sensing/repair. Interestingly, FA cells, either mutated in one of the proteins composing the FA core complex or in the downstream FA protein FANCD2, exhibited a partial intra-S checkpoint defect in response to crosslinked DNA. Most importantly, the FA proteins work in the ATR-NBS1 branch of the ICL-induced checkpoint pathway as demonstrated by knocking-down CHK1 or MRE11 expression in a FA background. Even though our data disclose a clear functional role for the FA proteins in the intra-S checkpoint response it does not give a definite answer on what FA proteins do in this process and how they participate in the suppression/restart of DNA synthesis. It seems conceivable that FA proteins participate in the process involved in the recovery of stalled replication forks, a common event in proliferating cells, possibly ensuring correct replication fork repair by homologous recombination. PMID:15136767

  14. A Monitor for Bud Emergence in the Yeast Morphogenesis Checkpoint

    PubMed Central

    Theesfeld, Chandra L.; Zyla, Trevin R.; Bardes, Elaine G.S.; Lew, Daniel J.

    2003-01-01

    Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation. PMID:12925763

  15. Berkeley Lab Checkpoint/Restart (BLCR) for Linux Clusters

    SciTech Connect

    Hargrove, Paul H.; Duell, Jason C.

    2006-07-26

    This article describes the motivation, design andimplementation of Berkeley Lab Checkpoint/Restart (BLCR), a system-levelcheckpoint/restart implementation for Linux clusters that targets thespace of typical High Performance Computing applications, including MPI.Application-level solutions, including both checkpointing andfault-tolerant algorithms, are recognized as more time and spaceefficient than system-level checkpoints, which cannot make use of anyapplication-specific knowledge. However, system-level checkpointingallows for preemption, making it suitable for responding to "faultprecursors" (for instance, elevated error rates from ECC memory ornetwork CRCs, or elevated temperature from sensors). Preemption can alsoincrease the efficiency of batch scheduling; for instance reducing idlecycles (by allowing for shutdown without any queue draining period orreallocation of resources to eliminate idle nodes when better fittingjobs are queued), and reducing the average queued time (by limiting largejobs to running during off-peak hours, without the need to limit thelength of such jobs). Each of these potential uses makes BLCR a valuabletool for efficient resource management in Linux clusters.

  16. Targeting lung cancer through inhibition of checkpoint kinases

    PubMed Central

    Syljuåsen, Randi G.; Hasvold, Grete; Hauge, Sissel; Helland, Åslaug

    2015-01-01

    Inhibitors of checkpoint kinases ATR, Chk1, and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed. We discuss how checkpoint kinase inhibition in principle can lead to selective killing of lung cancer cells while sparing the surrounding normal tissues. Several features of lung cancer may potentially be exploited for targeting through inhibition of checkpoint kinases, including mutated p53, low ERCC1 levels, amplified Myc, tumor hypoxia and presence of lung cancer stem cells. Synergistic effects have also been reported between inhibitors of ATR/Chk1/Wee1 and conventional lung cancer treatments, such as gemcitabine, cisplatin, or radiation. Altogether, inhibitors of ATR, Chk1, and Wee1 are emerging as new cancer treatment agents, likely to be useful in lung cancer treatment. However, as lung tumors are very diverse, the inhibitors are unlikely to be effective in all patients, and more work is needed to determine how such inhibitors can be utilized in the most optimal ways. PMID:25774168

  17. Complex Commingling: Nucleoporins and the Spindle Assembly Checkpoint

    PubMed Central

    Mossaid, Ikram; Fahrenkrog, Birthe

    2015-01-01

    The segregation of the chromosomes during mitosis is an important process, in which the replicated DNA content is properly allocated into two daughter cells. To ensure their genomic integrity, cells present an essential surveillance mechanism known as the spindle assembly checkpoint (SAC), which monitors the bipolar attachment of the mitotic spindle to chromosomes to prevent errors that would result in chromosome mis-segregation and aneuploidy. Multiple components of the nuclear pore complex (NPC), a gigantic protein complex that forms a channel through the nuclear envelope to allow nucleocytoplasmic exchange of macromolecules, were shown to be critical for faithful cell division and implicated in the regulation of different steps of the mitotic process, including kinetochore and spindle assembly as well as the SAC. In this review, we will describe current knowledge about the interconnection between the NPC and the SAC in an evolutional perspective, which primarily relies on the two mitotic checkpoint regulators, Mad1 and Mad2. We will further discuss the role of NPC constituents, the nucleoporins, in kinetochore and spindle assembly and the formation of the mitotic checkpoint complex during mitosis and interphase. PMID:26540075

  18. Spectral infrared hemispherical reflectance measurements for LDEF tray clamps

    NASA Technical Reports Server (NTRS)

    Wood, Bobby E.; Cromwell, Brian K.; Pender, Charles W.; Shepherd, Seth D.

    1992-01-01

    This paper describes infrared hemispherical reflectance measurements (2-15 microns) that were made on 58 chromic acid anodized tray clamps retrieved from the LDEF spacecraft. These clamps were used for maintaining the experiments in place and were located at various locations about the spacecraft. Changes in reflectance of the tray clamps at these locations were compared with atomic oxygen fluxes at the same locations. A decrease in absorption band depth was seen for the surfaces exposed to space indicating that there was some surface layer erosion. In all of the surfaces measured, little evidence of contamination was observed and none of the samples showed evidence of the brown nicotine stain that was so prominent in other experiments. Total emissivity values were calculated for both exposed and unexposed tray clamp surfaces. Only small differences, usually less than 1 percent, were observed. The spectral reflectances were measured using a hemi-ellipsoidal mirror reflectometer matched with an interferometer spectrometer. The rapid scanning capability of the interferometer allowed the reflectance measurements to be made in a timely fashion. The ellipsoidal mirror has its two foci separated by 2 inches and located on the major axis. A blackbody source was located at one focus while the tray clamp samples were located at the conjugate focus. The blackbody radiation was modulated and then focused by the ellipsoid onto the tray clamps. Radiation reflected from the tray clamp was sampled by the interferometer by viewing through a hole in the ellipsoid. A gold mirror (reflectance approximately 98 percent) was used as the reference surface.

  19. Measuring beta-cell function relative to insulin sensitivity in youth: Does the hyperglycemic clamp suffice?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To compare beta-cell function relative to insulin sensitivity, disposition index (DI), calculated from two clamps (2cDI, insulin sensitivity from the hyperinsulinemic-euglycemic clamp and first-phase insulin from the hyperglycemic clamp) with the DI calculated from the hyperglycemic clamp alone (hcD...

  20. Dynamic Clamp in Cardiac and Neuronal Systems Using RTXI

    PubMed Central

    Ortega, Francis A.; Butera, Robert J.; Christini, David J.; White, John A.; Dorval, Alan D.

    2016-01-01

    The injection of computer-simulated conductances through the dynamic clamp technique has allowed researchers to probe the intercellular and intracellular dynamics of cardiac and neuronal systems with great precision. By coupling computational models to biological systems, dynamic clamp has become a proven tool in electrophysiology with many applications, such as generating hybrid networks in neurons or simulating channelopathies in cardiomyocytes. While its applications are broad, the approach is straightforward: synthesizing traditional patch clamp, computational modeling, and closed-loop feedback control to simulate a cellular conductance. Here, we present two example applications: artificial blocking of the inward rectifier potassium current in a cardiomyocyte and coupling of a biological neuron to a virtual neuron through a virtual synapse. The design and implementation of the necessary software to administer these dynamic clamp experiments can be difficult. In this chapter, we provide an overview of designing and implementing a dynamic clamp experiment using the Real-Time eXperiment Interface (RTXI), an open- source software system tailored for real-time biological experiments. We present two ways to achieve this using RTXI’s modular format, through the creation of a custom user-made module and through existing modules found in RTXI’s online library. PMID:25023319

  1. Emodnet Med Sea Check-Point - Indicators for decision- maker

    NASA Astrophysics Data System (ADS)

    Besnard, Sophie; Claverie, Vincent; Blanc, Frédérique

    2015-04-01

    The Emodnet Checkpoint projects aim is to assess the cost-effectiveness, reliability and utility of the existing monitoring at the sea basin level. This involves the development of monitoring system indicators and a GIS Platform to perform the assessment and make it available. Assessment or production of Check-Point information is made by developing targeted products based on the monitoring data and determining whether the products are meeting the needs of industry and public authorities. Check-point users are the research community, the 'institutional' policy makers for IMP and MSFD implementation, the 'intermediate users', i.e., users capable to understand basic raw data but that benefit from seeing the Checkpoint targeted products and the assessment of the fitness for purpose. We define assessment criteria aimed to characterize/depict the input datasets in terms of 3 territories capable to show performance and gaps of the present monitoring system, appropriateness, availability and fitness for purpose. • Appropriateness: What is made available to users? What motivate/decide them to select this observation rather than this one. • Availability: How this is made available to the user? Place to understand the readiness and service performance of the EU infrastructure • Fitness for use / fitness for purpose: Ability for non-expert user to appreciate the data exploitability (feedback on efficiency & reliability of marine data) For each territory (appropriateness, Availability and Fitness for purpose / for use), we define several indicators. For example, for Availability we define Visibility, Accessibility and Performance. And Visibility is itself defined by "Easily found" and "EU service". So these indicators can be classified according to their territory and sub-territory as seen above, but also according to the complexity to build them. Indicators are built from raw descriptors in 3 stages:  Stage 1: to give a neutral and basic status directly computed from

  2. Self-locking clamping tool with swivel jaws

    NASA Technical Reports Server (NTRS)

    Redmon, Jr., John W. (Inventor); Jankowski, Fred (Inventor)

    1989-01-01

    A plier-like tool (11) having two plier-like members (13, 15) pivotally joined togther intermediate of their ends and having handle portions (17, 18) and swivel jaw members (29,30). An automatic locking mechanism (27) extending between the members permits an user to clamp the handle portions together so as to clamp the jaw members on an object (25) but holds the position so reached if the clamping action of the user is removed. A release device (65) is provided so that the jaw members may be opened up again. A compression spring (23) extending between the members (19, 20) assists in the opening of the jaw members. The swivel jaw members (29, 30) permit the user to rotate the plier-like members (13,15) relative to the object (25) being grasped.

  3. Hysteresis modeling of clamp band joint with macro-slip

    NASA Astrophysics Data System (ADS)

    Qin, Zhaoye; Cui, Delin; Yan, Shaoze; Chu, Fulei

    2016-01-01

    Clamp band joints are commonly used to connect spacecrafts with launch vehicles. Due to the frictional slippage between the joint components, hysteresis behavior might occur at joint interfaces under cyclic loading. The joint hysteresis will bring friction damping into the launching systems. In this paper, a closed-form hysteresis model for the clamp band joint is developed based on theoretical and numerical analyses of the interactions of the joint components. Then, the hysteresis model is applied to investigating the dynamic response of a payload fastened by the clamp band joint, where the nonlinearity and friction damping effects of the joint is evaluated. The proposed analytical model, which is validated by both finite element analyses and quasi-static experiments, has a simple form with sound accuracy and can be incorporated into the dynamic models of launching systems conveniently.

  4. Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells

    PubMed Central

    Gassmann, Reto; Holland, Andrew J.; Varma, Dileep; Wan, Xiaohu; Çivril, Filiz; Cleveland, Don W.; Oegema, Karen; Salmon, Edward D.; Desai, Arshad

    2010-01-01

    The spindle checkpoint generates a “wait anaphase” signal at unattached kinetochores to prevent premature anaphase onset. Kinetochore-localized dynein is thought to silence the checkpoint by transporting checkpoint proteins from microtubule-attached kinetochores to spindle poles. Throughout metazoans, dynein recruitment to kinetochores requires the protein Spindly. Here, we identify a conserved motif in Spindly that is essential for kinetochore targeting of dynein. Spindly motif mutants, expressed following depletion of endogenous Spindly, target normally to kinetochores but prevent dynein recruitment. Spindly depletion and Spindly motif mutants, despite their similar effects on kinetochore dynein, have opposite consequences on chromosome alignment and checkpoint silencing. Spindly depletion delays chromosome alignment, but Spindly motif mutants ameliorate this defect, indicating that Spindly has a dynein recruitment-independent role in alignment. In Spindly depletions, the checkpoint is silenced following delayed alignment by a kinetochore dynein-independent mechanism. In contrast, Spindly motif mutants are retained on microtubule-attached kinetochores along with checkpoint proteins, resulting in persistent checkpoint signaling. Thus, dynein-mediated removal of Spindly from microtubule-attached kinetochores, rather than poleward transport per se, is the critical reaction in checkpoint silencing. In the absence of Spindly, a second mechanism silences the checkpoint; this mechanism is likely evolutionarily ancient, as fungi and higher plants lack kinetochore dynein. PMID:20439434

  5. Defective Cell Cycle Checkpoint Functions in Melanoma Are Associated with Altered Patterns of Gene Expression

    PubMed Central

    Kaufmann, William K.; Nevis, Kathleen R.; Qu, Pingping; Ibrahim, Joseph G.; Zhou, Tong; Zhou, Yingchun; Simpson, Dennis A.; Helms-Deaton, Jennifer; Cordeiro-Stone, Marila; Moore, Dominic T.; Thomas, Nancy E.; Hao, Honglin; Liu, Zhi; Shields, Janiel M.; Scott, Glynis A.; Sharpless, Norman E.

    2009-01-01

    Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wild-type N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression. PMID:17597816

  6. Cutaneous autoimmune effects in the setting of therapeutic immune checkpoint inhibition for metastatic melanoma.

    PubMed

    Mochel, Mark C; Ming, Michael E; Imadojemu, Sotonye; Gangadhar, Tara C; Schuchter, Lynn M; Elenitsas, Rosalie; Payne, Aimee S; Chu, Emily Y

    2016-09-01

    Therapeutic immune checkpoint blockade for metastatic melanoma has been associated with vitiligo, pruritus and morbilliform eruptions. Reports of other autoimmune skin disease in this setting are rare. We sought to expand the spectrum of cutaneous immune-mediated effects related to immune checkpoint inhibitor therapy. In this report, we describe two unusual cutaneous reactions related to checkpoint inhibitor therapy, namely bullous pemphigoid (BP) and dermatitis herpetiformis. The development of BP and dermatitis herpetiformis in the context of checkpoint inhibitor therapy is consistent with previous investigations supporting the importance of effector and regulatory T cells in the pathogenesis of these diseases. PMID:27161449

  7. Action-oriented use of ergonomic checkpoints for healthy work design in different settings.

    PubMed

    Kogi, Kazutaka

    2007-12-01

    Recent experiences in the action-oriented use of ergonomic checkpoints in different work settings are reviewed. The purpose is to know what features are useful for healthy work design adjusted to each local situation. Based on the review results, common features of ergonomic checkpoints used in participatory training programs for improving workplace conditions in small enterprises, construction sites, home work and agriculture in industrially developing countries in Asia are discussed. These checkpoints generally compile practical improvement options in a broad range of technical areas, such as materials handling, workstation design, physical environment and work organization. Usually, "action checklists" comprising the tiles of the checkpoints are used together. A clear focus is placed on readily applicable low-cost options. Three common features of these various checkpoints appear to be important. First, the checkpoints represent typical good practices in multiple areas. Second, each how-to section of these checkpoints presents simple improvements reflecting basic ergonomic principles. Examples of these principles include easy reach, fewer and faster transport, elbow-level work, coded displays, isolated or screened hazards and shared teamwork. Third, the illustrated checkpoints accompanied by corresponding checklists are used as group work tools in short-term training courses. Many practical improvements achieved are displayed in websites for inter-country work improvement networks. It is suggested to promote the use of locally adjusted checkpoints in various forms of participatory action-oriented training in small-scale workplaces and in agriculture particularly in industrially developing countries. PMID:18572793

  8. McrEngine: A Scalable Checkpointing System Using Data-Aware Aggregation and Compression

    DOE PAGESBeta

    Islam, Tanzima Zerin; Mohror, Kathryn; Bagchi, Saurabh; Moody, Adam; de Supinski, Bronis R.; Eigenmann, Rudolf

    2013-01-01

    High performance computing (HPC) systems use checkpoint-restart to tolerate failures. Typically, applications store their states in checkpoints on a parallel file system (PFS). As applications scale up, checkpoint-restart incurs high overheads due to contention for PFS resources. The high overheads force large-scale applications to reduce checkpoint frequency, which means more compute time is lost in the event of failure. We alleviate this problem through a scalable checkpoint-restart system, mcrEngine. McrEngine aggregates checkpoints from multiple application processes with knowledge of the data semantics available through widely-used I/O libraries, e.g., HDF5 and netCDF, and compresses them. Our novel scheme improves compressibility ofmore » checkpoints up to 115% over simple concatenation and compression. Our evaluation with large-scale application checkpoints show that mcrEngine reduces checkpointing overhead by up to 87% and restart overhead by up to 62% over a baseline with no aggregation or compression.« less

  9. [Pedicular clamping in major hepatectomies: clamping "of principle" or "of necessity"? A comparative study].

    PubMed

    Le Treut, Y P; Christophe, M; Banti, J C; Berthet, B; Bricot, R

    1995-02-01

    Fifty-two consecutive patients undergoing major hepatic resection for liver tumor were divided into two groups according to the operative procedure. Group A consisted of 34 patients in whom vascular inflow occlusion was performed "de principle" during parenchymal division and intrahepatic approach of the portal structures; the mean duration of the portal triad clamping was 43 mn (ranged 17 to 70 mn). Group B patients (18 cases) had hilar division of the structures of that portion of the liver due to be removed, prior to parenchymal division was performed without vascular arrest, except in five "de necessitate" cases during 5 to 22 mn. Groups A and B were comparable in terms of patient age or status, of king of liver tumors and extent of resection. Mean operating duration (215 vs 263 mn), volume of intraoperative blood transfusion (557 vs 1019 ml), intensive care (2.5 vs 4.2 days) and total hospital stays (19.6 vs 30.5 days) were significantly reduced in group A. A higher but transient increase of amino-transferase level was the only biochemical consequence of liver ischemia in group A, whereas postoperative disturbance in serum bilirubin, prothrombin time, fibrinogen, and total protein were significantly greater in group B, probably because of the greater volume of blood transfusion in this group. Thus, routine vascular inflow occlusion with transhepatic approach of the portal structures may be an effective and innocuous procedure for major liver resection. PMID:7751341

  10. Plasma temperature clamping in filamentation laser induced breakdown spectroscopy

    SciTech Connect

    Harilal, Sivanandan S.; Yeak, J.; Phillips, Mark C.

    2015-10-19

    Ultrafast laser filament induced breakdown spectroscopy is a very promising method for remote material detection. We present characteristics of plasmas generated in a metal target by laser filaments in air. Our measurements show that the temperature of the ablation plasma is clamped along the filamentation channel due to intensity clamping in a filament. Nevertheless, significant changes in radiation intensity are noticeable, and this is essentially due to variation in the number density of emitting atoms. The present results also partly explains the reason for the occurrence of atomic plume during fs LIBS in air compared to long-pulse ns LIBS.

  11. Plasma temperature clamping in filamentation laser induced breakdown spectroscopy.

    PubMed

    Harilal, S S; Yeak, J; Phillips, M C

    2015-10-19

    Ultrafast laser filament induced breakdown spectroscopy is a very promising method for remote material detection. We present characteristics of plasmas generated in a metal target by laser filaments in air. Our measurements show that the temperature of the ablation plasma is clamped along the filament channel due to intensity clamping in a filament. Nevertheless, significant changes in radiation intensity are noticeable, and this is essentially due to variation in the number density of emitting atoms. The present results also explain the near absence of ion emission but strong atomic neutral emission from plumes produced during fs LIBS in air. PMID:26480372

  12. p31comet promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

    PubMed Central

    Teichner, Adar; Eytan, Esther; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Dumin, Elena; Gromis, Jonathan; Hershko, Avram

    2011-01-01

    Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31comet in this process. It is known that p31comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC. PMID:21300909

  13. Learning from the "tsunami" of immune checkpoint inhibitors in 2015.

    PubMed

    Kourie, Hampig Raphael; Awada, Gil; Awada, Ahmad Hussein

    2016-05-01

    2015 was marked by the tsunami of immune checkpoint inhibitors revealed by numerous FDA approvals, publications and abstracts in relation with these drugs in different cancers and settings. First, we reported all new indications of anti-CTLA4 and anti-PD1 approved by the FDA, the positive clinical trials published and the abstracts with promising results at important scientific meetings during 2015. Then, we discussed different critical issues of these new agents going from their predictive factors, combination therapies, tumor response patterns, efficacy in particular settings, side effect management to cost and economic burden. PMID:27051042

  14. A band clamp with a spring toggle lever

    NASA Technical Reports Server (NTRS)

    Simmonds, M.

    1974-01-01

    Clamp could have several applications, as it provides tolerance for both expansion and contraction. It might be useful with firemen's breathing apparatus and luggage racks and other freight-carrying equipment. Also, using same piece as handle and spring reduces production costs by reducing number of parts.

  15. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... APPROVAL OF MINING PRODUCTS ELECTRIC MOTOR-DRIVEN MINE EQUIPMENT AND ACCESSORIES Construction and Design...) cables to prevent strain on the cable terminals of a machine. Also insulated clamps shall be provided to prevent strain on both ends of each cable or cord leading from a machine to a detached or...

  16. Mechanical stability of multidomain proteins and novel mechanical clamps.

    PubMed

    Sikora, Mateusz; Cieplak, Marek

    2011-06-01

    We estimate the size of mechanostability for 318 multidomain proteins which are single-chain and contain up to 1021 amino acids. We predict existence of novel types of mechanical clamps in which interdomain contacts play an essential role. Mechanical clamps are structural regions which are the primary source of a protein's resistance to pulling. Among these clamps there is one that opposes tensile stress due to two domains swinging apart. This movement strains and then ruptures the contacts that hold the two domains together. Another clamp also involves tensile stress but it originates from an immobilization of a structural region by a surrounding knot-loop (without involving any disulfide bonds). Still another mechanism involves shear between helical regions belonging to two domains. We also consider the amyloid-prone cystatin C which provides an example of a two-chain 3D domain-swapped protein. We predict that this protein should withstand remarkably large stress, perhaps of order 800 pN, when inducing a shearing strain. The survey is generated through molecular dynamics simulations performed within a structure-based coarse grained model. PMID:21465555

  17. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Carotid artery clamp. 882.5175 Section 882.5175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... (the principal artery in the neck that supplies blood to the brain) and has a removable...

  18. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Carotid artery clamp. 882.5175 Section 882.5175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... (the principal artery in the neck that supplies blood to the brain) and has a removable...

  19. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Carotid artery clamp. 882.5175 Section 882.5175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... (the principal artery in the neck that supplies blood to the brain) and has a removable...

  20. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carotid artery clamp. 882.5175 Section 882.5175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... (the principal artery in the neck that supplies blood to the brain) and has a removable...

  1. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Carotid artery clamp. 882.5175 Section 882.5175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... (the principal artery in the neck that supplies blood to the brain) and has a removable...

  2. Management of Senile Ptosis with Levator Muscle Resection Using the Putterman Clamp

    PubMed Central

    2016-01-01

    Summary: Putterman clamp, a muscle clamp, is commonly used in conjunctival müllerectomies. We report 3 cases of senile ptosis repaired with levator muscle resection using the Putterman clamp. The redundant levator aponeurosis was removed with electrocautery after clamping with the Putterman clamp. The levator muscle was refixed to the tarsus with three 4-0 Vicryl stitches after adjusting the height of the eyelid fissure. No intraoperative difficulties were encountered. Ecchymosis and edema were limited in the immediate postoperative period. No complications were noted during the follow-up. The benefits of using the Putterman clamp in levator muscle resection are illustrated in these cases. PMID:27482474

  3. Management of Senile Ptosis with Levator Muscle Resection Using the Putterman Clamp.

    PubMed

    Yang, Ju-Wen

    2016-06-01

    Putterman clamp, a muscle clamp, is commonly used in conjunctival müllerectomies. We report 3 cases of senile ptosis repaired with levator muscle resection using the Putterman clamp. The redundant levator aponeurosis was removed with electrocautery after clamping with the Putterman clamp. The levator muscle was refixed to the tarsus with three 4-0 Vicryl stitches after adjusting the height of the eyelid fissure. No intraoperative difficulties were encountered. Ecchymosis and edema were limited in the immediate postoperative period. No complications were noted during the follow-up. The benefits of using the Putterman clamp in levator muscle resection are illustrated in these cases. PMID:27482474

  4. Twitter as a Tool to Warn Others about Sobriety Checkpoints: A Pilot Observational Study

    ERIC Educational Resources Information Center

    Seitz, Christopher M.; Orsini, Muhsin Michael; Fearnow-Kenney, Melodie; Hatzudis, Kiki; Wyrick, David L.

    2012-01-01

    Anecdotal evidence suggests that young people use the website Twitter as a tool to warn drivers about the locations of sobriety checkpoints. Researchers investigated this claim by independently analyzing the website's content regarding a sample of 10 sobriety checkpoints that were conducted in cities throughout the United States during the weekend…

  5. Centrosome-associated regulators of the G2/M checkpoint as targets for cancer therapy

    PubMed Central

    Wang, Yingmei; Ji, Ping; Liu, Jinsong; Broaddus, Russell R; Xue, Fengxia; Zhang, Wei

    2009-01-01

    In eukaryotic cells, control mechanisms have developed that restrain cell-cycle transitions in response to stress. These regulatory pathways are termed cell-cycle checkpoints. The G2/M checkpoint prevents cells from entering mitosis when DNA is damaged in order to afford these cells an opportunity to repair the damaged DNA before propagating genetic defects to the daughter cells. If the damage is irreparable, checkpoint signaling might activate pathways that lead to apoptosis. Since alteration of cell-cycle control is a hallmark of tumorigenesis, cell-cycle regulators represent potential targets for therapy. The centrosome has recently come into focus as a critical cellular organelle that integrates G2/M checkpoint control and repairs signals in response to DNA damage. A growing number of G2/M checkpoint regulators have been found in the centrosome, suggesting that centrosome has an important role in G2/M checkpoint function. In this review, we discuss centrosome-associated regulators of the G2/M checkpoint, the dysregulation of this checkpoint in cancer, and potential candidate targets for cancer therapy. PMID:19216791

  6. Space Reclamation for Uncoordinated Checkpointing in Message-Passing Systems. Ph.D. Thesis

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min

    1993-01-01

    Checkpointing and rollback recovery are techniques that can provide efficient recovery from transient process failures. In a message-passing system, the rollback of a message sender may cause the rollback of the corresponding receiver, and the system needs to roll back to a consistent set of checkpoints called recovery line. If the processes are allowed to take uncoordinated checkpoints, the above rollback propagation may result in the domino effect which prevents recovery line progression. Traditionally, only obsolete checkpoints before the global recovery line can be discarded, and the necessary and sufficient condition for identifying all garbage checkpoints has remained an open problem. A necessary and sufficient condition for achieving optimal garbage collection is derived and it is proved that the number of useful checkpoints is bounded by N(N+1)/2, where N is the number of processes. The approach is based on the maximum-sized antichain model of consistent global checkpoints and the technique of recovery line transformation and decomposition. It is also shown that, for systems requiring message logging to record in-transit messages, the same approach can be used to achieve optimal message log reclamation. As a final topic, a unifying framework is described by considering checkpoint coordination and exploiting piecewise determinism as mechanisms for bounding rollback propagation, and the applicability of the optimal garbage collection algorithm to domino-free recovery protocols is demonstrated.

  7. Science Competencies for Exit Level and Checkpoint Competencies for Levels 3, 5, & 8.

    ERIC Educational Resources Information Center

    New Mexico State Dept. of Education, Santa Fe.

    Restatements of the educational standards and the science competencies established by the New Mexico State Board of Education are provided in this document. Identified as basic and process skills, these checkpoint competencies are tied directly to the exit competencies for science. It is suggested that the checkpoint competencies be used as a…

  8. Spindle Checkpoint Protein Xmad1 Recruits Xmad2 to Unattached Kinetochores

    PubMed Central

    Chen, Rey-Huei; Shevchenko, Andrej; Mann, Matthias; Murray, Andrew W.

    1998-01-01

    The spindle checkpoint prevents the metaphase to anaphase transition in cells containing defects in the mitotic spindle or in chromosome attachment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentration fails to restore the checkpoint, suggesting that other checkpoint component(s) were depleted from the extract through their association with Xmad2. Mass spectrometry provided peptide sequences from an 85-kD protein that coimmunoprecipitates with Xmad2 from egg extracts. This information was used to clone XMAD1, which encodes a homologue of the budding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint in egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and to those kinetochores that are not bound to spindle microtubules during mitosis. Adding an anti-Xmad1 antibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xmad2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to recruit Xmad2 to kinetochores that have not bound microtubules. PMID:9786942

  9. Cytotoxic T lymphocyte antigen-4 and immune checkpoint blockade.

    PubMed

    Buchbinder, Elizabeth; Hodi, F Stephen

    2015-09-01

    The relationship between cancer and the immune system is complex and provides unique therapeutic opportunities. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a regulatory molecule that suppresses T cell effector function following initial activation by costimulatory signals. Fully human monoclonal antibodies targeting CTLA-4 have been shown to increase T cell function and antitumor responses in patients with advanced metastatic melanoma. Responses observed with such immune checkpoint therapy can follow a different pattern from that seen with cytotoxic chemotherapy or targeted therapy and may continue after therapy is discontinued. In addition, the toxicities that are associated with anti-CTLA-4 therapy may differ from those of conventional therapies and consist of inflammatory events in parts of the body that do not contain cancerous cells. Early recognition of these inflammatory events and intervention is important, and the identification of predictive biomarkers continues to be an unfulfilled need in the field of immunotherapy. Combinatorial approaches with targeted therapies, radiation therapy, chemotherapy, or other immune checkpoint agonists/antagonists have the potential to increase the efficacy of CTLA-4 blockade. PMID:26325034

  10. Mitotic Checkpoint Regulators Control Insulin Signaling and Metabolic Homeostasis.

    PubMed

    Choi, Eunhee; Zhang, Xiangli; Xing, Chao; Yu, Hongtao

    2016-07-28

    Insulin signaling regulates many facets of animal physiology. Its dysregulation causes diabetes and other metabolic disorders. The spindle checkpoint proteins MAD2 and BUBR1 prevent precocious chromosome segregation and suppress aneuploidy. The MAD2 inhibitory protein p31(comet) promotes checkpoint inactivation and timely chromosome segregation. Here, we show that whole-body p31(comet) knockout mice die soon after birth and have reduced hepatic glycogen. Liver-specific ablation of p31(comet) causes insulin resistance, hyperinsulinemia, glucose intolerance, and hyperglycemia and diminishes the plasma membrane localization of the insulin receptor (IR) in hepatocytes. MAD2 directly binds to IR and facilitates BUBR1-dependent recruitment of the clathrin adaptor AP2 to IR. p31(comet) blocks the MAD2-BUBR1 interaction and prevents spontaneous clathrin-mediated IR endocytosis. BUBR1 deficiency enhances insulin sensitivity in mice. BUBR1 depletion in hepatocytes or the expression of MAD2-binding-deficient IR suppresses the metabolic phenotypes of p31(comet) ablation. Our findings establish a major IR regulatory mechanism and link guardians of chromosome stability to nutrient metabolism. PMID:27374329

  11. Rituximab does not reset defective early B cell tolerance checkpoints

    PubMed Central

    Chamberlain, Nicolas; Massad, Christopher; Oe, Tyler; Cantaert, Tineke; Herold, Kevan C.; Meffre, Eric

    2015-01-01

    Type 1 diabetes (T1D) patients show abnormalities in early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in their blood. Treatment with rituximab, an anti-CD20 mAb that depletes B cells, has been shown to preserve β cell function in T1D patients and improve other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. However, it remains largely unknown how anti–B cell therapy thwarts autoimmunity in these pathologies. Here, we analyzed the reactivity of Abs expressed by single, mature naive B cells from 4 patients with T1D before and 52 weeks after treatment to determine whether rituximab resets early B cell tolerance checkpoints. We found that anti–B cell therapy did not alter the frequencies of autoreactive and polyreactive B cells, which remained elevated in the blood of all patients after rituximab treatment. Moreover, the limited proliferative history of autoreactive B cells after treatment revealed that these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. We conclude that anti–B cell therapy may provide a temporary dampening of autoimmune processes through B cell depletion. However, repletion with autoreactive B cells may explain the relapse that occurs in many autoimmune patients after anti–B cell therapy. PMID:26642366

  12. Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins▿

    PubMed Central

    Ball, Heather L.; Ehrhardt, Mark R.; Mordes, Daniel A.; Glick, Gloria G.; Chazin, Walter J.; Cortez, David

    2007-01-01

    The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP. PMID:17339343

  13. Cdc7 kinase mediates Claspin phosphorylation in DNA replication checkpoint.

    PubMed

    Kim, J M; Kakusho, N; Yamada, M; Kanoh, Y; Takemoto, N; Masai, H

    2008-05-29

    Cdc7 kinase is evolutionarily conserved and is involved in initiation and progression of DNA replication. However, roles of Cdc7 in checkpoint responses remain largely unknown. In this study, we show that deletion of the Cdc7 genes in mouse embryonic stem (ES) cells abrogates hydroxyurea (HU)- or UV-induced activation of Chk1. HU-induced Chk1 activation is also impaired in human cancer cell lines in which Cdc7 is depleted by siRNA, and Cdc7-depleted cells are more sensitive to HU treatment. In contrast, ATR and Rad17 are relocated to chromatin in these cells following HU treatment, indicating that stalled DNA replication forks are detected normally. Cdc7-depleted cells exhibit defects in chromatin association and phosphorylation of Claspin, suggesting that Cdc7 exerts its effect at least partially through Claspin. Consistent with this prediction, Cdc7 interacts with and phosphorylates Claspin. We propose that Cdc7 is required for activation of the ATR-Chk1 checkpoint pathway through regulation of Claspin. PMID:18084324

  14. Identification of inhibitors of checkpoint kinase 1 through template screening.

    PubMed

    Matthews, Thomas P; Klair, Suki; Burns, Samantha; Boxall, Kathy; Cherry, Michael; Fisher, Martin; Westwood, Isaac M; Walton, Michael I; McHardy, Tatiana; Cheung, Kwai-Ming J; Van Montfort, Rob; Williams, David; Aherne, G Wynne; Garrett, Michelle D; Reader, John; Collins, Ian

    2009-08-13

    Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells. PMID:19572549

  15. Vibration control of a flexible clamped-clamped plate based on an improved FULMS algorithm and laser displacement measurement

    NASA Astrophysics Data System (ADS)

    Xie, Lingbo; Qiu, Zhi-cheng; Zhang, Xian-min

    2016-06-01

    This paper presents a novel active resonant vibration control experiment of a flexible clamped-clamped plate using an improved filtered-U least mean square (FULMS) algorithm and laser displacement measurement. Different from the widely used PZT sensors or acceleration transducers, the vibration of the flexible clamped-clamped plate is measured by a non-contact laser displacement measurement sensor with higher measurement accuracy and without additional load to the plate. The conventional FULMS algorithm often uses fixed step size and needs reference signal related to the external disturbance signal. However, the fixed step size method cannot obtain a fast convergence speed and it will result in a low residual error. Thus, a variable step size method is investigated. In addition, it is difficult to extract reference signal related to the vibration source directly in the practical application. Therefore, it is practically useful that a reference signal is constructed by both the controller parameters and the vibration residual signal. The experimental results demonstrate that the improved FULMS algorithm has better vibration control effect than the proportional derivative (PD) feedback control algorithm and the fixed step-size control algorithm.

  16. [Genetic Mutation Accumulation and Clinical Outcome of Immune Checkpoint Blockade Therapy].

    PubMed

    Takahashi, Masanobu

    2016-06-01

    Immune checkpoint blockade therapy has recently attracted great attention in the area of oncology. In Japan, since 2014, an anti-PD-1antibody nivolumab and anti-CTLA-4 antibody ipilimumab have been available for the treatment of patients with malignant melanoma, and nivolumab has been available for patients with non-small cell lung cancer. Clinical trials using these drugs and other immune checkpoint inhibitors are currently in progress worldwide. The immune checkpoint blockade therapy is a promising new cancer therapy; however, not all patients with cancer can benefit from this therapy. Recent evidence shows that markers reflecting the extent of genetic mutation accumulation, including mutation burden, non-synonymous mutation that produces neoantigen, and microsatellite instability, possibly serve as promising marker to predict who can benefit from the immune checkpoint blockade therapy. Here, I introduce the recent evidence and discuss the correlation between genetic mutation accumulation and clinical outcome of immune checkpoint blockade therapy. PMID:27306805

  17. mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development

    PubMed Central

    Brodsky, Michael H.; Sekelsky, Jeff J.; Tsang, Garson; Hawley, R. Scott; Rubin, Gerald M.

    2000-01-01

    Checkpoints block cell cycle progression in eukaryotic cells exposed to DNA damaging agents. We show that several Drosophila homologs of checkpoint genes, mei-41, grapes, and 14-3-3ε, regulate a DNA damage checkpoint in the developing eye. We have used this assay to show that the mutagen-sensitive gene mus304 is also required for this checkpoint. mus304 encodes a novel coiled-coil domain protein, which is targeted to the cytoplasm. Similar to mei-41, mus304 is required for chromosome break repair and for genomic stability. mus304 animals also exhibit three developmental defects, abnormal bristle morphology, decreased meiotic recombination, and arrested embryonic development. We suggest that these phenotypes reflect distinct developmental consequences of a single underlying checkpoint defect. Similar mechanisms may account for the puzzling array of symptoms observed in humans with mutations in the ATM tumor suppressor gene. PMID:10733527

  18. Identifying security checkpoints locations to protect the major U.S. urban areas

    SciTech Connect

    Cuellar-Hengartner, Leticia; Watkins, Daniel; Kubicek, Deborah A.; Rodriguez, Erick; Stroud, Phillip D.

    2015-09-01

    Transit networks are integral to the economy and to society, but at the same time they could allow terrorists to transport weapons of mass destruction into any city. Road networks are especially vulnerable, because they lack natural checkpoints unlike air networks that have security measures in place at all major airports. One approach to mitigate this risk is ensuring that every road route passes through at least one security checkpoint. Using the Ford-Fulkerson maximum-flow algorithm, we generate a minimum set of checkpoint locations within a ring-shaped buffer area surrounding the 50 largest US urban areas. We study how the number of checkpoints changes as we increase the buffer width to perform a cost-benefit analysis and to identify groups of cities that behave similarly. The set of required checkpoints is surprisingly small (10-124) despite the hundreds of thousands of road arcs in those areas, making it feasible to protect all major cities.

  19. Roles of different pools of the mitotic checkpoint complex and the mechanisms of their disassembly

    PubMed Central

    Eytan, Esther; Sitry-Shevah, Danielle; Teichner, Adar; Hershko, Avram

    2013-01-01

    The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis. When the checkpoint is turned on, the mitotic checkpoint complex (MCC) inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 associated with the APC/C activator Cdc20. The mechanisms of the assembly of MCC when the checkpoint is turned on, and of its disassembly when the checkpoint is inactivated, are not sufficiently understood. Previous reports indicated that APC/C-mediated polyubiquitylation of Cdc20 in MCC is required for the dissociation of APC/C-associated MCC, but not of free MCC. The pool of free MCC is disassembled by an ATP-dependent process stimulated by the Mad2-binding protein p31comet. It remained unknown whether free MCC is the precursor or the dissociation product of APC/C-bound MCC. By characterizing the mechanisms of the disassembly of APC/C-bound MCC in a purified system, we find that it cannot be the source of free MCC, because it is bound at high affinity and is released only in ubiquitylated or partially disassembled forms. By the use of a cell-free system from Xenopus eggs that reproduces the mitotic checkpoint, we show that MCC can be assembled in the absence of APC/C in a checkpoint-dependent manner. We propose that when the checkpoint is turned on, free MCC is the precursor of APC/C-bound MCC. When the mitotic checkpoint is extinguished, both APC/C-bound and free MCC pools have to be disassembled to release APC/C from inhibition. PMID:23754430

  20. Detailed Modeling, Design, and Evaluation of a Scalable Multi-level Checkpointing System

    SciTech Connect

    Moody, A T; Bronevetsky, G; Mohror, K M; de Supinski, B R

    2010-04-09

    High-performance computing (HPC) systems are growing more powerful by utilizing more hardware components. As the system mean-time-before-failure correspondingly drops, applications must checkpoint more frequently to make progress. However, as the system memory sizes grow faster than the bandwidth to the parallel file system, the cost of checkpointing begins to dominate application run times. A potential solution to this problem is to use multi-level checkpointing, which employs multiple types of checkpoints with different costs and different levels of resiliency in a single run. The goal is to design light-weight checkpoints to handle the most common failure modes and rely on more expensive checkpoints for less common, but more severe failures. While this approach is theoretically promising, it has not been fully evaluated in a large-scale, production system context. To this end we have designed a system, called the Scalable Checkpoint/Restart (SCR) library, that writes checkpoints to storage on the compute nodes utilizing RAM, Flash, or disk, in addition to the parallel file system. We present the performance and reliability properties of SCR as well as a probabilistic Markov model that predicts its performance on current and future systems. We show that multi-level checkpointing improves efficiency on existing large-scale systems and that this benefit increases as the system size grows. In particular, we developed low-cost checkpoint schemes that are 100x-1000x faster than the parallel file system and effective against 85% of our system failures. This leads to a gain in machine efficiency of up to 35%, and it reduces the the load on the parallel file system by a factor of two on current and future systems.

  1. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint.

    PubMed

    Brito, Daniela A; Rieder, Conly L

    2006-06-20

    In the presence of unattached/weakly attached kinetochores, the spindle assembly checkpoint (SAC) delays exit from mitosis by preventing the anaphase-promoting complex (APC)-mediated proteolysis of cyclin B, a regulatory subunit of cyclin-dependent kinase 1 (Cdk1). Like all checkpoints, the SAC does not arrest cells permanently, and escape from mitosis in the presence of an unsatisfied SAC requires that cyclin B/Cdk1 activity be inhibited. In yeast , and likely Drosophila, this occurs through an "adaptation" process involving an inhibitory phosphorylation on Cdk1 and/or activation of a cyclin-dependent kinase inhibitor (Cdki). The mechanism that allows vertebrate cells to escape mitosis when the SAC cannot be satisfied is unknown. To explore this issue, we conducted fluorescence microscopy studies on rat kangaroo (PtK) and human (RPE1) cells dividing in the presence of nocodazole. We find that in the absence of microtubules (MTs), escape from mitosis occurs in the presence of an active SAC and requires cyclin B destruction. We also find that cyclin B is progressively destroyed during the block by a proteasome-dependent mechanism. Thus, vertebrate cells do not adapt to the SAC. Rather, our data suggest that in normal cells, the SAC cannot prevent a slow but continuous degradation of cyclin B that ultimately drives the cell out of mitosis. PMID:16782009

  2. Review: The lord of the rings: Structure and mechanism of the sliding clamp loader.

    PubMed

    Kelch, Brian A

    2016-08-01

    Sliding clamps are ring-shaped polymerase processivity factors that act as master regulators of cellular replication by coordinating multiple functions on DNA to ensure faithful transmission of genetic and epigenetic information. Dedicated AAA+ ATPase machines called clamp loaders actively place clamps on DNA, thereby governing clamp function by controlling when and where clamps are used. Clamp loaders are also important model systems for understanding the basic principles of AAA+ mechanism and function. After nearly 30 years of study, the ATP-dependent mechanism of opening and loading of clamps is now becoming clear. Here I review the structural and mechanistic aspects of the clamp loading process, as well as comment on questions that will be addressed by future studies. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 532-546, 2016. PMID:26918303

  3. Parallelization and checkpointing of GPU applications through program transformation

    SciTech Connect

    Solano-Quinde, Lizandro Damian

    2012-01-01

    GPUs have emerged as a powerful tool for accelerating general-purpose applications. The availability of programming languages that makes writing general-purpose applications for running on GPUs tractable have consolidated GPUs as an alternative for accelerating general purpose applications. Among the areas that have benefited from GPU acceleration are: signal and image processing, computational fluid dynamics, quantum chemistry, and, in general, the High Performance Computing (HPC) Industry. In order to continue to exploit higher levels of parallelism with GPUs, multi-GPU systems are gaining popularity. In this context, single-GPU applications are parallelized for running in multi-GPU systems. Furthermore, multi-GPU systems help to solve the GPU memory limitation for applications with large application memory footprint. Parallelizing single-GPU applications has been approached by libraries that distribute the workload at runtime, however, they impose execution overhead and are not portable. On the other hand, on traditional CPU systems, parallelization has been approached through application transformation at pre-compile time, which enhances the application to distribute the workload at application level and does not have the issues of library-based approaches. Hence, a parallelization scheme for GPU systems based on application transformation is needed. Like any computing engine of today, reliability is also a concern in GPUs. GPUs are vulnerable to transient and permanent failures. Current checkpoint/restart techniques are not suitable for systems with GPUs. Checkpointing for GPU systems present new and interesting challenges, primarily due to the natural differences imposed by the hardware design, the memory subsystem architecture, the massive number of threads, and the limited amount of synchronization among threads. Therefore, a checkpoint/restart technique suitable for GPU systems is needed. The goal of this work is to exploit higher levels of parallelism and

  4. Patch Clamp Experiments under Conditions of Variable Graviy

    NASA Astrophysics Data System (ADS)

    Kohn, F. P. M.; Meissner, K.

    2013-02-01

    The cellular membrane is an intrinsic part of any cell. It has a complex composition of lipid molecules and proteins. The membrane is, among others, involved in excitation and signal transduction. Ion channels, as integral membrane proteins, play an important role. For the question of gravity sensitivity of biological systems, especially neuronal cells, ion channels are of high interest. Gravity might directly interact with the ion channel protein or it might change the thermodynamic membrane parameters, influencing the incorporated proteins indirectly. Detailed information about the effects of gravity on the function of single ion-channels can up to now only be acquired by electrophysiological approaches like the patch clamp technique. Today this technique is the preferentially used technique for single ion-channel studies. Consequently, experiments have been developed in recent years to investigate the interaction of gravity with single ion channel molecules utilizing the patch-clamp technology on different macro- and micro-gravity platforms.

  5. Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes

    PubMed Central

    L Brown, Austin; E. Johnson, Brandon; B. Goodman, Miriam

    2008-01-01

    Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands. PMID:19078941

  6. DNA Sliding Clamps: Just the Right Twist to Load onto DNA

    SciTech Connect

    Barsky, D; Venclovas, C

    2005-10-24

    Two recent papers illuminate a long sought step in DNA sliding clamp loading. One paper reveals the structure of the PCNA clamp wrapped around DNA--still open from being loaded--while a second paper discovers that the clamp may assist this process by forming a right-handed helix upon opening.

  7. Oscillations and latency in the clamped pupil light reflex

    NASA Astrophysics Data System (ADS)

    Milton, John G.; Ohira, Toru; Steck, Jeff; Crate, John; Longtin, Andre

    1993-11-01

    It is shown that the pupil latency can be estimated from pupil cycling measurements when the pupil light reflex is clamped with piecewise constant negative feedback. The solution of the mathematical model previously shown to describe these oscillations is utilized to develop a variety of strategies to estimate latency and to evaluate the effects of noise on these estimates. The results demonstrate that the pupil latency shows considerable variation.

  8. Acute aortic dissection from cross-clamp injury.

    PubMed

    Litchford, B; Okies, J E; Sugimura, S; Starr, A

    1976-11-01

    Acute dissection of the ascending aorta secondary to cross-clamp injury can be successfully managed if the problem is recognized immediately. Bypass must be instituted after recannulation at a point distal to the innominate artery so that proper exposure of the site of injury can be obtained. Systemic as well as local hypothermia for myocardial preservation are both necessary. Direct suture closure of all layers at the site of dissection over Teflon felt can terminate this process. PMID:979312

  9. From Galvani to patch clamp: the development of electrophysiology.

    PubMed

    Verkhratsky, Alexei; Krishtal, O A; Petersen, Ole H

    2006-12-01

    The development of electrophysiology is traced from the early beginnings represented by the work of the Dutch microscopist, Jan Swammerdam, in the 17th century through the first notion of an aqueous transmembrane pore as a substrate of excitability made by Luigi Galvani in late 18th century to the invention late in the 20th century of the patch-clamp technique by Erwin Neher and Bert Sakmann. PMID:17072639

  10. Force-clamp laser trapping of rapidly interacting molecules

    NASA Astrophysics Data System (ADS)

    Capitanio, Marco; Monico, Carina; Vanzi, Francesco; Pavone, Francesco S.

    2013-06-01

    Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay of only ˜10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  11. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  12. Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast.

    PubMed Central

    Sia, R A; Herald, H A; Lew, D J

    1996-01-01

    A morphogenesis checkpoint in budding yeast delays nuclear division (and subsequent cell cycle progression) in cells that have failed to make a bud. We show that the ability of this checkpoint to delay nuclear division requires the SWE1 gene, encoding a protein kinase that inhibits the master cell cycle regulatory kinase Cdc28. The timing of nuclear division in cells that cannot make a bud is exquisitely sensitive to the dosage of SWE1 and MIH1 genes, which control phosphorylation of Cdc28 at tyrosine 19. In contrast, the timing of nuclear division in budded cells does not rely on Cdc28 phosphorylation, suggesting that the morphogenesis checkpoint somehow turns on this regulatory pathway. We show that SWE1 mRNA levels fluctuate during the cell cycle and are elevated in cells that cannot make a bud. However, regulation of SWE1 mRNA levels by the checkpoint is indirect, acting through a feedback loop requiring Swe1 activity. Further, the checkpoint is capable of delaying nuclear division even when SWE1 transcription is deregulated. We propose that the checkpoint delays nuclear division through post-translational regulation of Swe1 and that transcriptional feedback loops enhance the efficacy of the checkpoint. Images PMID:8930890

  13. Src family kinases maintain the balance between replication stress and the replication checkpoint.

    PubMed

    Miura, Takahito; Fukumoto, Yasunori; Morii, Mariko; Honda, Takuya; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2016-01-01

    Progression of DNA replication is tightly controlled by replication checkpoints to ensure the accurate and rapid duplication of genetic information. Upon replication stress, the replication checkpoint slows global DNA replication by inhibiting the late-firing origins and by slowing replication fork progression. Activation of the replication checkpoint has been studied in depth; however, little is known about the termination of the replication checkpoint. Here, we show that Src family kinases promote the recovery from replication checkpoints. shRNA knockdown of a Src family kinase, Lyn, and acute chemical inhibition of Src kinases prevented inactivation of Chk1 after removal of replication stress. Consistently, Src inhibition slowed resumption of DNA replication, after the removal of replication blocks. The effect of Src inhibition was not observed in the presence of an ATM/ATR inhibitor caffeine. These data indicate that Src kinases promote the resumption of DNA replication by suppressing ATR-dependent replication checkpoints. Surprisingly, the resumption of replication was delayed by caffeine. In addition, Src inhibition delayed recovery from replication fork collapse. We propose that Src kinases maintain the balance between replication stress and the activity of the replication checkpoint. PMID:26194897

  14. Selective killing of G2 decatenation checkpoint defective colon cancer cells by catalytic topoisomerase II inhibitor.

    PubMed

    Jain, Chetan Kumar; Roychoudhury, Susanta; Majumder, Hemanta Kumar

    2015-05-01

    Cancer cells with defective DNA decatenation checkpoint can be selectively targeted by the catalytic inhibitors of DNA topoisomerase IIα (topo IIα) enzyme. Upon treatment with catalytic topo IIα inhibitors, cells with defective decatenation checkpoint fail to arrest their cell cycle in G2 phase and enter into M phase with catenated and under-condensed chromosomes resulting into impaired mitosis and eventually cell death. In the present work we analyzed decatenation checkpoint in five different colon cancer cell lines (HCT116, HT-29, Caco2, COLO 205 and SW480) and in one non-cancerous cell line (HEK293T). Four out of the five colon cancer cell lines i.e. HCT116, HT-29, Caco2, and COLO 205 were found to be compromised for the decatenation checkpoint function at different extents, whereas SW480 and HEK293T cell lines were found to be proficient for the checkpoint function. Upon treatment with ICRF193, decatenation checkpoint defective cell lines failed to arrest the cell cycle in G2 phase and entered into M phase without proper chromosomal decatenation, resulting into the formation of tangled mass of catenated and under-condensed chromosomes. Such cells underwent mitotic catastrophe and rapid apoptosis like cell death and showed higher sensitivity for ICRF193. Our study suggests that catalytic inhibitors of topoisomerase IIα are promising therapeutic agents for the treatment of colon cancers with defective DNA decatenation checkpoint. PMID:25746763

  15. Attachment issues: kinetochore transformations and spindle checkpoint silencing.

    PubMed

    Etemad, Banafsheh; Kops, Geert J P L

    2016-04-01

    Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetochores of all chromosomes are established. The anaphase inhibitor is generated at unattached kinetochores and inhibitor production is prevented when microtubules are captured. Understanding the molecular changes in the kinetochore that are evoked by microtubule attachments is crucial for understanding the mechanisms of SAC signaling and silencing. Here, we highlight the most recent findings on these events, pinpoint some remaining mysteries, and argue for incorporating holistic views of kinetochore dynamics in order to understand SAC silencing. PMID:26947988

  16. Targeting the spindle assembly checkpoint for breast cancer treatment.

    PubMed

    Marques, Sandra; Fonseca, Joana; Silva, Patrícia M A; Bousbaa, Hassan

    2015-01-01

    Breast cancer is the most common malignancy in women worldwide and the second leading cause of cancer deaths after lung cancer. As in other malignancies, aneuploidy is a common feature of breast cancer and influences its behavior. Aneuploidy has been linked to inappropriate activity of the spindle assembly checkpoint (SAC), a surveillance mechanism that, in normal cells, prevents anaphase onset until correct alignment of all chromosomes at the metaphase is achieved. Interestingly, the widely used anti-microtubule drugs, vinca alkaloids and taxanes, kill cancer cells through chronic arrest in mitosis as a consequence of chronic SAC activation. Deregulated SAC has been reported in breast cancer in many reports and presents an attractive therapeutic strategy. We present here a review of the current knowledge on the SAC defects and the underlying molecular mechanisms in breast cancer, and discuss the potential of SAC components as targets for breast cancer therapies. PMID:25731686

  17. OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases.

    PubMed

    Ji, Wenbin; Arnst, Christopher; Tipton, Aaron R; Bekier, Michael E; Taylor, William R; Yen, Tim J; Liu, Song-Tao

    2016-01-01

    OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays. PMID:27082996

  18. OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases

    PubMed Central

    Tipton, Aaron R.; Bekier, Michael E.; Taylor, William R.; Yen, Tim J.; Liu, Song-Tao

    2016-01-01

    OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays. PMID:27082996

  19. EMODnet MedSea Checkpoint for sustainable Blue Growth

    NASA Astrophysics Data System (ADS)

    Moussat, Eric; Pinardi, Nadia; Manzella, Giuseppe; Blanc, Frederique

    2016-04-01

    The EMODNET checkpoint is a wide monitoring system assessment activity aiming to support the sustainable Blue Growth at the scale of the European Sea Basins by: 1) Clarifying the observation landscape of all compartments of the marine environment including Air, Water, Seabed, Biota and Human activities, pointing out to the existing programs, national, European and international 2) Evaluating fitness for use indicators that will show the accessibility and usability of observation and modeling data sets and their roles and synergies based upon selected applications by the European Marine Environment Strategy 3) Prioritizing the needs to optimize the overall monitoring Infrastructure (in situ and satellite data collection and assembling, data management and networking, modeling and forecasting, geo-infrastructure) and release recommendations for evolutions to better meet the application requirements in view of sustainable Blue Growth The assessment is designed for : - Institutional stakeholders for decision making on observation and monitoring systems - Data providers and producers to know how their data collected once for a given purpose could fit other user needs - End-users interested in a regional status and possible uses of existing monitoring data Selected end-user applications are of paramount importance for: (i) the blue economy sector (offshore industries, fisheries); (ii) marine environment variability and change (eutrophication, river inputs and ocean climate change impacts); (iii) emergency management (oil spills); and (iv) preservation of natural resources and biodiversity (Marine Protected Areas). End-user applications generate innovative products based on the existing observation landscape. The fitness for use assessment is made thanks to the comparison of the expected product specifications with the quality of the product derived from the selected data. This involves the development of checkpoint information and indicators based on Data quality and

  20. Checkpoint inhibition for colorectal cancer: progress and possibilities.

    PubMed

    Paul, Barry; O'Neil, Bert H; McRee, Autumn J

    2016-06-01

    Colorectal cancer (CRC) remains the third most common cause of cancer death in the USA. Despite an increase in the repertoire of treatment options available for CRC, median overall survival has plateaued at approximately 2.5 years. Strategies that engage the patient's native immune system to overcome checkpoint inhibition have proven to be promising in subsets of CRCs, specifically those with mismatch repair deficiency. Further studies are required to determine combinations of standard therapies with immunotherapy drugs and to discover the best biomarkers to predict response. This review provides insight into the progress made in treating patients with advanced CRC with immunotherapeutics and the areas that demand further research to make these drugs more effective in this patient population. PMID:27197538

  1. Safety of immune checkpoint inhibitors in Chinese patients with melanoma.

    PubMed

    Wen, Xizhi; Wang, Yao; Ding, Ya; Li, Dandan; Li, Jingjing; Guo, Yiqun; Peng, Ruiqing; Zhao, Jingjing; Zhang, Xing; Zhang, Xiao-Shi

    2016-06-01

    This study aimed to determine the tolerability of Chinese melanoma patients, particularly those with hepatitis B virus (HBV) infection, to immune checkpoint inhibitor therapy. Patients with metastatic melanoma who received anti-cytotoxic T lymphocyte-associated antigen-4 antibody (ipilimumab) or anti-programmed death 1 antibody (pembrolizumab) therapy at our hospital between August 2012 and July 2015 were retrospectively reviewed. Adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0. Twenty-three patients with advanced melanoma were included; nine and 10 patients received infusions of ipilimumab and pembrolizumab, respectively, whereas four patients received concurrent ipilimumab and pembrolizumab therapy. There was no cessation of treatment because of agent-related adverse events in any patient. Immune-related adverse events were observed in 44% (4/9), 60% (6/10), 100% (4/4), and 61% (14/23) of patients receiving ipilimumab, pembrolizumab, concomitant ipilimumab and pembrolizumab, and any treatment, respectively. The most frequent immune-related adverse events were endocrine disorders (39%, 9/23), liver function abnormalities (22%, 5/23), and dermatological events (17%, 4/23). There were no gastrointestinal reactions. Toxicities were usually mild and easily managed; only 13% (3/23) of patients had grade 3 adverse events and none experienced grade 4 events or treatment-related death. No additional toxicity nor severe hepatotoxicity was observed in 11 patients who had previous HBV infection. The recommended anti-cytotoxic T lymphocyte-associated antigen-4 and anti-programmed death 1 antibody doses were well tolerated by Chinese patients. Thus, immune checkpoint inhibitors appear to be effective and safe in metastatic melanoma patients, including those with pre-existing HBV infection. PMID:27116334

  2. Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint

    PubMed Central

    Braunstein, Ilana; Miniowitz, Shirly; Moshe, Yakir; Hershko, Avram

    2007-01-01

    The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint [Sudakin V, Chan GKT, Yen TJ (2001) J Cell Biol 154:925–936]. We examined this problem by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5′-(γ-thio)triphosphate (ATP[γS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, we isolated inhibitory factors associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, we find that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration. PMID:17360335

  3. Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

    PubMed Central

    Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

    2015-01-01

    When cells undergo replication stress, proper checkpoint activation and deactivation are critical for genomic stability and cell survival and therefore must be highly regulated. Although mechanisms of checkpoint activation are well studied, mechanisms of checkpoint deactivation are far less understood. Previously, we reported that chromatin remodeling factors Isw2 and Ino80 attenuate the S-phase checkpoint activity in Saccharomyces cerevisiae, especially during recovery from hydroxyurea. In this study, we found that Isw2 and Ino80 have a more pronounced role in attenuating checkpoint activity during late S phase in the presence of methyl methanesulfonate (MMS). We therefore screened for checkpoint factors required for Isw2 and Ino80 checkpoint attenuation in the presence of MMS. Here we demonstrate that Isw2 and Ino80 antagonize checkpoint activators and attenuate checkpoint activity in S phase in MMS either through a currently unknown pathway or through RPA. Unexpectedly, we found that Isw2 and Ino80 increase chromatin accessibility around replicating regions in the presence of MMS through a novel mechanism. Furthermore, through growth assays, we provide additional evidence that Isw2 and Ino80 partially counteract checkpoint activators specifically in the presence of MMS. Based on these results, we propose that Isw2 and Ino80 attenuate S-phase checkpoint activity through a novel mechanism. PMID:25701287

  4. Thyroid Hormone Receptor Interacting Protein 13 (TRIP13) AAA-ATPase Is a Novel Mitotic Checkpoint-silencing Protein*

    PubMed Central

    Wang, Kexi; Sturt-Gillespie, Brianne; Hittle, James C.; Macdonald, Dawn; Chan, Gordon K.; Yen, Tim J.; Liu, Song-Tao

    2014-01-01

    The mitotic checkpoint (or spindle assembly checkpoint) is a fail-safe mechanism to prevent chromosome missegregation by delaying anaphase onset in the presence of defective kinetochore-microtubule attachment. The target of the checkpoint is the E3 ubiquitin ligase anaphase-promoting complex/cyclosome. Once all chromosomes are properly attached and bioriented at the metaphase plate, the checkpoint needs to be silenced. Previously, we and others have reported that TRIP13 AAA-ATPase binds to the mitotic checkpoint-silencing protein p31comet. Here we show that endogenous TRIP13 localizes to kinetochores. TRIP13 knockdown delays metaphase-to-anaphase transition. The delay is caused by prolonged presence of the effector for the checkpoint, the mitotic checkpoint complex, and its association and inhibition of the anaphase-promoting complex/cyclosome. These results suggest that TRIP13 is a novel mitotic checkpoint-silencing protein. The ATPase activity of TRIP13 is essential for its checkpoint function, and interference with TRIP13 abolished p31comet-mediated mitotic checkpoint silencing. TRIP13 overexpression is a hallmark of cancer cells showing chromosomal instability, particularly in certain breast cancers with poor prognosis. We suggest that premature mitotic checkpoint silencing triggered by TRIP13 overexpression may promote cancer development. PMID:25012665

  5. Self-tuning behavior of a clamped-clamped beam with sliding proof mass for broadband energy harvesting

    NASA Astrophysics Data System (ADS)

    Pillatsch, P.; Miller, L. M.; Halvorsen, E.; Wright, P. K.; Yeatman, E. M.; Holmes, A. S.

    2013-12-01

    Real world systems rarely vibrate at a single resonance frequency and the frequencies drift over time. Tunable devices exist, but generally need additional energy to achieve frequency adaptation. This means that the benefits in power output from this tuning need to be large enough to power the mechanism itself. Passively self-tuning systems go into resonance without requiring active control. This paper focuses on a passively self-tuning system with a proof mass that can slide freely along a clamped-clamped beam. Under external vibration, the slider moves along the beam until the system goes into resonance. A proof-of-concept design is introduced using either a copper or a steel beam and a 3D-printed ABS thermoplastic proof mass. Successful self-tuning is demonstrated in both cases. The frequencies range from 80 - 140 Hz at accelerations as low as 0.007 g rms. Results show the resonance of the beam and the position of the slider along the beam with time. Furthermore, the dynamic magnification and the proof mass position at resonance are discussed, together with the inherent non-linearities of double-clamped beam resonators. The findings support the hypothesis that the effect of the ratio between proof mass and beam mass outweighs the Duffing spring stiffening effects.

  6. Effect of various irrigant and autoclaving regimes on the fracture resistance of rubber dam clamps.

    PubMed

    Sutton, J; Saunders, W P

    1996-09-01

    Rubber dam clamps are known to break during clinical use in endodontics. This in-vitro study examined some of the variables which may contribute to the fracture. Stainless steel rubber dam clamps were subjected to various cleaning and autoclaving regimes and exposure to various solutions of sodium hypochlorite (NaOCl). Each clamp was examined after four cycles of cleaning and exposure to NaOCl. During environmental exposure to NaOCl, the clamp was stressed over a perspex rod to simulate placement onto the crown of a tooth. Clamps were examined after each test cycle visually and microscopically, or immediately after breakage. Results suggested that the fractures were because of a stress corrosion cracking phenomenon. There was evidence of intergranular and transgranular cracking of the metal. Corrosion spots were seen on the surface of the clamps and fracture occurred mainly through these spots. A number of recommendations to reduce breakage of clamps have been suggested. PMID:9206417

  7. Perspectives on the DNA damage and replication checkpoint responses in Saccharomyces cerevisiae

    PubMed Central

    Putnam, Christopher D.; Jaehnig, Eric J.; Kolodner, Richard D.

    2009-01-01

    The DNA damage and replication checkpoints are believed to primarily slow the progression of the cell cycle to allow DNA repair to occur. Here we summarize known aspects of the Saccharomyces cerevisiae checkpoints including how these responses are integrated into downstream effects on the cell cycle, chromatin, DNA repair, and cytoplasmic targets. Analysis of the transcriptional response demonstrates that it is far more complex and less relevant to the repair of DNA damage than the bacterial SOS response. We also address more speculative questions regarding potential roles of the checkpoint during the normal S-phase and how current evidence hints at a checkpoint activation mechanism mediated by positive feedback that amplifies initial damage signals above a minimium threshold. PMID:19477695

  8. Coupling end resection with the checkpoint response at DNA double-strand breaks.

    PubMed

    Villa, Matteo; Cassani, Corinne; Gobbini, Elisa; Bonetti, Diego; Longhese, Maria Pia

    2016-10-01

    DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3'-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast. PMID:27141941

  9. The spindle position checkpoint is coordinated by the Elm1 kinase

    PubMed Central

    Moore, Jeffrey K.; Chudalayandi, Prakash; Heil-Chapdelaine, Richard A.

    2010-01-01

    How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother–daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization. PMID:21041444

  10. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    NASA Technical Reports Server (NTRS)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  11. Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies.

    PubMed

    Sanmamed, M F; Chester, C; Melero, I; Kohrt, H

    2016-07-01

    The recent success of checkpoint blockers to treat cancer has demonstrated that the immune system is a critical player in the war against cancer. Historically, anticancer therapeutics have been tested in syngeneic mouse models (with a fully murine immune system) or in immunodeficient mice that allow the engraftment of human xenografts. Animal models with functioning human immune systems are critically needed to more accurately recapitulate the complexity of the human tumor microenvironment. Such models are integral to better predict tumor responses to both immunomodulatory agents and directly antineoplastic therapies. In this regard, the development of humanized models is a promising, novel strategy that offers the possibility of testing checkpoint blockers' capacity and their combination with other antitumor drugs. In this review, we discuss the strengths and weaknesses of the available animal models regarding their capacity to evaluate checkpoint blockers and checkpoint blocker-based combination immunotherapy. PMID:26912558

  12. Nonlinear vibrations of fluid-filled clamped circular cylindrical shells

    NASA Astrophysics Data System (ADS)

    Karagiozis, K. N.; Amabili, M.; Païdoussis, M. P.; Misra, A. K.

    2005-12-01

    In this study, the nonlinear vibrations are investigated of circular cylindrical shells, empty or fluid-filled, clamped at both ends and subjected to a radial harmonic force excitation. Two different theoretical models are developed. In the first model, the standard form of the Donnell's nonlinear shallow-shell equations is used; in the second, the equations of motion are derived by a variational approach which permits the inclusion of constraining springs at the shell extremities and taking in-plane inertial terms into account. In both cases, the solution includes both driven and companion modes, thus allowing for a travelling wave in the circumferential direction; they also include axisymmetric modes to capture the nonlinear inward shell contraction and the correct type (softening) nonlinear behaviour observed in experiments. In the first model, the clamped beam eigenfunctions are used to describe the axial variations of the shell deformation, automatically satisfying the boundary conditions, leading to a 7 degree-of-freedom (dof) expansion for the solution. In the second model, rotational springs are used at the ends of the shell, which when large enough reproduce a clamped end; the solution involves a sine series for axial variations of the shell deformation, leading to a 54 dof expansion for the solution. In both cases the modal expansions satisfy the boundary conditions and the circumferential continuity condition exactly. The Galerkin method is used to discretize the equations of motion, and AUTO to integrate the discretized equations numerically. When the shells are fluid-filled, the fluid is assumed to be incompressible and inviscid, and the fluid structure interaction is described by linear potential flow theory. The results from the two theoretical models are compared with existing experimental data, and in all cases good qualitative and quantitative agreement is observed.

  13. Immune checkpoint blockade in hepatocellular carcinoma: Current progress and future directions

    PubMed Central

    Hato, Tai; Goyal, Lipika; Greten, Tim F.; Duda, Dan G.; Zhu, Andrew X.

    2014-01-01

    Immune checkpoint blockade has recently emerged as a promising therapeutic approach for various malignancies including hepatocellular carcinoma (HCC). Preclinical and clinical studies have shown the potential benefit of modulating immunogenicity of HCC. In addition, recent advances in tumor immunology have broadened our understanding of the complex mechanism of immune evasion. In this review, we summarize the current knowledge on HCC immunology, and discuss the potential of immune checkpoint blockade as a novel HCC therapy from the basic, translational, and clinical perspectives. PMID:24912948

  14. Dissection of Rad9 BRCT Domain Function In The Mitotic Checkpoint Response To Telomere Uncapping

    PubMed Central

    Nnakwe, Chinonye C.; Altaf, Mohammed; Côté, Jacques; Kron, Stephen J.

    2009-01-01

    In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double-strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5' strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle. PMID:19880356

  15. A conserved checkpoint monitors meiotic chromosome synapsis inCaenorhabditis elegans

    SciTech Connect

    Bhalla, Needhi; Dernburg, Abby F.

    2005-07-14

    We report the discovery of a checkpoint that monitorssynapsis between homologous chromosomes to ensure accurate meioticsegregation. Oocytes containing unsynapsed chromosomes selectivelyundergo apoptosis even if agermline DNA damage checkpoint is inactivated.This culling mechanism isspecifically activated by unsynapsed pairingcenters, cis-acting chromosomesites that are also required to promotesynapsis in Caenorhabditis elegans. Apoptosis due to synaptic failurealso requires the C. elegans homolog of PCH2,a budding yeast pachytenecheckpoint gene, which suggests that this surveillance mechanism iswidely conserved.

  16. Intensity clamping in the filament of femtosecond laser radiation

    SciTech Connect

    Kandidov, V P; Fedorov, V Yu; Tverskoi, O V; Kosareva, O G; Chin, S L

    2011-04-30

    We have studied numerically the evolution of the light field intensity and induced refractive index of a medium upon filamentation of femtosecond laser radiation in air. It is shown that the intensity clamping results from the dynamic balance of optical powers of nonlinear lenses, induced by radiation due to the Kerr nonlinearity of air, and laser plasma produced during photoionisation. We have found the relation between the peak values of the light field intensity and the electron density in laser-produced plasma, as well as the transverse sizes of the filament and the plasma channel. (effects of laser radiation on matter)

  17. Vibration of clamped right triangular thin plates: Accurate simplified solutions

    NASA Astrophysics Data System (ADS)

    Saliba, H. T.

    1994-12-01

    Use of the superposition techniques in the free-vibration analyses of thin plates, as they were first introduced by Gorman, has provided simple and effective solutions to a vast number of rectangular plate problems. A modified superposition method is presented that is a noticeable improvement over existing techniques. It deals only with simple support conditions, leading to a simple, highly accurate, and very economical solution to the free-vibration problem of simply-supported right angle triangular plates. The modified method is also applicable to clamped-edge conditions.

  18. Normal-Pressure Tests of Circular Plates with Clamped Edges

    NASA Technical Reports Server (NTRS)

    Mcpherson, Albert E; Ramberg, Walter; Levy, Samuel

    1942-01-01

    A fixture is described for making normal-pressure tests of flat plates 5 inches in diameter in which particular care was taken to obtain rigid clamping at the edges. Results are given for 19 plates, ranging in thickness form 0.015 to 0.072 inch. The center deflections and the extreme-fiber stresses at low pressures were found to agree with theoretical values; the center deflections at high pressures were 4 to 12 percent greater than the theoretical values. Empirical curves are derived of the pressure for the beginning of the permanent set as a function of the dimensions of the plate and the tensile properties of the material.

  19. Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint*

    PubMed Central

    Fukumoto, Yasunori; Morii, Mariko; Miura, Takahito; Kubota, Sho; Ishibashi, Kenichi; Honda, Takuya; Okamoto, Aya; Yamaguchi, Noritaka; Iwama, Atsushi; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination. PMID:24634213

  20. TRIP13PCH-2 promotes Mad2 localization to unattached kinetochores in the spindle checkpoint response

    PubMed Central

    Nelson, Christian R.; Hwang, Tom; Chen, Pin-Hsi

    2015-01-01

    The spindle checkpoint acts during cell division to prevent aneuploidy, a hallmark of cancer. During checkpoint activation, Mad1 recruits Mad2 to kinetochores to generate a signal that delays anaphase onset. Yet, whether additional factors contribute to Mad2’s kinetochore localization remains unclear. Here, we report that the conserved AAA+ ATPase TRIP13PCH-2 localizes to unattached kinetochores and is required for spindle checkpoint activation in Caenorhabditis elegans. pch-2 mutants effectively localized Mad1 to unattached kinetochores, but Mad2 recruitment was significantly reduced. Furthermore, we show that the C. elegans orthologue of the Mad2 inhibitor p31(comet)CMT-1 interacts with TRIP13PCH-2 and is required for its localization to unattached kinetochores. These factors also genetically interact, as loss of p31(comet)CMT-1 partially suppressed the requirement for TRIP13PCH-2 in Mad2 localization and spindle checkpoint signaling. These data support a model in which the ability of TRIP13PCH-2 to disassemble a p31(comet)/Mad2 complex, which has been well characterized in the context of checkpoint silencing, is also critical for spindle checkpoint activation. PMID:26527744

  1. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    NASA Astrophysics Data System (ADS)

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  2. The human dynamic clamp as a paradigm for social interaction.

    PubMed

    Dumas, Guillaume; de Guzman, Gonzalo C; Tognoli, Emmanuelle; Kelso, J A Scott

    2014-09-01

    Social neuroscience has called for new experimental paradigms aimed toward real-time interactions. A distinctive feature of interactions is mutual information exchange: One member of a pair changes in response to the other while simultaneously producing actions that alter the other. Combining mathematical and neurophysiological methods, we introduce a paradigm called the human dynamic clamp (HDC), to directly manipulate the interaction or coupling between a human and a surrogate constructed to behave like a human. Inspired by the dynamic clamp used so productively in cellular neuroscience, the HDC allows a person to interact in real time with a virtual partner itself driven by well-established models of coordination dynamics. People coordinate hand movements with the visually observed movements of a virtual hand, the parameters of which depend on input from the subject's own movements. We demonstrate that HDC can be extended to cover a broad repertoire of human behavior, including rhythmic and discrete movements, adaptation to changes of pacing, and behavioral skill learning as specified by a virtual "teacher." We propose HDC as a general paradigm, best implemented when empirically verified theoretical or mathematical models have been developed in a particular scientific field. The HDC paradigm is powerful because it provides an opportunity to explore parameter ranges and perturbations that are not easily accessible in ordinary human interactions. The HDC not only enables to test the veracity of theoretical models, it also illuminates features that are not always apparent in real-time human social interactions and the brain correlates thereof. PMID:25114256

  3. Immune checkpoint inhibitors: a milestone in the treatment of melanoma.

    PubMed

    Wilden, Sophia M; Lang, Berenice M; Mohr, Peter; Grabbe, Stephan

    2016-07-01

    It has been known for decades that the immune system is able to detect and destroy tumor cells. In the past, this knowledge - mostly acquired through animal experiments - could not be used to benefit our patients, because immuno-oncological therapeutic approaches in humans had constantly failed over recent decades. With the exception of adjuvant interferon therapy, none of these approaches had found its way into everyday clinical practice, and only very few patients were able to enjoy long-term survival associated with good quality of life. With the advent of novel immunological approaches, the meaning of long-term survival as well as quality of life has been redefined for oncological patients. For the first time, a significant percentage of patients responds to treatment with immune checkpoint inhibitors, showing long-term remission and even cure. It has already become apparent that immunotherapy will in the future be one of the therapeutic mainstays in the treatment of metastatic melanoma as well as many other tumor types. The present review article presents the most important new treatment modalities, their mechanism of action, clinical data regarding treatment response, and adverse events to be expected. PMID:27373242

  4. Immunotherapy for Gastric Cancer: A Focus on Immune Checkpoints.

    PubMed

    Alsina, Maria; Moehler, Markus; Hierro, Cinta; Guardeño, Raquel; Tabernero, Josep

    2016-08-01

    Gastric cancer (GC) is a major world-wide health problem. It is the third leading cause of death from cancer. The treatment of advanced GC by chemotherapy has limited efficacy. The addition of some targeted therapies like trastuzumab and ramucirumab have added a modest benefit, but only in human epidermal growth factor receptor 2 (ERBB2 or HER2)-positive patients and in the second-line setting, respectively. The development of new and effective therapeutic strategies must consider the genetic complexity and heterogeneity of GC; prognostic and predictive biomarkers should be identified for clinical implementation. Immune deregulation has been associated with some GC subtypes, especially those that are associated with virus infection and those with a high mutational rate. Different mechanisms to prevent immunologic escape have been characterized during the last years; in particular the PD-1/PD-L1 inhibitors pembrolizumab, avelumab, durvalumab and atezolizumab have shown early sign of efficacy. Therefore, immunotherapeutic strategies may provide new opportunities for GC patients. This review will discuss (1) the main characteristics of GC treatment, (2) the immune response in GC, and (3) the current status of immune-related strategies in clinical development in GC patients, focusing on immune checkpoints therapies. PMID:26880697

  5. A Cdc2 dependent checkpoint maintains diploidy in Drosophila.

    PubMed

    Hayashi, S

    1996-04-01

    DNA replication in G2 does not normally occur due to the checkpoint control. To elucidate its mechanism, the functions of the escargot and Dmcdc2 genes of Drosophila were studied. When escargot function was eliminated, diploid imaginal cells that were arrested in G2 lost Cyclin A, a regulatory subunit of G2/M cdk, and entered an endocycle. escargot genetically interacted with Dmcdc2 which encodes a catalytic subunit of G2/M cdk. The mutant phenotypes of Dmcdc2 itself was similar to those of escargot: many diploid cells in imaginal discs, salivary glands and the central nervous system entered an endocycle and sometimes formed polytene chromosomes. Since mitotically quiescent abdominal histoblasts still required Dmcdc2 to remain diploid, the inhibitory activity of G2/M cdk on DNA replication appeared to be separable from its activity as the mitosis promoting factor. These results suggest that in G2, escargot is required to maintain a high level of G2/M cdk that actively inhibits the entry into S phase. PMID:8620832

  6. Identification of a novel EGF-sensitive cell cycle checkpoint

    SciTech Connect

    Walker, Francesca . E-mail: francesca.walker@ludwig.edu.au; Zhang Huihua; Burgess, Antony W.

    2007-02-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells.

  7. Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena

    PubMed Central

    Sandoval, Pamela Y.; Lee, Po-Hsuen; Meng, Xiangzhou; Kapler, Geoffrey M.

    2015-01-01

    The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress. PMID:26218270

  8. Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording.

    PubMed

    Harrison, Reid R; Kolb, Ilya; Kodandaramaiah, Suhasa B; Chubykin, Alexander A; Yang, Aimei; Bear, Mark F; Boyden, Edward S; Forest, Craig R

    2015-02-15

    Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation. PMID:25429119

  9. Delaying cord clamping until ventilation onset improves cardiovascular function at birth in preterm lambs

    PubMed Central

    Bhatt, Sasmira; Alison, Beth J; Wallace, Euan M; Crossley, Kelly J; Gill, Andrew W; Kluckow, Martin; te Pas, Arjan B; Morley, Colin J; Polglase, Graeme R; Hooper, Stuart B

    2013-01-01

    Delayed cord clamping improves circulatory stability in preterm infants at birth, but the underlying physiology is unclear. We investigated the effects of umbilical cord clamping, before and after ventilation onset, on cardiovascular function at birth. Prenatal surgery was performed on lambs (123 days) to implant catheters into the pulmonary and carotid arteries and probes to measure pulmonary (PBF), carotid (CaBF) and ductus arteriosus blood flows. Lambs were delivered at 126 ± 1 days and: (1) the umbilical cord was clamped at delivery and ventilation was delayed for about 2 min (Clamp 1st; n = 6), and (2) umbilical cord clamping was delayed for 3–4 min, until after ventilation was established (Vent 1st; n = 6). All lambs were subsequently ventilated for 30 min. In Clamp 1st lambs, cord clamping rapidly (within four heartbeats), but transiently, increased pulmonary and carotid arterial pressures (by ∼30%) and CaBF (from 30.2 ± 5.6 to 40.1 ± 4.6 ml min−1 kg−1), which then decreased again within 30–60 s. Following ventilation onset, these parameters rapidly increased again. In Clamp 1st lambs, cord clamping reduced heart rate (by ∼40%) and right ventricular output (RVO; from 114.6 ± 14.4 to 38.8 ± 9.7 ml min−1 kg−1), which were restored by ventilation. In Vent 1st lambs, cord clamping reduced RVO from 153.5 ± 3.8 to 119.2 ± 10.6 ml min−1 kg−1, did not affect heart rates and resulted in stable blood flows and pressures during transition. Delaying cord clamping for 3–4 min until after ventilation is established improves cardiovascular function by increasing pulmonary blood flow before the cord is clamped. As a result, cardiac output remains stable, leading to a smoother cardiovascular transition throughout the early newborn period. PMID:23401615

  10. Outward currents in voltage-clamped rat sympathetic neurones.

    PubMed Central

    Galvan, M; Sedlmeir, C

    1984-01-01

    Outward membrane currents were studied in neurones of the isolated rat superior cervical ganglion by using a two-micro-electrode or single-micro-electrode voltage-clamp technique. Under current clamp, depolarization elicited electrotonic potentials that displayed marked outward rectification. From negative resting potentials (-70 mV) a short latency, short duration outward rectification was observed. From more positive potentials (-40 mV) a longer latency persistent outward rectification could be demonstrated. Under voltage clamp, four distinct outward currents were observed: a delayed rectifier (IK); a transient outward current (IA); a Ca2+-activated current (IC) and the M-current (IM). The maximum amplitude of IK, IA and IC was 1-2 orders of magnitude greater than IM. Depolarizing from -40 mV to potentials more positive than -20 mV co-activated IK and IC, producing a characteristic N-shaped current voltage curve with a minimum at about +80 mV. Superfusion with Mn2+-containing solutions reduced outward current at all voltages and abolished the N-characteristic; the remaining current (IK) slowly inactivated (tau greater than 1 s). Raising [K+]o from 6 to 36 mmol/l reversed outward tail currents observed in normal solution. Addition of tetraethylammonium ions (1-3 mmol/l) strongly reduced the amplitude of IK and IC. IA was characterized by very rapid activation at potentials more positive than -60 mV and by fast and complete inactivation at potentials in the activation range. The amplitude of IA was dependent on [K+]o and was reduced by external 4-aminopyridine (1-3 mmol/l). The activation appeared to depend on the nature and concentration of divalent cations present in the superfusate. It is concluded that the soma membrane of rat sympathetic neurones, like many other vertebrate and invertebrate neurones, contains multiple populations of K+ channels. The possible functions of these in the control of ganglion cell excitability are discussed. PMID:6097667

  11. Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

    PubMed Central

    Pati, D; Keller, C; Groudine, M; Plon, S E

    1997-01-01

    A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast. PMID:9154802

  12. Triplex-stabilizing properties of parallel clamps carrying LNA derivatives at the Hoogsteen strand.

    PubMed

    Alvira, Margarita; Eritja, Ramon

    2010-02-01

    DNA Parallel clamps with a polypurine strand linked to a polypyrimidine Hoogsteen strand containing locked nucleic acids bind their corresponding polypyrimidine targets with high affinity. PMID:20151386

  13. Self-clamping arc light reflector for welding torch

    NASA Technical Reports Server (NTRS)

    Gordon, Stephen S. (Inventor)

    1987-01-01

    This invention is directed to a coaxial extending metal mirror reflector attached to the electrode housing or gas cup on a welding torch. An electric welding torch with an internal viewing system for robotic welding is provded with an annular arc light reflector to reflect light from the arc back onto the workpiece. The reflector has a vertical split or gap in its surrounding wall to permit the adjacent wall ends forming the split to be sprung open slightly to permit the reflector to be removed or slipped onto the torch housing or gas cup. The upper opening of the reflector is slightly smaller than the torch housing or gas cup and therefore, when placed on the torch housing or gas cup has that springiness to cause it to clamp tightly on the housing or gas cup. The split or gap also serves to permit the feed of weld wire through to the weld area,

  14. Non-Linear Vibration Characteristics of Clamped Laminated Shallow Shells

    NASA Astrophysics Data System (ADS)

    ABE, A.; KOBAYASHI, Y.; YAMADA, G.

    2000-07-01

    This paper examines non-linear free vibration characteristics of first and second vibration modes of laminated shallow shells with rigidly clamped edges. Non-linear equations of motion for the shells based on the first order shear deformation and classical shell theories are derived by means of Hamilton's principle. We apply Galerkin's procedure to the equations of motion in which eigenvectors for first and second modes of linear vibration obtained by the Ritz method are employed as trial functions. Then simultaneous non-linear ordinary differential equations are derived in terms of amplitudes of the first and second vibration modes. Backbone curves for the first and second vibration modes are solved numerically by the Gauss-Legendre integration method and the shooting method respectively. The effects of lamination sequences and transverse shear deformation on the behavior are discussed. It is also shown that the motion of the first vibration mode affects the response for the second vibration mode.

  15. Simple clamp pressure cell up to 30 kbar

    NASA Astrophysics Data System (ADS)

    Fujiwara, H.; Kadomatsu, H.; Tohma, K.

    1980-10-01

    A design of simple clamp type pressure apparatus utilized for measurements of magnetic susceptibility and electrical resistivity at low temperatures is presented. The cell consists of a WC piston and Be-Cu cylinder which was autofrettage-processed, and the sample cavity consists of a teflon bucket and an electrode plug. In a temperature range from 300 to 77 K, pressure was determined by a manganin gage calibrated by Bi I-II transition pressure at room temperature; the temperature dependence of pressure coefficient of manganin resistance was taken into account. As a result, the cell was capable of generating hydrostatic pressures up to 30 kbar at room temperature and at least up to 25 kbar at 4.2 K.

  16. Vibration of clamped right triangular thin plates: Accurate simplified solutions

    NASA Astrophysics Data System (ADS)

    Saliba, H. T.

    1994-12-01

    Use of the superposition techniques in the free-vibration analyses of thin plates, as they were first introduced by Gorman, has provided simple and effective solutions to a vast number of rectangular plate problems. The method has also been extended to nonrectangular plates such as triangular and trapezoidal plates. However, serious difficulties were encountered in some of these analyses. These difficulties were discussed and obviated in Salibra, 1990. This reference, however, dealt only with simple support conditions, leading to a simple, highly accurate, and very economical solution to the free-vibration problem of simply supported right angle triangular plates. The purpose of this Note is to show that the modified superposition method of Salibra, 1990 is also applicable to clamped-edge conditions. This is accomplished through the application of this method to the title problem.

  17. Whole-cell Patch-clamp Recordings in Brain Slices.

    PubMed

    Segev, Amir; Garcia-Oscos, Francisco; Kourrich, Saïd

    2016-01-01

    Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices, a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular, when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals

  18. The human dynamic clamp as a paradigm for social interaction

    PubMed Central

    Dumas, Guillaume; de Guzman, Gonzalo C.; Tognoli, Emmanuelle; Kelso, J. A. Scott

    2014-01-01

    Social neuroscience has called for new experimental paradigms aimed toward real-time interactions. A distinctive feature of interactions is mutual information exchange: One member of a pair changes in response to the other while simultaneously producing actions that alter the other. Combining mathematical and neurophysiological methods, we introduce a paradigm called the human dynamic clamp (HDC), to directly manipulate the interaction or coupling between a human and a surrogate constructed to behave like a human. Inspired by the dynamic clamp used so productively in cellular neuroscience, the HDC allows a person to interact in real time with a virtual partner itself driven by well-established models of coordination dynamics. People coordinate hand movements with the visually observed movements of a virtual hand, the parameters of which depend on input from the subject’s own movements. We demonstrate that HDC can be extended to cover a broad repertoire of human behavior, including rhythmic and discrete movements, adaptation to changes of pacing, and behavioral skill learning as specified by a virtual “teacher.” We propose HDC as a general paradigm, best implemented when empirically verified theoretical or mathematical models have been developed in a particular scientific field. The HDC paradigm is powerful because it provides an opportunity to explore parameter ranges and perturbations that are not easily accessible in ordinary human interactions. The HDC not only enables to test the veracity of theoretical models, it also illuminates features that are not always apparent in real-time human social interactions and the brain correlates thereof. PMID:25114256

  19. Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC)

    PubMed Central

    Tipton, Aaron R; Tipton, Michael; Yen, Tim

    2011-01-01

    The mitotic checkpoint is a specialized signal transduction pathway that monitors kinetochore-microtubule attachment to achieve faithful chromosome segregation. MAD2 is an evolutionarily conserved mitotic checkpoint protein that exists in open (O) and closed (C) conformations. The increase of intracellular C-MAD2 level during mitosis, through O→C-MAD2 conversion as catalyzed by unattached kinetochores, is a critical signaling event for the mitotic checkpoint. However, it remains controversial whether MAD2 is an integral component of the effector of the mitotic checkpoint—the mitotic checkpoint complex (MCC). We show here that endogenous human MCC is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2 and then selectively incorporating C-MAD2 during mitosis. Nevertheless, MCC can be induced to form in G1/S cells by expressing a C-conformation locked MAD2 mutant, indicating intracellular level of C-MAD2 as a major limiting factor for MCC assembly. In addition, a recombinant MCC containing C-MAD2 exhibits effective inhibitory activity toward APC/C isolated from mitotic HeLa cells, while a recombinant BUBR1:BUB3:CDC20 ternary complex is ineffective at comparable concentrations despite association with APC/C. These results help establish a direct connection between a major signal transducer (C-MAD2) and the potent effector (MCC) of the mitotic checkpoint, and provide novel insights into protein-protein interactions during assembly of a functional MCC. PMID:22037211

  20. Identifying security checkpoints locations to protect the major U.S. urban areas

    DOE PAGESBeta

    Cuellar-Hengartner, Leticia; Watkins, Daniel; Kubicek, Deborah A.; Rodriguez, Erick; Stroud, Phillip D.

    2015-09-01

    Transit networks are integral to the economy and to society, but at the same time they could allow terrorists to transport weapons of mass destruction into any city. Road networks are especially vulnerable, because they lack natural checkpoints unlike air networks that have security measures in place at all major airports. One approach to mitigate this risk is ensuring that every road route passes through at least one security checkpoint. Using the Ford-Fulkerson maximum-flow algorithm, we generate a minimum set of checkpoint locations within a ring-shaped buffer area surrounding the 50 largest US urban areas. We study how the numbermore » of checkpoints changes as we increase the buffer width to perform a cost-benefit analysis and to identify groups of cities that behave similarly. The set of required checkpoints is surprisingly small (10-124) despite the hundreds of thousands of road arcs in those areas, making it feasible to protect all major cities.« less

  1. Scalable Transparent Checkpoint-Restart of Global Address Space Applications on Virtual Machines over Infiniband

    SciTech Connect

    Villa, Oreste; Krishnamoorthy, Sriram; Nieplocha, Jaroslaw; Brown, David ML

    2009-05-18

    Checkpoint-Restart is one of the most used software approaches to achieve fault-tolerance in high-end clusters. While standard techniques typically focus on user-level solutions, the advent of virtualization software has enabled efficient and transparent system-level approaches. In this paper, we present a scalable transparent system-level solution to address fault-tolerance for applications based on global address space (GAS) programming models on Infiniband clusters. In addition to handling communication, the solution addresses transparent checkpoint of user-generated files. We exploit the support for the Infiniband network in the Xen virtual machine environment. We have developed a version of the Aggregate Remote Memory Copy Interface (ARMCI) one-sided communication library capable of suspending and resuming applications. We present efficient and scalable mechanisms to distribute checkpoint requests and to backup virtual machines memory images and file systems. We tested our approach in the context of NWChem, a popular computational chemistry suite. We demonstrated that NWChem can be executed, without any modification to the source code, on a virtualized 8-node cluster with very little overhead (below 3%). We observe that the total checkpoint time is limited by disk I/O. Finally, we measured system-size depended components of the checkpoint time on up to 1024 cores (128 nodes), demonstrating the scalability of our approach in medium/large-scale systems.

  2. Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

    PubMed Central

    Wan, Xiaohu; Cheerambathur, Dhanya; Gassmann, Reto; Suzuki, Aussie; Lawrimore, Josh; Desai, Arshad; Salmon, E.D.

    2013-01-01

    Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation. PMID:23979716

  3. Immune Checkpoint Blockade to Improve Tumor Infiltrating Lymphocytes for Adoptive Cell Therapy

    PubMed Central

    Kodumudi, Krithika N.; Siegel, Jessica; Weber, Amy M.; Scott, Ellen; Sarnaik, Amod A.; Pilon-Thomas, Shari

    2016-01-01

    Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function. PMID:27050669

  4. Immune Checkpoint inhibitors: An introduction to the next-generation cancer immunotherapy.

    PubMed

    Lee, Lucy; Gupta, Manish; Sahasranaman, Srikumar

    2016-02-01

    Activating the immune system to eliminate cancer cells and produce clinically relevant responses has been a long-standing goal of cancer research. Most promising therapeutic approaches to activating antitumor immunity include immune checkpoint inhibitors. Immune checkpoints are numerous inhibitory pathways hardwired in the immune system. They are critical for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage. Tumors regulate certain immune checkpoint pathways as a major mechanism of immune resistance. Because immune checkpoints are initiated by ligand-receptor interactions, blockade by antibodies provides a rational therapeutic approach. Although targeted therapies are clinically successful, they are often short-lived due to rapid development of resistance. Immunotherapies offer one notable advantage. Enhancing the cell-mediated immune response against tumor cells leads to generation of a long-term memory lymphocyte population patrolling the body to attack growth of any new tumor cells, thereby sustaining the therapeutic effects. Furthermore, early clinical results suggest that combination immunotherapies offer even more potent antitumor activity. This review is intended to provide an introduction to immune checkpoint inhibitors and discusses the scientific overview of cancer immunotherapy, mechanisms of the inhibitors, clinical pharmacology considerations, advances in combination therapies, and challenges in drug development. PMID:26183909

  5. Analysis of Bub3 spindle checkpoint function in Xenopus egg extracts.

    PubMed

    Campbell, Leigh; Hardwick, Kevin G

    2003-02-15

    The spindle checkpoint delays the onset of anaphase if there are any defects in the interactions between spindle microtubules and kinetochores. This checkpoint has been reconstituted in vitro in Xenopus egg extracts, and here we use antibodies to Xenopus Bub3 (XBub3) to show that this protein is required for both the activation and the maintenance of a spindle checkpoint arrest in egg extracts. We detect two forms of XBub3 in egg extracts and find both to be complexed with the XBub1 and XBubR1 kinases. Only one form of XBub3 is apparent in Xenopus tissue culture (XTC) cells, and localisation studies reveal that, unlike the Mad proteins, which are concentrated at the nuclear periphery, XBub3 is diffusely localised throughout the nucleus during interphase. During early prophase it is recruited to kinetochores, where it remains until chromosomes align at the metaphase plate. We discuss the mechanism by which our alpha-XBub3 antibodies interfere with the checkpoint and possible roles for XBub3 in the spindle checkpoint pathway. PMID:12538762

  6. Mouse Pachytene Checkpoint 2 (Trip13) Is Required for Completing Meiotic Recombination but Not Synapsis

    PubMed Central

    Li, Xin; Schimenti, John C

    2007-01-01

    In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice. PMID:17696610

  7. Centrosome-Dependent Bypass of the DNA Damage Checkpoint by the Polo Kinase Cdc5.

    PubMed

    Ratsima, Hery; Serrano, Diego; Pascariu, Mirela; D'Amours, Damien

    2016-02-16

    Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage. PMID:26832404

  8. Analysis of impactor residues in tray clamps from the Long Duration Exposure Facility. Part 1: Clamps from Bay A of the satellite

    NASA Technical Reports Server (NTRS)

    Zolensky, Michael E.; Bernhard, Ronald P.

    1993-01-01

    The Long Duration Exposure Facility (LDEF) was placed in low Earth orbit (LEO) in 1984 and was recovered 5.7 years later. The LDEF was host to several individual experiments that were specifically designed to characterize critical aspects of meteoroid and debris environment in LEO. It was realized from the beginning, however, that the most efficient use of the satellite would be to examine the entire surface of the Earth for impact features. In this regard, particular interest has centered on common exposed materials that faced in all LDEF pointing directions. Among the most important of these materials is the tray clamps. Therefore, in an effort to understand the nature of particulates in LEO and their effects on spacecraft hardware better, we are analyzing residues found in impact features on LDEF tray clamp surfaces. This catalog presents all data from clamps from Bay A of the LDEF. Subsequent catalogs will include clamps from succeeding bays of the satellite.

  9. Resistance Mechanisms to Immune-Checkpoint Blockade in Cancer: Tumor-Intrinsic and -Extrinsic Factors.

    PubMed

    Pitt, Jonathan M; Vétizou, Marie; Daillère, Romain; Roberti, María Paula; Yamazaki, Takahiro; Routy, Bertrand; Lepage, Patricia; Boneca, Ivo Gomperts; Chamaillard, Mathias; Kroemer, Guido; Zitvogel, Laurence

    2016-06-21

    Inhibition of immune regulatory checkpoints, such as CTLA-4 and the PD-1-PD-L1 axis, is at the forefront of immunotherapy for cancers of various histological types. However, such immunotherapies fail to control neoplasia in a significant proportion of patients. Here, we review how a range of cancer-cell-autonomous cues, tumor-microenvironmental factors, and host-related influences might account for the heterogeneous responses and failures often encountered during therapies using immune-checkpoint blockade. Furthermore, we describe the emerging evidence of how the strong interrelationship between the immune system and the host microbiota can determine responses to cancer therapies, and we introduce a concept by which prior or concomitant modulation of the gut microbiome could optimize therapeutic outcomes upon immune-checkpoint blockade. PMID:27332730

  10. Integrating S-phase Checkpoint Signaling with Trans-Lesion Synthesis of Bulky DNA Adducts

    PubMed Central

    Barkley, Laura R.; Ohmori, Haruo; Vaziri, Cyrus

    2011-01-01

    Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression. PMID:17652783

  11. Immune checkpoints aberrations and gastric cancer; assessment of prognostic value and evaluation of therapeutic potentials.

    PubMed

    Abdel-Rahman, Omar

    2016-01-01

    Till now, the prognosis of advanced gastric cancer looked dreadful; thus the search for newer better approaches for this lethal disease has been a strategic target for cancer researchers. In recent years, important immunobiological aspects of the tumor have been revealed with the subsequent proposal of immune check point inhibitors to target these pathways. Clinically, unselected use of immune checkpoint inhibitors in gastric cancer has been deemed with failure; in contrast to the clear success of more recent studies reporting on the use of pembrolizumab in molecularly selected patients. This may illustrate that any future use of immune checkpoint inhibitors in gastric cancer has to be molecularly supported. This review provides a delicate dissection of the clinical and immunobiological considerations underlying the use of these agents in addition to a thorough review of the published clinical data of immune checkpoint inhibitors in gastric cancer. PMID:26321371

  12. Beyond CTLA-4 and PD-1, the Generation Z of Negative Checkpoint Regulators

    PubMed Central

    Le Mercier, Isabelle; Lines, J. Louise; Noelle, Randolph J.

    2015-01-01

    In the last two years, clinical trials with blocking antibodies to the negative checkpoint regulators CTLA-4 and PD-1 have rekindled the hope for cancer immunotherapy. Multiple negative checkpoint regulators protect the host against autoimmune reactions but also restrict the ability of T cells to effectively attack tumors. Releasing these brakes has emerged as an exciting strategy for cancer treatment. Conversely, these pathways can be manipulated to achieve durable tolerance for treatment of autoimmune diseases and transplantation. In the future, treatment may involve combination therapy to target multiple cell types and stages of the adaptive immune responses. In this review, we describe the current knowledge on the recently discovered negative checkpoint regulators, future targets for immunotherapy. PMID:26347741

  13. Evaluation of checkpoint kinase targeting therapy in acute myeloid leukemia with complex karyotype.

    PubMed

    Didier, Christine; Demur, Cécile; Grimal, Fanny; Jullien, Denis; Manenti, Stéphane; Ducommun, Bernard

    2012-03-01

    There has been considerable interest in targeting cell cycle checkpoints particularly in emerging and alternative anticancer strategies. Here, we show that checkpoint abrogation by AZD7762, a potent and selective CHK1/2 kinase inhibitor enhances genotoxic treatment efficacy in immature KG1a leukemic cell line and in AML patient samples, particularly those with a complex karyotype, which display major genomic instability and chemoresistance. Furthermore, these data suggest that constitutive DNA-damage level might be useful markers to select AML patients susceptible to receive checkpoint inhibitor in combination with conventional chemotherapy. Moreover, this study demonstrates for the first time that AZD7762 inhibitor targets the CD34(+)CD38(-)CD123(+) primitive leukemic progenitors, which are responsible for the majority of AML patients relapse. Finally, CHK1 inhibition does not seem to affect clonogenic potential of normal hematopoietic progenitors. PMID:22258035

  14. Association between cellular radiosensitivity and G1/G2 checkpoint proficiencies in human cholangiocarcinoma cell lines.

    PubMed

    Hematulin, Arunee; Sagan, Daniel; Sawanyawisuth, Kanlayanee; Seubwai, Wunchana; Wongkham, Sopit

    2014-09-01

    Cholangiocarcinoma is a destructive malignancy with a poor prognosis and lack of effective medical treatment. Radiotherapy is an alternative treatment for patients with unresectable cholangiocarcinoma. However, there are limited data on the radiation responsiveness of individual cholangiocarcinoma cells, which is a key factor that influences radiation treatment outcome. In this study, we found that cholangiocarcinoma cell lines differ remarkably in their radiosensitivity. The variation of radiosensitivity of cholangiocarcinoma cells correlates with their p53 status and existing G1 and/or G2 checkpoint defects. We also demonstrated the potential of checkpoint kinase Chk1/2 inhibition on the enhancement of the radiosensitivity of cholangiocarcinoma cells. Thus, this study provides useful information for predicting radiation response and provides evidence for the enchantment of radiotherapeutic efficiency by targeting checkpoint kinase Chk1/2 in some subpopulations of cholangiocarcinoma patients. PMID:24969815

  15. Use of checkpoint-restart for complex HEP software on traditional architectures and Intel MIC

    NASA Astrophysics Data System (ADS)

    Arya, Kapil; Cooperman, Gene; Dotti, Andrea; Elmer, Peter

    2014-06-01

    Process checkpoint-restart is a technology with great potential for use in HEP workflows. Use cases include debugging, reducing the startup time of applications both in offline batch jobs and the High Level Trigger, permitting job preemption in environments where spare CPU cycles are being used opportunistically and efficient scheduling of a mix of multicore and single-threaded jobs. We report on tests of checkpoint-restart technology using CMS software, Geant4-MT (multi-threaded Geant4), and the DMTCP (Distributed Multithreaded Checkpointing) package. We analyze both single- and multi-threaded applications and test on both standard Intel x86 architectures and on Intel MIC. The tests with multi-threaded applications on Intel MIC are used to consider scalability and performance. These are considered an indicator of what the future may hold for many-core computing.

  16. A comparison of the performance and application differences between manual and automated patch-clamp techniques.

    PubMed

    Yajuan, Xiao; Xin, Liang; Zhiyuan, Li

    2012-01-01

    The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the interaction between a drug and a specific ion channel at different conformational states to understand the ion channel modulators' mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp systems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Therefore, further improvements in flexibility with regard to cell types and data quality will broaden the applications of the automated patch clamp systems in both academia and industry. PMID:23346269

  17. 30 CFR 75.605 - Clamping of trailing cables to equipment.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Clamping of trailing cables to equipment. 75.605 Section 75.605 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.605 Clamping of trailing cables to equipment....

  18. 30 CFR 75.605 - Clamping of trailing cables to equipment.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Clamping of trailing cables to equipment. 75.605 Section 75.605 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.605 Clamping of trailing cables to equipment....

  19. 30 CFR 75.605 - Clamping of trailing cables to equipment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Clamping of trailing cables to equipment. 75.605 Section 75.605 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.605 Clamping of trailing cables to equipment....

  20. 30 CFR 75.605 - Clamping of trailing cables to equipment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Clamping of trailing cables to equipment. 75.605 Section 75.605 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.605 Clamping of trailing cables to equipment....

  1. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori.

    PubMed

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; Kumari, Nilima; Gourinath, Samudrala

    2016-01-01

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp. PMID:27499105

  2. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori

    PubMed Central

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; kumari, Nilima; Gourinath, Samudrala

    2016-01-01

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp. PMID:27499105

  3. Chronic exposure to particulate chromate induces spindle assembly checkpoint bypass in human lung cells.

    PubMed

    Wise, Sandra S; Holmes, Amie L; Xie, Hong; Thompson, W Douglas; Wise, John Pierce

    2006-11-01

    One of the hallmarks of lung cancer is chromosome instability (CIN), particularly a tetraploid phenotype, which is normally prevented by the spindle assembly checkpoint. Hexavalent chromium Cr(VI) is an established human lung carcinogen, and Cr(VI) induces tumors at lung bifurcation sites where Cr(VI) particles impact and persist. However, the effects of Cr(VI) on the spindle assembly checkpoint are unknown and little is known about prolonged exposure to particulate Cr(VI). Accordingly, we investigated particulate Cr(VI)-induced bypass of the spindle assembly checkpoint after several days of exposure in WHTBF-6 cells. We found that lead chromate indeed induces spindle assembly checkpoint bypass in human lung cells, as 72, 96, and 120 h treatments with 0.5 or 1 microg/cm2 lead chromate induced significant increases in the percentage of cells with aberrant mitotic figures. For example, treatment with 1 microg/cm2 lead chromate for 96 h induced 11, 12.3, and 14% of cells with premature anaphase, centromere spreading and premature centromere division, respectively. In addition, we found a disruption of mitosis with more cells accumulating in anaphase; cells treated for 96 h increased from 18% in controls to 31% in cells treated with lead chromate. To confirm involvement of the spindle assembly checkpoint, Mad2 expression was used as a marker. Mad2 expression was decreased in cells exposed to chronic treatments of lead chromate, consistent with disruption of the checkpoint. We also found concentration- and time-dependent increases in tetraploid cells, which continued to grow and form colonies. When cells were treated with chronic lead alone there was no increase in aberrant mitotic cells or polyploidy; however, chronic exposure to a soluble Cr(VI) showed an increase in aberrant mitotic cells and polyploidy. These data suggest that lead chromate does induce CIN and may be one mechanism in the development of Cr(VI)-induced lung cancer. PMID:17112237

  4. Note: High-efficiency energy harvester using double-clamped piezoelectric beams

    SciTech Connect

    Zheng, Yingmei; Wu, Xuan; Parmar, Mitesh; Lee, Dong-weon

    2014-02-15

    In this study, an improvement in energy conversion efficiency has been reported, which is realized by using a double-clamped piezoelectric beam, based on uniaxial stretching strain. The buckling mechanism is applied to maximize axial stress in the double-clamped beam. The voltage generated by using the double-clamped piezoelectric beam is higher than that generated by using other conventional structures, such as bending cantilevers coated/sandwiched with piezoelectric film, which is proven both theoretically and experimentally. The power generation efficiency is enhanced by further optimizing the double-clamped structure. The optimized high-efficiency energy harvester utilizing double-clamped piezoelectric beams generates a peak output power of 80 μW, under an acceleration of 0.1g.

  5. Disassembly of mitotic checkpoint complexes by the joint action of the AAA-ATPase TRIP13 and p31comet

    PubMed Central

    Eytan, Esther; Wang, Kexi; Miniowitz-Shemtov, Shirly; Sitry-Shevah, Danielle; Kaisari, Sharon; Yen, Tim J.; Liu, Song-Tao; Hershko, Avram

    2014-01-01

    The mitotic (or spindle assembly) checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is active, a Mitotic Checkpoint Complex (MCC) assembles and inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C). MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3 associated with the APC/C activator Cdc20. When the checkpoint signal is turned off, MCC is disassembled and the checkpoint is inactivated. The mechanisms of the disassembly of MCC are not sufficiently understood. We have previously observed that ATP hydrolysis is required for the action of the Mad2-binding protein p31comet to disassemble MCC. We now show that HeLa cell extracts contain a factor that promotes ATP- and p31comet-dependent disassembly of a Cdc20–Mad2 subcomplex and identify it as Thyroid Receptor Interacting Protein 13 (TRIP13), an AAA-ATPase known to interact with p31comet. The joint action of TRIP13 and p31comet also promotes the release of Mad2 from MCC, participates in the complete disassembly of MCC and abrogates checkpoint inhibition of APC/C. We propose that TRIP13 plays centrally important roles in the sequence of events leading to MCC disassembly and checkpoint inactivation. PMID:25092294

  6. Immune Checkpoint Modulation in Colorectal Cancer: What's New and What to Expect

    PubMed Central

    Jacobs, Julie; Smits, Evelien; Lardon, Filip; Pauwels, Patrick; Deschoolmeester, Vanessa

    2015-01-01

    Colorectal cancer (CRC), as one of the most prevalent types of cancer worldwide, is still a leading cause of cancer related mortality. There is an urgent need for more efficient therapies in metastatic disease. Immunotherapy, a rapidly expanding field of oncology, is designed to boost the body's natural defenses to fight cancer. Of the many approaches currently under study to improve antitumor immune responses, immune checkpoint inhibition has thus far been proven to be the most effective. This review will outline the treatments that take advantage of our growing understanding of the role of the immune system in cancer, with a particular emphasis on immune checkpoint molecules, involved in CRC pathogenesis. PMID:26605342

  7. Clamp ultrastructure of the basal monogenean Chimaericola leptogaster (Leuckart, 1830) (Polyopisthocotylea: Chimaericolidae).

    PubMed

    Poddubnaya, Larisa G; Hemmingsen, Willy; Gibson, David I

    2014-11-01

    The ultrastructure of the haptoral clamps of the chimaericolid monogenean Chimaericola leptogaster, a basal polyopisthocotylean from the gills of a holocephalan fish, is described. These clamps are characterized by the presence of two muscle blocks interrupted mid-anteriorly and mid-posteriorly and different kinds of hard structures: a single median and paired lateral sclerites embedded in the clamp wall; six spine-like structures directed towards the clamp lumen; and electron dense surface structures along the internal surface of the anterior clamp lips and along the luminal surface of the tegument of the clamp lumen. The lateral sclerites are situated deep within muscular tissue and are closely bounded by radial myofibrils, possessing a uniform electron dense matrix within which are hollow areas of different sizes. The median sclerite occupies an area between the clamp wall myofibrils and the luminal epithelium, is surrounded by a basement lamina and is composed of a heterogeneous matrix comprising two different morphological layers related to variations in the type and concentration of fibrils. Four of the spine-like structures are extensions of the margins of the two spindle-like muscle blocks in the clamps, i.e. the two anterior and two posterior structures, and the two others are situated at the lateral constrictions of the left and right muscle blocks. The electron dense surface structures are derivations of the clamp tegument or, to be more precise, its outer, densely fibrous region. These results are discussed in relation to the evidence that the haptoral clamps of C. leptogaster are apparently ancient origin. PMID:25112214

  8. Defective DNA repair increases susceptibility to senescence through extension of Chk1-mediated G2 checkpoint activation

    PubMed Central

    Johmura, Yoshikazu; Yamashita, Emiri; Shimada, Midori; Nakanishi, Keiko; Nakanishi, Makoto

    2016-01-01

    Susceptibility to senescence caused by defective DNA repair is a major hallmark of progeroid syndrome patients, but molecular mechanisms of how defective DNA repair predisposes to senescence are largely unknown. We demonstrate here that suppression of DNA repair pathways extends the duration of Chk1-dependent G2 checkpoint activation and sensitizes cells to senescence through enhancement of mitosis skipping. Extension of G2 checkpoint activation by introduction of the TopBP1 activation domain and the nondegradable mutant of Claspin sensitizes cells to senescence. In contrast, a shortening of G2 checkpoint activation by expression of SIRT6 or depletion of OTUB2 reduces susceptibility to senescence. Fibroblasts from progeroid syndromes tested shows a correlation between an extension of G2 checkpoint activation and an increase in the susceptibility to senescence. These results suggest that extension of G2 checkpoint activation caused by defective DNA repair is critical for senescence predisposition in progeroid syndrome patients. PMID:27507734

  9. Defective DNA repair increases susceptibility to senescence through extension of Chk1-mediated G2 checkpoint activation.

    PubMed

    Johmura, Yoshikazu; Yamashita, Emiri; Shimada, Midori; Nakanishi, Keiko; Nakanishi, Makoto

    2016-01-01

    Susceptibility to senescence caused by defective DNA repair is a major hallmark of progeroid syndrome patients, but molecular mechanisms of how defective DNA repair predisposes to senescence are largely unknown. We demonstrate here that suppression of DNA repair pathways extends the duration of Chk1-dependent G2 checkpoint activation and sensitizes cells to senescence through enhancement of mitosis skipping. Extension of G2 checkpoint activation by introduction of the TopBP1 activation domain and the nondegradable mutant of Claspin sensitizes cells to senescence. In contrast, a shortening of G2 checkpoint activation by expression of SIRT6 or depletion of OTUB2 reduces susceptibility to senescence. Fibroblasts from progeroid syndromes tested shows a correlation between an extension of G2 checkpoint activation and an increase in the susceptibility to senescence. These results suggest that extension of G2 checkpoint activation caused by defective DNA repair is critical for senescence predisposition in progeroid syndrome patients. PMID:27507734

  10. Position clamping in a holographic counterpropagating optical trap.

    PubMed

    Bowman, Richard; Jesacher, Alexander; Thalhammer, Gregor; Gibson, Graham; Ritsch-Marte, Monika; Padgett, Miles

    2011-05-01

    Optical traps consisting of two counterpropagating, divergent beams of light allow relatively high forces to be exerted along the optical axis by turning off one beam, however the axial stiffness of the trap is generally low due to the lower numerical apertures typically used. Using a high speed spatial light modulator and CMOS camera, we demonstrate 3D servocontrol of a trapped particle, increasing the stiffness from 0.004 to 1.5 μN m(-1). This is achieved in the "macro-tweezers" geometry [Thalhammer, J. Opt. 13, 044024 (2011); Pitzek, Opt. Express 17, 19414 (2009)], which has a much larger field of view and working distance than single-beam tweezers due to its lower numerical aperture requirements. Using a 10×, 0.2 NA objective, active feedback produces a trap with similar effective stiffness to a conventional single-beam gradient trap, of order 1 μN m(-1) in 3D. Our control loop has a round-trip latency of 10 ms, leading to a resonance at 20 Hz. This is sufficient bandwidth to reduce the position fluctuations of a 10 μm bead due to Brownian motion by two orders of magnitude. This approach can be trivially extended to multiple particles, and we show three simultaneously position-clamped beads. PMID:21643247

  11. Dynamic Clamp Analysis of Synaptic Integration in Sympathetic Ganglia

    PubMed Central

    Horn, J. P.; Kullmann, P. H. M.

    2008-01-01

    Advances in modern neuroscience require the identification of principles that connect different levels of experimental analysis, from molecular mechanisms to explanations of cellular functions, then to circuits, and, ultimately, to systems and behavior. Here, we examine how synaptic organization of the sympathetic ganglia may enable them to function as use-dependent amplifiers of preganglionic activity and how the gain of this amplification may be modulated by metabotropic signaling mechanisms. The approach combines a general computational model of ganglionic integration together with experimental tests of the model using the dynamic clamp method. In these experiments, we recorded intracellularly from dissociated bullfrog sympathetic neurons and then mimicked physiological synapses with virtual computer-generated synapses. It thus became possible to analyze the synaptic gain by recording cellular responses to complex patterns of synaptic activity that normally arise in vivo from convergent nicotinic and muscarinic synapses. The results of these studies are significant because they illustrate how gain generated through ganglionic integration may contribute to the feedback control of important autonomic behaviors, in particular to the control of the blood pressure. We dedicate this paper to the memory of Professor Vladimir Skok, whose rich legacy in synaptic physiology helped establish the modern paradigm for connecting multiple levels of analysis in studies of the nervous system. PMID:19756262

  12. One-channel Cell-attached Patch-clamp Recording

    PubMed Central

    Maki, Bruce A.; Cummings, Kirstie A.; Paganelli, Meaghan A.; Murthy, Swetha E.; Popescu, Gabriela K.

    2014-01-01

    Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel’s conductance properties and the temporal sequence of open-closed conformations that make up the channel’s activation mechanism, thus helping to understand their functions in health and disease. PMID:24961614

  13. Stochastic modal models of slender uncertain curved beams preloaded through clamping

    NASA Astrophysics Data System (ADS)

    Avalos, Javier; Richter, Lanae A.; Wang, X. Q.; Murthy, Raghavendra; Mignolet, Marc P.

    2015-01-01

    This paper addresses the stochastic modeling of the stiffness matrix of slender uncertain curved beams that are forced fit into a clamped-clamped fixture designed for straight beams. Because of the misfit with the clamps, the final shape of the clamped-clamped beams is not straight and they are subjected to an axial preload. Both of these features are uncertain given the uncertainty on the initial, undeformed shape of the beams and affect significantly the stiffness matrix associated with small motions around the clamped-clamped configuration. A modal model using linear modes of the straight clamped-clamped beam with a randomized stiffness matrix is employed to characterize the linear dynamic behavior of the uncertain beams. This stiffness matrix is modeled using a mixed nonparametric-parametric stochastic model in which the nonparametric (maximum entropy) component is used to model the uncertainty in final shape while the preload is explicitly, parametrically included in the stiffness matrix representation. Finally, a maximum likelihood framework is proposed for the identification of the parameters associated with the uncertainty level and the mean model, or part thereof, using either natural frequencies only or natural frequencies and mode shape information of the beams around their final clamped-clamped state. To validate these concepts, three simulated, computational experiments were conducted within Nastran to produce populations of natural frequencies and mode shapes of uncertain slender curved beams after clamping. The three experiments differed from each other by the nature of the clamping condition in the in-plane direction. One experiment assumed a no-slip condition (zero in-plane displacement), another a perfect slip (no in-plane force), while the third one invoked friction. The first two experiments gave distributions of frequencies with similar features while the latter one yielded a strong deterministic dependence of the frequencies on each other, a

  14. Experimental Evaluation of the Static Strain on the Clamping Bolt in the Structure of a Bolt-Clamped Langevin-Type Transducer

    NASA Astrophysics Data System (ADS)

    Takahashi, Toru; Adachi, Kazunari

    2008-06-01

    Bolt-clamped Langevin-type transducers (BLTs) used in high-power ultrasonics are required to realize various characteristics depending on the technical field where they are used. Specifically for high amplitude operation, the static prestress or bearing stress imposed on the piezoelectric elements in the transducer by clamping should be large enough to compensate for their low tensile strength. The authors previously calculated prestress by the finite element method (FEM), but the numerical results have not been experimentally confirmed yet because of the difficulty of directly measuring of the prestress. In this study, the authors measured the strain on the surface of the clamping bolt using strain gauges pasted on it and compared the results with those of the numerical analysis by FEM in order to confirm the validity of the calculation. The measurement has been conducted for three BLTs of identical shape. The results of the measurement show reasonable agreement with those of the numerical analysis, and thus the authors have found that the measurement of the strain on the clamping bolt gives us a practical method for indirect evaluation of the prestress actually imposed on the piezoelectric elements that changes with the turning angle of the metal block in the clamping.

  15. The double transverse microvascular clamp: a new instrument for microsurgical anastomoses.

    PubMed

    El-Shazly, Mohamed

    2012-11-01

    Since the introduction of microvascular surgeries, the sophisticated ideas and techniques of tissue transplantations are continually advancing and searching for the best work conditions to present the best outcomes in these critical interferences. Every tissue transplant has its donor vessels, artery and vein, which should be anastomosed to recipient vessels. A new instrument, the double transverse microvascular clamp (DTMC), has been developed to be applied simultaneously, as one clamp, to both the artery and its accompanying vein. The transverse design of this clamp keeps the artery separate from its vein, allowing each anastomosis to be performed more easily. The limited clamp surface area minimizes the glazing and blurring effects. Applying only one clamp to the two vessels presents more work space and overcomes the crowdedness caused by the use of two single clamps. Using a DTMC on both the recipient and donor vessels provides optimal suture maneuverability and ideal work situation compared with the use of two double approximating clamps. We believe this DTMC would be a valuable addition to the microsurgical instruments market. PMID:22711201

  16. Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy*S⃞

    PubMed Central

    Laurence, Ted A.; Kwon, Youngeun; Johnson, Aaron; Hollars, Christopher W.; O'Donnell, Mike; Camarero, Julio A.; Barsky, Daniel

    2008-01-01

    DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III β subunit or β clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the β clamp diffusing along DNA is on the order of 10–14 m2/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the β clamp remains at the 3′ end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA. PMID:18556658

  17. Solutions for transients in arbitrarily branching cables: III. Voltage clamp problems.

    PubMed Central

    Major, G

    1993-01-01

    Branched cable voltage recording and voltage clamp analytical solutions derived in two previous papers are used to explore practical issues concerning voltage clamp. Single exponentials can be fitted reasonably well to the decay phase of clamped synaptic currents, although they contain many underlying components. The effective time constant depends on the fit interval. The smoothing effects on synaptic clamp currents of dendritic cables and series resistance are explored with a single cylinder + soma model, for inputs with different time courses. "Soma" and "cable" charging currents cannot be separated easily when the soma is much smaller than the dendrites. Subtractive soma capacitance compensation and series resistance compensation are discussed. In a hippocampal CA1 pyramidal neurone model, voltage control at most dendritic sites is extremely poor. Parameter dependencies are illustrated. The effects of series resistance compound those of dendritic cables and depend on the "effective capacitance" of the cell. Plausible combinations of parameters can cause order-of-magnitude distortions to clamp current waveform measures of simulated Schaeffer collateral inputs. These voltage clamp problems are unlikely to be solved by the use of switch clamp methods. PMID:8369450

  18. Immune checkpoint inhibitor-related hypophysitis and endocrine dysfunction: clinical review.

    PubMed

    Joshi, M N; Whitelaw, B C; Palomar, M T P; Wu, Y; Carroll, P V

    2016-09-01

    Immune checkpoint inhibitors are a new and effective class of cancer therapy, with ipilimumab being the most established drug in this category. The drugs' mechanism of action includes promoting the effector T cell response to tumours and therefore increased autoimmunity is a predictable side effect. The endocrine effects of these drugs include hypophysitis and thyroid dysfunction, with rare reports of adrenalitis. The overall incidence of hypophysitis with these medications is up to 9%. Primary thyroid dysfunction occurs in up to 15% of patients, with adrenalitis reported in approximately 1%. The mean onset of endocrine side effects is 9 weeks after initiation (range 5-36 weeks). Investigation and/or screening for hypophysitis requires biochemical and radiological assessment. Hypopituitarism is treated with replacement doses of deficient hormones. Since the endocrine effects of immune checkpoint inhibitors are classed as toxic adverse events, most authors recommend both discontinuation of the immune checkpoint inhibiting medication and 'high-dose' glucocorticoid treatment. However, this has been challenged by some authors, particularly if the endocrine effects can be managed (e.g. pituitary hormone deficiency), and the therapy is proving effective as an anticancer agent. This review describes the mechanism of action of immune checkpoint inhibitors and details the key clinical endocrine-related consequences of this novel class of immunotherapies. PMID:26998595

  19. Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes

    SciTech Connect

    Bhatt, Shweta; Gupta, Manoj; Khamaisi, Mogher; Martinez, Rachael; Gritsenko, Marina A.; Wagner, Bridget; Guye, Patrick; Busskamp, Volker; Shirakawa, Jun; Wu, Gongxiong; Liew, Chong Wee; Clauss, Therese RW; Valdez, Ivan; EL Ouaaman, Abdelfattah; Dirice, Ercument; Takatani, Tomozumi; Keenan, Hillary; Smith, Richard D.; Church, George; Weiss, Ron; Wagers, Amy J.; Qian, Weijun; King, George L.; Kulkami, Rohit N.

    2015-08-04

    Themechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D(disease durationR50 years) with severe (Medalist +C) or absent to mild complications (Medalist *C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist *C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage.WeproposemiR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.

  20. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

    PubMed

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-11-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  1. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

    PubMed Central

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A.; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-01-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)–dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  2. Genetic variation in the major mitotic checkpoint genes associated with chromosomal aberrations in healthy humans.

    PubMed

    Försti, Asta; Frank, Christoph; Smolkova, Bozena; Kazimirova, Alena; Barancokova, Magdalena; Vymetalkova, Veronika; Kroupa, Michal; Naccarati, Alessio; Vodickova, Ludmila; Buchancova, Janka; Dusinska, Maria; Musak, Ludovit; Vodicka, Pavel; Hemminki, Kari

    2016-10-01

    Non-specific chromosomal aberrations (CAs) are microscopically detected in about 1% of lymphocytes drawn from healthy persons. Causes of CAs in general population are not known but they may be related to risk of cancer. In view of the importance of the mitotic checkpoint machinery on maintaining chromosomal integrity we selected 9 variants in main checkpoint related genes (BUB1B, BUB3, MAD2L1, CENPF, ESPL1/separase, NEK2, PTTG1/securin, ZWILCH and ZWINT) for a genotyping study on samples from healthy individuals (N = 330 to 729) whose lymphocytes had an increased number of CAs compared to persons with a low number of CAs. Genetic variation in individual genes played a minor importance, consistent with the high conservation and selection pressure of the checkpoint system. However, gene pairs were significantly associated with CAs: PTTG1-ZWILCH and PTTG1-ZWINT. MAD2L1 and PTTG1 were the most common partners in any of the two-way interactions. The results suggest that interactions at the level of cohesin (PTTG1) and kinetochore function (ZWINT, ZWILCH and MAD2L1) contribute to the frequency of CAs, suggesting that gene variants at different checkpoint functions appeared to be required for the formation of CAs. PMID:27424524

  3. The mitosis-differentiation checkpoint, another guardian of the epidermal genome.

    PubMed

    Gandarillas, Alberto; Molinuevo, Rut; Freije, Ana; Alonso-Lecue, Pilar

    2015-01-01

    The role of p53, the original "guardian of the genome", in skin has remained elusive. We have explored p53 function in human epidermal cells and demonstrated the importance of a mitosis-differentiation checkpoint to suppress potentially precancerous cells. This model places epidermal endoreplication as an antioncogenic mechanism in the face of irreparable genetic alterations. PMID:27308487

  4. Spindle assembly checkpoint proteins regulate and monitor meiotic synapsis in C. elegans

    PubMed Central

    Bohr, Tisha; Nelson, Christian R.; Klee, Erin

    2015-01-01

    Homologue synapsis is required for meiotic chromosome segregation, but how synapsis is initiated between chromosomes is poorly understood. In Caenorhabditis elegans, synapsis and a checkpoint that monitors synapsis depend on pairing centers (PCs), cis-acting loci that interact with nuclear envelope proteins, such as SUN-1, to access cytoplasmic microtubules. Here, we report that spindle assembly checkpoint (SAC) components MAD-1, MAD-2, and BUB-3 are required to negatively regulate synapsis and promote the synapsis checkpoint response. Both of these roles are independent of a conserved component of the anaphase-promoting complex, indicating a unique role for these proteins in meiotic prophase. MAD-1 and MAD-2 localize to the periphery of meiotic nuclei and interact with SUN-1, suggesting a role at PCs. Consistent with this idea, MAD-1 and BUB-3 require full PC function to inhibit synapsis. We propose that SAC proteins monitor the stability of pairing, or tension, between homologues to regulate synapsis and elicit a checkpoint response. PMID:26483555

  5. Importance of immunopharmacogenomics in cancer treatment: Patient selection and monitoring for immune checkpoint antibodies.

    PubMed

    Choudhury, Noura; Nakamura, Yusuke

    2016-02-01

    In the last 5 years, immune checkpoint antibodies have become established as anticancer agents for various types of cancer. These antibody drugs, namely cytotoxic T-lymphocyte-associated antigen, programmed death-1, and programmed death ligand-1 antibodies, have revealed relatively high response rates, the ability to induce durable responses, and clinical efficacy in malignancies not previously thought to be susceptible to immune-based strategies. However, because of its unique mechanisms of activating the host immune system against cancer as well as expensive cost, immune checkpoint blockade faces novel challenges in selecting appropriate patient populations, monitoring clinical responses, and predicting immune adverse events. The development of objective criteria for selecting patient populations that are likely to have benefit from these therapies has been vigorously investigated but still remains unclear. In this review, we describe immune checkpoint inhibition-specific challenges with patient selection and monitoring, and focus on approaches to remedy these challenges. We also discuss applications of the emerging field of immunopharmacogenomics for guiding selection and monitoring for anti-immune checkpoint treatment. PMID:26678880

  6. The mitosis-differentiation checkpoint, another guardian of the epidermal genome

    PubMed Central

    Gandarillas, Alberto; Molinuevo, Rut; Freije, Ana; Alonso-Lecue, Pilar

    2015-01-01

    The role of p53, the original “guardian of the genome”, in skin has remained elusive. We have explored p53 function in human epidermal cells and demonstrated the importance of a mitosis-differentiation checkpoint to suppress potentially precancerous cells. This model places epidermal endoreplication as an antioncogenic mechanism in the face of irreparable genetic alterations.

  7. Structure and Substrate Recruitment of the Human Spindle Checkpoint Kinase Bub1

    SciTech Connect

    Kang, Jungseog; Yang, Maojun; Li, Bing; Qi, Wei; Zhang, Chao; Shokat, Kevan M.; Tomchick, Diana R.; Machius, Mischa; Yu, Hongtao

    2009-11-10

    In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

  8. Cdc18/CDC6 activates the Rad3-dependent checkpoint in the fission yeast.

    PubMed

    Fersht, Naomi; Hermand, Damien; Hayles, Jacqueline; Nurse, Paul

    2007-01-01

    A screen for genes that can ectopically activate a Rad3-dependent checkpoint block over mitosis in fission yeast has identified the DNA replication initiation factor cdc18 (known as CDC6 in other organisms). Either a stabilized form of Cdc18, the Cdc18-T6A phosphorylation mutant, or overexpression of wild type Cdc18, activate the Rad3-dependent S-M checkpoint in the apparent absence of detectable replication structures and gross DNA damage. This cell cycle block relies on the Rad checkpoint pathway and requires Chk1 phosphorylation and activation. Unexpectedly, Cdc18-T6A induces changes in the mobility of Chromosome III, affecting the size of a restriction fragment containing rDNA repeats and producing aberrant nucleolar structures. Recombination events within the rDNA appear to contribute at least in part to the cell cycle delay. We propose that an elevated level of Cdc18 activates the Rad3-dependent checkpoint either directly or indirectly, and additionally causes expansion of the rDNA repeats on Chromosome III. PMID:17690116

  9. [A data interface based on USB bus technology for full auto patch-clamp system].

    PubMed

    Liu, Youlin; Hu, Yang; Qu, Anlian

    2006-04-01

    A USB bus based data interface technology for full auto Patch-Clamp system is discussed in the article. The main controller is CY2131QC (Cypress) and the logic controller is EPM3256A (Altera). Optocouplers are used to get rid of the noise from the interface. It makes the installation of the Patch-Clamp system easier by using the USB bus, and is suitable for the new generation of the Patch-Clamp system with a high speed of 1M bytes/s. PMID:16706338

  10. Voltage clamping single cells in intact malpighian tubules of mosquitoes.

    PubMed

    Masia, R; Aneshansley, D; Nagel, W; Nachman, R J; Beyenbach, K W

    2000-10-01

    Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P < 0.001). In the presence of Ba(2+), leucokinin-VIII (1 microM) increased V(pc) to -101.8 mV (P < 0.001) and reduced R(pc) to 340.2 kOmega (P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652. 0 kOmega; apical membrane resistance, 340.2 kOmega; shunt resistance (R(sh)), 344.3 kOmega; transcellular resistance, 992.2 kOmega. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R(sh) (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways. PMID:10997925

  11. Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling.

    PubMed

    Sedgwick, Garry G; Larsen, Marie Sofie Yoo; Lischetti, Tiziana; Streicher, Werner; Jersie-Christensen, Rosa Rakownikow; Olsen, Jesper V; Nilsson, Jakob

    2016-01-01

    The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation. PMID:26986935

  12. Response of the G2-prophase checkpoint to genotoxic drugs in lymphocytes from healthy individuals.

    PubMed

    Pincheira, Juana; de la Torre, Consuelo; Rodríguez, Natalie; Valenzuela, Carlos Y

    2012-01-01

    We analyzed the in vitro effects of the anti-tumoral drugs doxorubicin, cytosine arabinoside and hydroxyurea on the G2-prophase checkpoint in lymphocytes from healthy individuals. At biologically equivalent concentrations, the induced DNA damage activated the corresponding checkpoint. Thus: i) there was a concentration-dependent delay of G2 time and an increase of both the total DNA lesions produced and repaired before metaphase and; ii) G2-checkpoint adaptation took place as chromosome aberrations (CAs) started to appear in the metaphase, indicating the presence of unrepaired double-strand breaks (DSBs) in the previous G2. The checkpoint ATM/ATR kinases are involved in DSB repair, since the recorded frequency of CAs increased when both kinases were caffeine-abrogated. In genotoxic-treated cells about three-fold higher repair activity was observed in relation to the endogenous background level of DNA lesions. The maximum rate of DNA repaired was 3.4 CAs/100 metaphases/hour, this rise being accompanied by a modest 1.3 fold lengthening of late G2 prophase timing. Because of mitotic chromosome condensation, no DSBs repair can take place until the G1 phase of the next cell cycle, when it occurs by DNA non-homologous end joining (NHEJ). Chromosomal rearrangements formed as a consequence of these error-prone DSB repairs ensure the development of genome instability through the DNA-fusion-bridge cycle. Hence, adaptation of the G2 checkpoint supports the appearance of secondary neoplasia in patients pretreated with genotoxic drugs. PMID:23096362

  13. 5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

    PubMed Central

    LUCIANI, M. GLORIA; CAMPREGHER, CHRISTOPH; FORTUNE, JOHN M.; KUNKEL, THOMAS A.; GASCHE, CHRISTOPH

    2007-01-01

    Background & Aims Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53−/−, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis. PMID:17241873

  14. Inhibition of Chk1 by the G[subscript 2] DNA damage checkpoint inhibitor isogranulatimide

    SciTech Connect

    Jiang, Xiuxian; Zhao, Baoguang; Britton, Robert; Lim, Lynette Y.; Leong, Dan; Sanghera, Jasbinder S.; Zhou, Bin-Bing S.; Piers, Edward; Andersen, Raymond J.; Roberge, Michel

    2008-07-01

    Inhibitors of the G{sub 2} DNA damage checkpoint can selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Isogranulatimide is a G{sub 2} checkpoint inhibitor containing a unique indole/maleimide/imidazole skeleton identified in a phenotypic cell-based screen; however, the mechanism of action of isogranulatimide is unknown. Using natural and synthetic isogranulatimide analogues, we show that the imide nitrogen and a basic nitrogen at position 14 or 15 in the imidazole ring are important for checkpoint inhibition. Isogranulatimide shows structural resemblance to the aglycon of UCN-01, a potent bisindolemaleimide inhibitor of protein kinase C{beta} (IC{sub 50}, 0.001 micromol/L) and of the checkpoint kinase Chk1 (IC{sub 50}, 0.007 micromol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC{sub 50}, 0.1 {micro}mol/L) but not protein kinase C{beta}. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3{beta} (IC{sub 50}, 0.5 {micro}mol/L). We determined the crystal structure of the Chk1 catalytic domain complexed with isogranulatimide. Like UCN-01, isogranulatimide binds in the ATP-binding pocket of Chk1 and hydrogen bonds with the backbone carbonyl oxygen of Glu{sup 85} and the amide nitrogen of Cys{sup 87}. Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu{sub 17}, causing a conformation change in the kinase glycine-rich loop that may contribute importantly to inhibition. The mechanism by which isogranulatimide inhibits Chk1 and its favorable kinase selectivity profile make it a promising candidate for modulating checkpoint responses in tumors for therapeutic benefit.

  15. Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition

    PubMed Central

    Shen, John Paul; Srivas, Rohith; Gross, Andrew; Li, Jianfeng; Jaehnig, Eric J.; Sun, Su Ming; Bojorquez-Gomez, Ana; Licon, Katherine; Sivaganesh, Vignesh; Xu, Jia L.; Klepper, Kristin; Yeerna, Huwate; Pekin, Daniel; Qiu, Chu Ping; van Attikum, Haico; Sobol, Robert W.; Ideker, Trey

    2015-01-01

    Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function. PMID:26437225

  16. Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition.

    PubMed

    Shen, John Paul; Srivas, Rohith; Gross, Andrew; Li, Jianfeng; Jaehnig, Eric J; Sun, Su Ming; Bojorquez-Gomez, Ana; Licon, Katherine; Sivaganesh, Vignesh; Xu, Jia L; Klepper, Kristin; Yeerna, Huwate; Pekin, Daniel; Qiu, Chu Ping; van Attikum, Haico; Sobol, Robert W; Ideker, Trey

    2015-11-01

    Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function. PMID:26437225

  17. Understanding checkpointing overheads on massive-scale systems : analysis of the IBM Blue Gene/P system.

    SciTech Connect

    Gupta, R.; Naik, H.; Beckman, P.

    2011-05-01

    Providing fault tolerance in high-end petascale systems, consisting of millions of hardware components and complex software stacks, is becoming an increasingly challenging task. Checkpointing continues to be the most prevalent technique for providing fault tolerance in such high-end systems. Considerable research has focussed on optimizing checkpointing; however, in practice, checkpointing still involves a high-cost overhead for users. In this paper, we study the checkpointing overhead seen by various applications running on leadership-class machines like the IBM Blue Gene/P at Argonne National Laboratory. In addition to studying popular applications, we design a methodology to help users understand and intelligently choose an optimal checkpointing frequency to reduce the overall checkpointing overhead incurred. In particular, we study the Grid-Based Projector-Augmented Wave application, the Carr-Parrinello Molecular Dynamics application, the Nek5000 computational fluid dynamics application and the Parallel Ocean Program application-and analyze their memory usage and possible checkpointing trends on 65,536 processors of the Blue Gene/P system.

  18. Substrate Clamping Effects on Irreversible Domain Wall Dynamics in Lead Zirconate Titanate Thin Films

    SciTech Connect

    Griggio, Flavio; Jesse, Stephen; Kumar, Amit; Ovchinnikov, Oleg S; Kim, H.; Jackson, T. N.; Damjanovic, Dragan; Kalinin, Sergei V; Trolier-Mckinstry, Susan E

    2012-01-01

    The role of long-range strain interactions on domain wall dynamics is explored through macroscopic and local measurements of nonlinear behavior in mechanically clamped and released polycrystalline lead zirconate-titanate (PZT) films. Released films show a dramatic change in the global dielectric nonlinearity and its frequency dependence as a function of mechanical clamping. Furthermore, we observe a transition from strong clustering of the nonlinear response for the clamped case to almost uniform nonlinearity for the released film. This behavior is ascribed to increased mobility of domain walls. These results suggest the dominant role of collective strain interactions mediated by the local and global mechanical boundary conditions on the domain wall dynamics. The work presented in this Letter demonstrates that measurements on clamped films may considerably underestimate the piezoelectric coefficients and coupling constants of released structures used in microelectromechanical systems, energy harvesting systems, and microrobots.

  19. Finite element analysis on factors influencing the clamping force in an electrostatic chuck

    NASA Astrophysics Data System (ADS)

    Xingkuo, Wang; Jia, Cheng; Kesheng, Wang; Yiyong, Yang; Yuchun, Sun; Minglu, Cao; Linhong, Ji

    2014-09-01

    As one of the core components of IC manufacturing equipment, the electrostatic chuck (ESC) has been widely applied in semiconductor processing such as etching, PVD and CVD. The clamping force of the ESC is one of the most important technical indicators. A multi-physics simulation software COMSOL is used to analyze the factors influencing the clamping force. The curves between the clamping force and the main parameters such as DC voltage, electrode thickness, electrode radius, dielectric thickness and helium gap are obtained. Moreover, the effects of these factors on the clamping force are investigated by means of orthogonal experiments. The results show that the factors can be ranked in order of voltage, electrode radius, helium gap and dielectric thickness according to their importance, which may offer certain reference for the design of ESCs.

  20. Acoustic plane waves incident on an oblique clamped panel in a rectangular duct

    NASA Technical Reports Server (NTRS)

    Unz, H.; Roskam, J.

    1980-01-01

    The theory of acoustic plane waves incident on an oblique clamped panel in a rectangular duct was developed from basic theoretical concepts. The coupling theory between the elastic vibrations of the panel (plate) and the oblique incident acoustic plane wave in infinite space was considered in detail, and was used for the oblique clamped panel in the rectangular duct. The partial differential equation which governs the vibrations of the clamped panel (plate) was modified by adding to it stiffness (spring) forces and damping forces. The Transmission Loss coefficient and the Noise Reduction coefficient for oblique incidence were defined and derived in detail. The resonance frequencies excited by the free vibrations of the oblique finite clamped panel (plate) were derived and calculated in detail for the present case.

  1. The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

    PubMed Central

    Rudokas, Michael W.; Varga, Zoltan; Schubert, Angela R.; Asaro, Alexandra B.; Silva, Jonathan R.

    2014-01-01

    The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule. PMID:24637712

  2. The TCF C-clamp DNA binding domain expands the Wnt transcriptome via alternative target recognition

    PubMed Central

    Hoverter, Nate P.; Zeller, Michael D.; McQuade, Miriam M.; Garibaldi, Angela; Busch, Anke; Selwan, Elizabeth M.; Hertel, Klemens J.; Baldi, Pierre; Waterman, Marian L.

    2014-01-01

    LEF/TCFs direct the final step in Wnt/β-catenin signalling by recruiting β-catenin to genes for activation of transcription. Ancient, non-vertebrate TCFs contain two DNA binding domains, a High Mobility Group box for recognition of the Wnt Response Element (WRE; 5′-CTTTGWWS-3′) and the C-clamp domain for recognition of the GC-rich Helper motif (5′-RCCGCC-3′). Two vertebrate TCFs (TCF-1/TCF7 and TCF-4/TCF7L2) use the C-clamp as an alternatively spliced domain to regulate cell-cycle progression, but how the C-clamp influences TCF binding and activity genome-wide is not known. Here, we used a doxycycline inducible system with ChIP-seq to assess how the C-clamp influences human TCF1 binding genome-wide. Metabolic pulse-labeling of nascent RNA with 4′Thiouridine was used with RNA-seq to connect binding to the Wnt transcriptome. We find that the C-clamp enables targeting to a greater number of gene loci for stronger occupancy and transcription regulation. The C-clamp uses Helper sites concurrently with WREs for gene targeting, but it also targets TCF1 to sites that do not have readily identifiable canonical WREs. The coupled ChIP-seq/4′Thiouridine-seq analysis identified new Wnt target genes, including additional regulators of cell proliferation. Thus, C-clamp containing isoforms of TCFs are potent transcriptional regulators with an expanded transcriptome directed by C-clamp-Helper site interactions. PMID:25414359

  3. Modeling CICR in rat ventricular myocytes: voltage clamp studies

    PubMed Central

    2010-01-01

    Background The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo). Methods The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ channels (trigger-channel and release-channel). It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo. Results Our model reproduces measured VC data published by several laboratories, and generates graded Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its refractory characteristics

  4. Comparative analysis of Drosophila and mammalian complexins as fusion clamps and facilitators of neurotransmitter release

    PubMed Central

    Cho, Richard W.; Song, Yun; Littleton, J. Troy

    2010-01-01

    The SNARE-binding protein complexin (Cpx) has been demonstrated to regulate synaptic vesicle fusion. Previous studies are consistent with Cpx functioning either as a synaptic vesicle fusion clamp to prevent premature exocytosis, or as a facilitator to directly stimulate release. Here we examined conserved roles of invertebrate and mammalian Cpx isoforms in the regulation of neurotransmitter release using the Drosophila neuromuscular junction as a model synapse. We find that SNARE binding by Cpx is required for its role as a fusion clamp. All four mammalian Cpx proteins (mCpx), which have been demonstrated to facilitate release, also function as fusion clamps when expressed in Drosophilacpx null mutants, though their clamping abilities varies between isoforms. Moreover, expression of mCpx I, II or III isoforms dramatically enhance evoked release compared to mCpx IV or Drosophila Cpx. Differences in the clamping and facilitating properties of complexin isoforms can be partially attributed to differences in the C-terminal membrane tethering domain. Our findings indicate that the function of complexins as fusion clamps and facilitators of fusion are conserved across evolution, and that these roles are genetically separable within an isoform and across different isoforms. PMID:20678575

  5. The sliding clamp tethers the endonuclease domain of MutL to DNA

    PubMed Central

    Pillon, Monica C.; Babu, Vignesh M. P.; Randall, Justin R.; Cai, Jiudou; Simmons, Lyle A.; Sutton, Mark D.; Guarné, Alba

    2015-01-01

    The sliding clamp enhances polymerase processivity and coordinates DNA replication with other critical DNA processing events including translesion synthesis, Okazaki fragment maturation and DNA repair. The relative binding affinity of the sliding clamp for its partners determines how these processes are orchestrated and is essential to ensure the correct processing of newly replicated DNA. However, while stable clamp interactions have been extensively studied; dynamic interactions mediated by the sliding clamp remain poorly understood. Here, we characterize the interaction between the bacterial sliding clamp (β-clamp) and one of its weak-binding partners, the DNA mismatch repair protein MutL. Disruption of this interaction causes a mild mutator phenotype in Escherichia coli, but completely abrogates mismatch repair activity in Bacillus subtilis. We stabilize the MutL-β interaction by engineering two cysteine residues at variable positions of the interface. Using disulfide bridge crosslinking, we have stabilized the E. coli and B. subtilis MutL-β complexes and have characterized their structures using small angle X-ray scattering. We find that the MutL-β interaction greatly stimulates the endonuclease activity of B. subtilis MutL and supports this activity even in the absence of the N-terminal region of the protein. PMID:26384423

  6. Mechanism of polymerase collision release from sliding clamps on the lagging strand

    PubMed Central

    Georgescu, Roxana E; Kurth, Isabel; Yao, Nina Y; Stewart, Jelena; Yurieva, Olga; O'Donnell, Mike

    2009-01-01

    Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the ‘collision release' model, the lagging strand polymerase collides with the 5′ terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5′ terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA-binding elements are involved, the τ subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The τ subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA. PMID:19696739

  7. The sliding clamp tethers the endonuclease domain of MutL to DNA.

    PubMed

    Pillon, Monica C; Babu, Vignesh M P; Randall, Justin R; Cai, Jiudou; Simmons, Lyle A; Sutton, Mark D; Guarné, Alba

    2015-12-15

    The sliding clamp enhances polymerase processivity and coordinates DNA replication with other critical DNA processing events including translesion synthesis, Okazaki fragment maturation and DNA repair. The relative binding affinity of the sliding clamp for its partners determines how these processes are orchestrated and is essential to ensure the correct processing of newly replicated DNA. However, while stable clamp interactions have been extensively studied; dynamic interactions mediated by the sliding clamp remain poorly understood. Here, we characterize the interaction between the bacterial sliding clamp (β-clamp) and one of its weak-binding partners, the DNA mismatch repair protein MutL. Disruption of this interaction causes a mild mutator phenotype in Escherichia coli, but completely abrogates mismatch repair activity in Bacillus subtilis. We stabilize the MutL-β interaction by engineering two cysteine residues at variable positions of the interface. Using disulfide bridge crosslinking, we have stabilized the E. coli and B. subtilis MutL-β complexes and have characterized their structures using small angle X-ray scattering. We find that the MutL-β interaction greatly stimulates the endonuclease activity of B. subtilis MutL and supports this activity even in the absence of the N-terminal region of the protein. PMID:26384423

  8. A novel monolithic piezoelectric actuated flexure-mechanism based wire clamp for microelectronic device packaging.

    PubMed

    Liang, Cunman; Wang, Fujun; Tian, Yanling; Zhao, Xingyu; Zhang, Hongjie; Cui, Liangyu; Zhang, Dawei; Ferreira, Placid

    2015-04-01

    A novel monolithic piezoelectric actuated wire clamp is presented in this paper to achieve fast, accurate, and robust microelectronic device packaging. The wire clamp has compact, flexure-based mechanical structure and light weight. To obtain large and robust jaw displacements and ensure parallel jaw grasping, a two-stage amplification composed of a homothetic bridge type mechanism and a parallelogram leverage mechanism was designed. Pseudo-rigid-body model and Lagrange approaches were employed to conduct the kinematic, static, and dynamic modeling of the wire clamp and optimization design was carried out. The displacement amplification ratio, maximum allowable stress, and natural frequency were calculated. Finite element analysis (FEA) was conducted to evaluate the characteristics of the wire clamp and wire electro discharge machining technique was utilized to fabricate the monolithic structure. Experimental tests were carried out to investigate the performance and the experimental results match well with the theoretical calculation and FEA. The amplification ratio of the clamp is 20.96 and the working mode frequency is 895 Hz. Step response test shows that the wire clamp has fast response and high accuracy and the motion resolution is 0.2 μm. High speed precision grasping operations of gold and copper wires were realized using the wire clamper. PMID:25933896

  9. A novel monolithic piezoelectric actuated flexure-mechanism based wire clamp for microelectronic device packaging

    NASA Astrophysics Data System (ADS)

    Liang, Cunman; Wang, Fujun; Tian, Yanling; Zhao, Xingyu; Zhang, Hongjie; Cui, Liangyu; Zhang, Dawei; Ferreira, Placid

    2015-04-01

    A novel monolithic piezoelectric actuated wire clamp is presented in this paper to achieve fast, accurate, and robust microelectronic device packaging. The wire clamp has compact, flexure-based mechanical structure and light weight. To obtain large and robust jaw displacements and ensure parallel jaw grasping, a two-stage amplification composed of a homothetic bridge type mechanism and a parallelogram leverage mechanism was designed. Pseudo-rigid-body model and Lagrange approaches were employed to conduct the kinematic, static, and dynamic modeling of the wire clamp and optimization design was carried out. The displacement amplification ratio, maximum allowable stress, and natural frequency were calculated. Finite element analysis (FEA) was conducted to evaluate the characteristics of the wire clamp and wire electro discharge machining technique was utilized to fabricate the monolithic structure. Experimental tests were carried out to investigate the performance and the experimental results match well with the theoretical calculation and FEA. The amplification ratio of the clamp is 20.96 and the working mode frequency is 895 Hz. Step response test shows that the wire clamp has fast response and high accuracy and the motion resolution is 0.2 μm. High speed precision grasping operations of gold and copper wires were realized using the wire clamper.

  10. Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes

    PubMed Central

    Das, Debasis; Krantz, Bryan A.

    2016-01-01

    Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins—protective antigen (PA), lethal factor (LF), and edema factor—translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity. PMID:27506790

  11. Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes.

    PubMed

    Das, Debasis; Krantz, Bryan A

    2016-08-23

    Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins-protective antigen (PA), lethal factor (LF), and edema factor-translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity. PMID:27506790

  12. Analysis of impactor residues in tray clamps from the Long Duration Exposure Facility. Part 2: Clamps from Bay B of the satellite

    NASA Technical Reports Server (NTRS)

    Bernhard, Ronald P.; Zolensky, Michael E.

    1994-01-01

    The Long Duration Exposure Facility (LDEF) was placed in low-Earth orbit (LEO) in 1984 and recovered 5.7 years later. The LDEF was host to several individual experiments specifically designed to characterize critical aspects of meteoroid and debris environment in LEO. However, it was realized from the beginning that the most efficient use of the satellite would be to examine the entire surface for impact features. In this regard, particular interest centered on common exposed materials that faced in all LDEF pointing directions. Among the most important of these materials was the tray clamps. Therefore, in an effort to better understand the nature of particulates in LEO and their effects on spacecraft hardware, residues found in impact features on LDEF tray clamp surfaces are being analyzed. This catalog presents all data from clamps from Bay B of the LDEF. NASA Technical Memorandum 104759 has cataloged impacts that occurred on Bay B (published March 1993). Subsequent catalogs will include clamps from succeeding bays of the satellite.

  13. Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells

    PubMed Central

    Kim, KiBem; Skora, Andrew D.; Li, Zhaobo; Liu, Qiang; Tam, Ada J.; Blosser, Richard L.; Diaz, Luis A.; Papadopoulos, Nickolas; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2014-01-01

    Impressive responses have been observed in patients treated with checkpoint inhibitory anti–programmed cell death-1 (PD-1) or anti–cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibodies. However, immunotherapy against poorly immunogenic cancers remains a challenge. Here we report that treatment with both anti–PD-1 and anti–CTLA-4 antibodies was unable to eradicate large, modestly immunogenic CT26 tumors or metastatic 4T1 tumors. Cotreatment with epigenetic-modulating drugs and checkpoint inhibitors markedly improved treatment outcomes, curing more than 80% of the tumor-bearing mice. Functional studies revealed that the primary targets of the epigenetic modulators were myeloid-derived suppressor cells (MDSCs). A PI3K inhibitor that reduced circulating MDSCs also eradicated 4T1 tumors in 80% of the mice when combined with immune checkpoint inhibitors. Thus, cancers resistant to immune checkpoint blockade can be cured by eliminating MDSCs. PMID:25071169

  14. Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

    SciTech Connect

    Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko; Nakanishi, Makoto; Yoshida, Takashi; Aiba, Hirofumi; Murakami, Hiroshi

    2009-10-23

    The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

  15. Modulating Mek1 kinase alters outcomes of meiotic recombination and the stringency of the recombination checkpoint response

    PubMed Central

    Hsin-Yen, Wu; Hsuan-Chung, Ho; Burgess, Sean M.

    2010-01-01

    Summary Background During meiosis, recombination between homologous chromosomes promotes their proper segregation. In budding yeast, programmed double-strand breaks (DSBs) promote recombination between homologs versus sister chromatids by dimerizing and activating Mek1, a chromosome axis-associated kinase. Mek1 is also a proposed effector kinase in the recombination checkpoint that arrests exit from pachytene in response to aberrant DNA/axis structures. Elucidating a role for Mek1 in the recombination checkpoint has been difficult since in mek1 loss-of-function mutants DSBs are rapidly repaired using a sister chromatid thereby bypassing formation of checkpoint-activating lesions. Here we tested the hypothesis that a MEK1 gain-of-function allele would enhance interhomolog bias and the recombination checkpoint response. Results When Mek1 activation was artificially maintained through GST-mediated dimerization, there was an enhanced skew toward interhomolog recombination and reduction of intersister events including multi-chromatid joint molecules. Increased interhomolog events were specifically repaired as noncrossovers rather than crossovers. Ectopic Mek1 dimerization was also sufficient to impose interhomolog bias in the absence of recombination checkpoint functions, thereby uncoupling these two processes. Finally, the stringency of the recombination checkpoint was enhanced in weak meiotic recombination mutants by blocking prophase exit in a subset of cells where arrest is not absolute. Conclusions We propose that Mek1 plays dual roles during meiotic prophase I by phosphorylating targets directly involved in the recombination checkpoint as well as targets involved in sister chromatid recombination. We discuss how regulation of pachytene exit by Mek1 or similar kinases could influence checkpoint stringency, which may differ among species and between sexes. PMID:20888230

  16. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    SciTech Connect

    Iida, Tetsushi; Iida, Naoko; Tsutsui, Yasuhiro; Yamao, Fumiaki; Kobayashi, Takehiko

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  17. Social and ethical checkpoints for bottom-up synthetic biology, or protocells.

    PubMed

    Bedau, Mark A; Parke, Emily C; Tangen, Uwe; Hantsche-Tangen, Brigitte

    2009-12-01

    An alternative to creating novel organisms through the traditional "top-down" approach to synthetic biology involves creating them from the "bottom up" by assembling them from non-living components; the products of this approach are called "protocells." In this paper we describe how bottom-up and top-down synthetic biology differ, review the current state of protocell research and development, and examine the unique ethical, social, and regulatory issues raised by bottom-up synthetic biology. Protocells have not yet been developed, but many expect this to happen within the next five to ten years. Accordingly, we identify six key checkpoints in protocell development at which particular attention should be given to specific ethical, social and regulatory issues concerning bottom-up synthetic biology, and make ten recommendations for responsible protocell science that are tied to the achievement of these checkpoints. PMID:19816801

  18. Targeting KIT on innate immune cells to enhance the antitumor activity of checkpoint inhibitors.

    PubMed

    Stahl, Maximilian; Gedrich, Richard; Peck, Ronald; LaVallee, Theresa; Eder, Joseph Paul

    2016-06-01

    Innate immune cells such as mast cells and myeloid-derived suppressor cells are key components of the tumor microenvironment. Recent evidence indicates that levels of myeloid-derived suppressor cells in melanoma patients are associated with poor survival to checkpoint inhibitors. This suggests that targeting both the innate and adaptive suppressive components of the immune system will maximize clinical benefit and elicit more durable responses in cancer patients. Preclinical data suggest that targeting signaling by the receptor tyrosine kinase KIT, particularly on mast cells, may modulate innate immune cell numbers and activity in tumors. Here, we review data highlighting the importance of the KIT signaling in regulating antitumor immune responses and the potential benefit of combining selective KIT inhibitors with immune checkpoint inhibitors. PMID:27349976

  19. Severe acute interstitial nephritis after combination immune-checkpoint inhibitor therapy for metastatic melanoma.

    PubMed

    Murakami, Naoka; Borges, Thiago J; Yamashita, Michifumi; Riella, Leonardo V

    2016-06-01

    Immune-checkpoint inhibitors are emerging as revolutionary drugs for certain malignancies. However, blocking the co-inhibitory signals may lead to immune-related adverse events, mainly in the spectrum of autoimmune diseases including colitis, endocrinopathies and nephritis. Here, we report a case of a 75-year-old man with metastatic malignant melanoma treated with a combination of nivolumab (anti-PD1-antibody) and ipilimumab (anti-CTLA-4 antibody) who developed systemic rash along with severe acute tubulointerstitial nephritis after two doses of combination therapy. Kidney biopsy and peripheral blood immune profile revealed highly proliferative and cytotoxic T cell features. Herein, we discuss the pathophysiology and management of immune checkpoint blockade-related adverse events. PMID:27274826

  20. PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease.

    PubMed

    Baruch, Kuti; Deczkowska, Aleksandra; Rosenzweig, Neta; Tsitsou-Kampeli, Afroditi; Sharif, Alaa Mohammad; Matcovitch-Natan, Orit; Kertser, Alexander; David, Eyal; Amit, Ido; Schwartz, Michal

    2016-02-01

    Systemic immune suppression may curtail the ability to mount the protective, cell-mediated immune responses that are needed for brain repair. By using mouse models of Alzheimer's disease (AD), we show that immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway evokes an interferon (IFN)-γ-dependent systemic immune response, which is followed by the recruitment of monocyte-derived macrophages to the brain. When induced in mice with established pathology, this immunological response leads to clearance of cerebral amyloid-β (Aβ) plaques and improved cognitive performance. Repeated treatment sessions were required to maintain a long-lasting beneficial effect on disease pathology. These findings suggest that immune checkpoints may be targeted therapeutically in AD. PMID:26779813

  1. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis.

    PubMed

    Dubin, Krista; Callahan, Margaret K; Ren, Boyu; Khanin, Raya; Viale, Agnes; Ling, Lilan; No, Daniel; Gobourne, Asia; Littmann, Eric; Huttenhower, Curtis; Pamer, Eric G; Wolchok, Jedd D

    2016-01-01

    The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is challenging, because clinically detectable inflammation and its treatment can alter the microbiota's composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-mediated colitis. Here we conduct a prospective study of patients with metastatic melanoma undergoing ipilimumab treatment and correlate the pre-inflammation faecal microbiota and microbiome composition with subsequent colitis development. We demonstrate that increased representation of bacteria belonging to the Bacteroidetes phylum is correlated with resistance to the development of checkpoint-blockade-induced colitis. Furthermore, a paucity of genetic pathways involved in polyamine transport and B vitamin biosynthesis is associated with an increased risk of colitis. Identification of these biomarkers may enable interventions to reduce the risk of inflammatory complications following cancer immunotherapy. PMID:26837003

  2. RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

    PubMed Central

    Davey, S; Beach, D

    1995-01-01

    We have identified a novel human gene by virtue of its ability to complement the rad1-1 checkpoint mutant of Schizosaccharomyces pombe. This gene, called RACH2, rescues the temperature-sensitive lethality of a rad1-1 wee1-50 double mutant of S. pombe. Expression of RACH2 in S. pombe rad1-1 strains partially restores UV resistance to the rad1-1 mutant strain. Expression of RACH2 in a rad1-1 cdc25-22 double mutant partially restores the dose-dependent delay in mitotic entry after irradiation that is lost in rad1-1 checkpoint-deficient mutants. Overexpression of RACH2 in human tissue culture cells induces apoptosis. Images PMID:8573795

  3. Cloud object store for checkpoints of high performance computing applications using decoupling middleware

    DOEpatents

    Bent, John M.; Faibish, Sorin; Grider, Gary

    2016-04-19

    Cloud object storage is enabled for checkpoints of high performance computing applications using a middleware process. A plurality of files, such as checkpoint files, generated by a plurality of processes in a parallel computing system are stored by obtaining said plurality of files from said parallel computing system; converting said plurality of files to objects using a log structured file system middleware process; and providing said objects for storage in a cloud object storage system. The plurality of processes may run, for example, on a plurality of compute nodes. The log structured file system middleware process may be embodied, for example, as a Parallel Log-Structured File System (PLFS). The log structured file system middleware process optionally executes on a burst buffer node.

  4. An Overview of the Spindle Assembly Checkpoint Status in Oral Cancer

    PubMed Central

    Teixeira, José Henrique; Silva, Patrícia Manuela; Reis, Rita Margarida; Moura, Inês Moranguinho; Marques, Sandra; Fonseca, Joana; Monteiro, Luís Silva; Bousbaa, Hassan

    2014-01-01

    Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the core components of SAC and its target Cdc20. PMID:24995269

  5. Severe acute interstitial nephritis after combination immune-checkpoint inhibitor therapy for metastatic melanoma

    PubMed Central

    Murakami, Naoka; Borges, Thiago J.; Yamashita, Michifumi; Riella, Leonardo V.

    2016-01-01

    Immune-checkpoint inhibitors are emerging as revolutionary drugs for certain malignancies. However, blocking the co-inhibitory signals may lead to immune-related adverse events, mainly in the spectrum of autoimmune diseases including colitis, endocrinopathies and nephritis. Here, we report a case of a 75-year-old man with metastatic malignant melanoma treated with a combination of nivolumab (anti-PD1-antibody) and ipilimumab (anti-CTLA-4 antibody) who developed systemic rash along with severe acute tubulointerstitial nephritis after two doses of combination therapy. Kidney biopsy and peripheral blood immune profile revealed highly proliferative and cytotoxic T cell features. Herein, we discuss the pathophysiology and management of immune checkpoint blockade-related adverse events. PMID:27274826

  6. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis

    PubMed Central

    Dubin, Krista; Callahan, Margaret K.; Ren, Boyu; Khanin, Raya; Viale, Agnes; Ling, Lilan; No, Daniel; Gobourne, Asia; Littmann, Eric; Huttenhower, Curtis; Pamer, Eric G.; Wolchok, Jedd D.

    2016-01-01

    The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is challenging, because clinically detectable inflammation and its treatment can alter the microbiota's composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-mediated colitis. Here we conduct a prospective study of patients with metastatic melanoma undergoing ipilimumab treatment and correlate the pre-inflammation faecal microbiota and microbiome composition with subsequent colitis development. We demonstrate that increased representation of bacteria belonging to the Bacteroidetes phylum is correlated with resistance to the development of checkpoint-blockade-induced colitis. Furthermore, a paucity of genetic pathways involved in polyamine transport and B vitamin biosynthesis is associated with an increased risk of colitis. Identification of these biomarkers may enable interventions to reduce the risk of inflammatory complications following cancer immunotherapy. PMID:26837003

  7. Essential functions for ID proteins at multiple checkpoints in natural killer T cell development

    PubMed Central

    Verykokakis, Mihalis; Krishnamoorthy, Veena; Iavarone, Antonio; Lasorella, Anna; Sigvardsson, Mikael; Kee, Barbara L.

    2013-01-01

    Invariant natural killer T (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. Here we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the post-selection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development. PMID:24244015

  8. The Next Immune-Checkpoint Inhibitors: PD-1/PD-L1 Blockade in Melanoma

    PubMed Central

    Mahoney, Kathleen M.; Freeman, Gordon J.; McDermott, David F.

    2015-01-01

    Purpose Blocking the interaction between the programmed cell death (PD)-1 protein and one of its ligands, PD-L1, has been reported to have impressive antitumor responses. Therapeutics targeting this pathway are currently in clinical trials. Pembrolizumab and nivolumab are the first of this anti-PD-1 pathway family of checkpoint inhibitors to gain accelerated approval from the US Food and Drug Administration (FDA) for the treatment of ipilimumab-refractory melanoma. Nivolumab has been associated with improved overall survival compared with dacarbazine in patients with previously untreated wild-type serine/threonine-protein kinase B-raf proto-oncogene BRAF melanoma. Although the most mature data are in the treatment of melanoma, the FDA has granted approval of nivolumab for squamous cell lung cancer and the breakthrough therapy designation to immune-checkpoint inhibitors for use in other cancers: nivolumab, an anti-PD-1 monoclonal antibody, for Hodgkin lymphoma, and MPDL-3280A, an anti-PD-L1 monoclonal antibody, for bladder cancer and non–small cell lung cancer. Here we review the literature on PD-1 and PD-L1 blockade and focus on the reported clinical studies that have included patients with melanoma. Methods PubMed was searched to identify relevant clinical studies of PD-1/PD-L1–targeted therapies in melanoma. A review of data from the current trials on clinicaltrial.gov was incorporated, as well as data presented in abstracts at the 2014 annual meeting of the American Society of Clinical Oncology, given the limited number of published clinical trials on this topic. Findings The anti-PD-1 and anti-PD-L1 agents have been reported to have impressive antitumor effects in several malignancies, including melanoma. The greatest clinical activity in unselected patients has been seen in melanoma. Tumor expression of PD-L1 is a suggestive, but inadequate, biomarker predictive of response to immune-checkpoint blockade. However, tumors expressing little or no PD-L1 are

  9. Immune Checkpoint Inhibitors: A New Opportunity in the Treatment of Ovarian Cancer?

    PubMed Central

    Mittica, Gloria; Genta, Sofia; Aglietta, Massimo; Valabrega, Giorgio

    2016-01-01

    Epithelial ovarian cancer (EOC) is the leading cause of death for gynecological cancer. The standard treatment for advanced stage is the combination of optimal debulking surgery and platinum-based chemotherapy. Nevertheless, recurrence is frequent (around 70%) and prognosis is globally poor. New therapeutic agents are needed to improve survival. Since EOC is strongly immunogenic, immune checkpoint inhibitors are under evaluation for their capacity to contrast the “turn off” signals expressed by the tumor to escape the immune system and usually responsible for self-tolerance maintenance. This article reviews the literature on anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), anti-PD-1, anti-PD-L1, and anti-PD-L2 antibodies in EOC and highlights their possible lines of development. Further studies are needed to better define the prognostic role of the immune checkpoint inhibitors, to identify predictors of response and the optimal clinical setting in EOC. PMID:27447625

  10. Negative immune checkpoints on T lymphocytes and their relevance to cancer immunotherapy.

    PubMed

    Śledzińska, Anna; Menger, Laurie; Bergerhoff, Katharina; Peggs, Karl S; Quezada, Sergio A

    2015-12-01

    The term 'inhibitory checkpoint' refers to the broad spectrum of co-receptors expressed by T cells that negatively regulate T cell activation thus playing a crucial role in maintaining peripheral self-tolerance. Co-inhibitory receptor ligands are highly expressed by a variety of malignancies allowing evasion of anti-tumour immunity. Recent studies demonstrate that manipulation of these co-inhibitory pathways can remove the immunological brakes that impede endogenous immune responses against tumours. Antibodies that block the interactions between co-inhibitory receptors and their ligands have delivered very promising clinical responses, as has been shown by recent successful trials targeting the CTLA-4 and PD-1 pathways. In this review, we discuss the mechanisms of action and expression pattern of co-inhibitory receptors on different T cells subsets, emphasising differences between CD4(+) and CD8(+) T cells. We also summarise recent clinical findings utilising immune checkpoint blockade. PMID:26578451

  11. Immunotherapy with checkpoint inhibitors for lung cancer: novel agents, biomarkers and paradigms.

    PubMed

    Monteiro, Inês-de-Paula; Califano, Raffaele; Mountzios, Giannis; de Mello, Ramon Andrade

    2016-02-01

    Despite recent advances, prognosis of patients with advanced lung cancer remains dismal. Owing to a better understanding of the interactions between immune system and tumor cells, immunotherapy has emerged as a promising therapeutic strategy. After the recent approval of nivolumab and the promising results with other immune checkpoint inhibitors, combination strategies are now subject of intensive research. Notwithstanding these successes, immunotherapy still holds significant drawbacks. As the target shifts from tumor cells to the tumor microenvironment, treatment paradigms are changing and several improvements are needed for optimal use in clinical practice. Robust biomarkers for patient selection and a reliable way of evaluating treatment response are high priorities. Herein we review current data on immune checkpoint inhibitors for lung cancer treatment. PMID:26776915

  12. Immune Checkpoint Inhibitors: A New Opportunity in the Treatment of Ovarian Cancer?

    PubMed

    Mittica, Gloria; Genta, Sofia; Aglietta, Massimo; Valabrega, Giorgio

    2016-01-01

    Epithelial ovarian cancer (EOC) is the leading cause of death for gynecological cancer. The standard treatment for advanced stage is the combination of optimal debulking surgery and platinum-based chemotherapy. Nevertheless, recurrence is frequent (around 70%) and prognosis is globally poor. New therapeutic agents are needed to improve survival. Since EOC is strongly immunogenic, immune checkpoint inhibitors are under evaluation for their capacity to contrast the "turn off" signals expressed by the tumor to escape the immune system and usually responsible for self-tolerance maintenance. This article reviews the literature on anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), anti-PD-1, anti-PD-L1, and anti-PD-L2 antibodies in EOC and highlights their possible lines of development. Further studies are needed to better define the prognostic role of the immune checkpoint inhibitors, to identify predictors of response and the optimal clinical setting in EOC. PMID:27447625

  13. ARID1A Deficiency Impairs the DNA Damage Checkpoint and Sensitizes Cells to PARP Inhibitors

    PubMed Central

    Shen, Jianfeng; Peng, Yang; Wei, Leizhen; Zhang, Wei; Yang, Lin; Lan, Li; Kapoor, Prabodh; Ju, Zhenlin; Mo, Qianxing; Shih, Ie-Ming; Uray, Ivan P.; Wu, Xiangwei; Brown, Powel H.; Shen, Xuetong; Mills, Gordon B.; Peng, Guang

    2015-01-01

    ARID1A, a chromatin remodeler of the SWI/SNF family, is a recently identified tumor suppressor that is mutated in a broad spectrum of human cancers. Thus, it is of fundamental clinical importance to understand its molecular functions and determine whether ARID1A deficiency can be exploited therapeutically. In this manuscript, we report a key function of ARID1A in regulating the DNA damage checkpoint. ARID1A is recruited to DNA double strand breaks (DSBs) via its interaction with the upstream DNA damage checkpoint kinase ATR. At the molecular level, ARID1A facilitates efficient processing of DSB to single strand ends, and sustains DNA damage signaling. Importantly, ARID1A deficiency sensitizes cancer cells to PARP inhibitors in vitro and in vivo providing a potential therapeutic strategy for patients with ARID1A-mutant tumors. PMID:26069190

  14. Intermittent selective clamping improves rat liver regeneration by attenuating oxidative and endoplasmic reticulum stress

    PubMed Central

    Ben Mosbah, I; Duval, H; Mbatchi, S-F; Ribault, C; Grandadam, S; Pajaud, J; Morel, F; Boudjema, K; Compagnon, P; Corlu, A

    2014-01-01

    Intermittent clamping of the portal trial is an effective method to avoid excessive blood loss during hepatic resection, but this procedure may cause ischemic damage to liver. Intermittent selective clamping of the lobes to be resected may represent a good alternative as it exposes the remnant liver only to the reperfusion stress. We compared the effect of intermittent total or selective clamping on hepatocellular injury and liver regeneration. Entire hepatic lobes or only lobes to be resected were subjected twice to 10 min of ischemia followed by 5 min of reperfusion before hepatectomy. We provided evidence that the effect of intermittent clamping can be damaging or beneficial depending to its mode of application. Although transaminase levels were similar in all groups, intermittent total clamping impaired liver regeneration and increased apoptosis. In contrast, intermittent selective clamping improved liver protein secretion and hepatocyte proliferation when compared with standard hepatectomy. This beneficial effect was linked to better adenosine-5′-triphosphate (ATP) recovery, nitric oxide production, antioxidant activities and endoplasmic reticulum adaptation leading to limit mitochondrial damage and apoptosis. Interestingly, transient and early chaperone inductions resulted in a controlled activation of the unfolded protein response concomitantly to endothelial nitric oxide synthase, extracellular signal-regulated kinase-1/2 (ERK1/2) and p38 MAPK activation that favors liver regeneration. Endoplasmic reticulum stress is a central target through which intermittent selective clamping exerts its cytoprotective effect and improves liver regeneration. This procedure could be applied as a powerful protective modality in the field of living donor liver transplantation and liver surgery. PMID:24603335

  15. Can selective arterial clamping with fluorescence imaging preserve kidney function during robotic partial nephrectomy?

    PubMed Central

    McClintock, Tyler R.; Bjurlin, Marc A.; Wysock, James S.; Borofsky, Michael S.; Marien, Tracy P.; Okoro, Chinonyerem; Stifelman, Michael D.

    2015-01-01

    Objectives To compare renal functional outcomes in robotic partial nephrectomy (RPN) with selective arterial clamping guided by near infrared fluorescence (NIRF) imaging to a matched cohort of patients who underwent RPN without selective arterial clamping and NIRF imaging. Methods From April 2011 to December 2012, NIRF imaging-enhanced RPN with selective clamping was utilized in 42 cases. Functional outcomes of successful cases were compared with a cohort of patients, matched by tumor size, preoperative eGFR, functional kidney status, age, sex, body mass index, and American Society of Anesthesiologists score, who underwent RPN without selective clamping and NIRF imaging. Results In matched-pair analysis, selective clamping with NIRF was associated with superior kidney function at discharge, as demonstrated by postoperative eGFR (78.2 vs 68.5 ml/min per 1.73m2; P=0.04), absolute reduction of eGFR (−2.5 vs −14.0 ml/min per 1.73m2; P<0.01) and percent change in eGFR (−1.9% vs −16.8%, P<0.01). Similar trends were noted at three month follow up but these differences became non-significant (P[eGFR]=0.07], P[absolute reduction of eGFR]=0.10, and P[percent change in eGFR]=0.07). In the selective clamping group, a total of four perioperative complications occurred in three patients, all of which were Clavien I-III. Conclusion Utilization of NIRF imaging was associated with improved short-term renal functional outcomes when compared to RPN without selective arterial clamping and NIRF imaging. With this effect attenuated at later follow-up, randomized prospective studies and long-term assessment of kidney-specific functional outcomes are needed to further assess the benefits of this technology. PMID:24909960

  16. Yielding of the clamped-wire system in the Ilizarov external fixator.

    PubMed

    Watson, M A; Matthias, K J; Maffulli, N; Hukins, D W L

    2003-01-01

    This study demonstrates that the clamped-wire system used to suspend bones within an Ilizarov external fixator yields when the perpendicular load exceeds 50 N per wire. Cyclic loading was applied to tensioned wires clamped within an Ilizarov ring component, with steadily increasing load amplitude. Wires were tested at four initial tension settings. The amount of energy lost within the clamped-wire system per load cycle was calculated for every test. The results showed that there was a consistent trend to increasing non-recoverable energy loss per load cycle when peak loads exceed 50 N for all initial tension settings. A finite element (FE) model replicating the experimental conditions was performed to investigate the levels of stress within the loaded wires. The FE model analyses showed that high stresses were generated in the wires close to the clamping sites, and that the stress levels could reasonably be expected to exceed the material yield stress when loaded to about 55 N, for all initial tension settings. The results show that material yield, accompanied by some wire slippage through the clamps, is responsible for system yield, in agreement with previous studies. Although the initial wire tension has an appreciable effect on the wire stiffness, it did not affect the elastic load range of the clamped-wire system. To prevent yield of the clamped-wire system in practice, the fixator should be assembled with sufficient wires to ensure that the load transmitted to each wire by the patient does not exceed 50 N. PMID:14558649

  17. Intermittent selective clamping improves rat liver regeneration by attenuating oxidative and endoplasmic reticulum stress.

    PubMed

    Ben Mosbah, I; Duval, H; Mbatchi, S-F; Ribault, C; Grandadam, S; Pajaud, J; Morel, F; Boudjema, K; Compagnon, P; Corlu, A

    2014-01-01

    Intermittent clamping of the portal trial is an effective method to avoid excessive blood loss during hepatic resection, but this procedure may cause ischemic damage to liver. Intermittent selective clamping of the lobes to be resected may represent a good alternative as it exposes the remnant liver only to the reperfusion stress. We compared the effect of intermittent total or selective clamping on hepatocellular injury and liver regeneration. Entire hepatic lobes or only lobes to be resected were subjected twice to 10 min of ischemia followed by 5 min of reperfusion before hepatectomy. We provided evidence that the effect of intermittent clamping can be damaging or beneficial depending to its mode of application. Although transaminase levels were similar in all groups, intermittent total clamping impaired liver regeneration and increased apoptosis. In contrast, intermittent selective clamping improved liver protein secretion and hepatocyte proliferation when compared with standard hepatectomy. This beneficial effect was linked to better adenosine-5'-triphosphate (ATP) recovery, nitric oxide production, antioxidant activities and endoplasmic reticulum adaptation leading to limit mitochondrial damage and apoptosis. Interestingly, transient and early chaperone inductions resulted in a controlled activation of the unfolded protein response concomitantly to endothelial nitric oxide synthase, extracellular signal-regulated kinase-1/2 (ERK1/2) and p38 MAPK activation that favors liver regeneration. Endoplasmic reticulum stress is a central target through which intermittent selective clamping exerts its cytoprotective effect and improves liver regeneration. This procedure could be applied as a powerful protective modality in the field of living donor liver transplantation and liver surgery. PMID:24603335

  18. A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

    PubMed

    Spoerri, Loredana; Brooks, Kelly; Chia, KeeMing; Grossman, Gavriel; Ellis, Jonathan J; Dahmer-Heath, Mareike; Škalamera, Dubravka; Pavey, Sandra; Burmeister, Bryan; Gabrielli, Brian

    2016-05-01

    Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected. PMID:26854966

  19. The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

    PubMed Central

    Negishi, Takahiro; Veis, Jiri; Hollenstein, David; Sekiya, Mizuho; Ammerer, Gustav

    2016-01-01

    The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation. PMID:26729465

  20. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

    PubMed Central

    Mahale, Sagar P.; Sharma, Amit; Mylavarapu, Sivaram V. S.

    2016-01-01

    The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC). However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division. PMID:27441562

  1. The Bub1–Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation

    PubMed Central

    Jia, Luying; Li, Bing; Yu, Hongtao

    2016-01-01

    The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy. A critical checkpoint inhibitor of APC/CCdc20 is the mitotic checkpoint complex (MCC). It is unclear whether MCC suffices to inhibit all cellular APC/C. Here we show that human checkpoint kinase Bub1 not only directly phosphorylates Cdc20, but also scaffolds Plk1-mediated phosphorylation of Cdc20. Phosphorylation of Cdc20 by Bub1–Plk1 inhibits APC/CCdc20 in vitro and is required for checkpoint signalling in human cells. Bub1–Plk1-dependent Cdc20 phosphorylation is regulated by upstream checkpoint signals and is dispensable for MCC assembly. A phospho-mimicking Cdc20 mutant restores nocodazole-induced mitotic arrest in cells depleted of Mad2 or BubR1. Thus, Bub1–Plk1-mediated phosphorylation of Cdc20 constitutes an APC/C-inhibitory mechanism that is parallel, but not redundant, to MCC formation. Both mechanisms are required to sustain mitotic arrest in response to spindle defects. PMID:26912231

  2. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    SciTech Connect

    Fukumoto, Yasunori Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  3. DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae.

    PubMed

    Wang, H; Elledge, S J

    1999-03-30

    In addition to DNA polymerase complexes, DNA replication requires the coordinate action of a series of proteins, including regulators Cdc28/Clb and Dbf4/Cdc7 kinases, Orcs, Mcms, Cdc6, Cdc45, and Dpb11. Of these, Dpb11, an essential BRCT repeat protein, has remained particularly enigmatic. The Schizosaccharomyces pombe homolog of DPB11, cut5, has been implicated in the DNA replication checkpoint as has the POL2 gene with which DPB11 genetically interacts. Here we describe a gene, DRC1, isolated as a dosage suppressor of dpb11-1. DRC1 is an essential cell cycle-regulated gene required for DNA replication. We show that both Dpb11 and Drc1 are required for the S-phase checkpoint, including the proper activation of the Rad53 kinase in response to DNA damage and replication blocks. Dpb11 is the second BRCT-repeat protein shown to control Rad53 function, possibly indicating a general function for this class of proteins. DRC1 and DPB11 show synthetic lethality and reciprocal dosage suppression. The Drc1 and Dpb11 proteins physically associate and function together to coordinate DNA replication and the cell cycle. PMID:10097122

  4. Regulation of zygotic genome activation and DNA damage checkpoint acquisition at the mid-blastula transition

    PubMed Central

    Zhang, Maomao; Kothari, Priyanka; Mullins, Mary; Lampson, Michael A.

    2014-01-01

    Following fertilization, oviparous embryos undergo rapid, mostly transcriptionally silent cleavage divisions until the mid-blastula transition (MBT), when large-scale developmental changes occur, including zygotic genome activation (ZGA) and cell cycle remodeling, via lengthening and checkpoint acquisition. Despite their concomitant appearance, whether these changes are co-regulated is unclear. Three models have been proposed to account for the timing of (ZGA). One model implicates a threshold nuclear to cytoplasmic (N:C) ratio, another stresses the importance cell cycle elongation, while the third model invokes a timer mechanism. We show that precocious Chk1 activity in pre-MBT zebrafish embryos elongates cleavage cycles, thereby slowing the increase in the N:C ratio. We find that cell cycle elongation does not lead to transcriptional activation. Rather, ZGA slows in parallel with the N:C ratio. We show further that the DNA damage checkpoint program is maternally supplied and independent of ZGA. Although pre-MBT embryos detect damage and activate Chk2 after induction of DNA double-strand breaks, the Chk1 arm of the DNA damage response is not activated, and the checkpoint is nonfunctional. Our results are consistent with the N:C ratio model for ZGA. Moreover, the ability of precocious Chk1 activity to delay pre-MBT cell cycles indicate that lack of Chk1 activity limits checkpoint function during cleavage cycles. We propose that Chk1 gain-of-function at the MBT underlies cell cycle remodeling, whereas ZGA is regulated independently by the N:C ratio. PMID:25558827

  5. A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

    PubMed Central

    Wang, Xiao Min; Zhai, Ye; Ferrell, James E.

    1997-01-01

    The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells. PMID:9128253

  6. Burden of Nonsynonymous Mutations among TCGA Cancers and Candidate Immune Checkpoint Inhibitor Responses.

    PubMed

    Colli, Leandro M; Machiela, Mitchell J; Myers, Timothy A; Jessop, Lea; Yu, Kai; Chanock, Stephen J

    2016-07-01

    Immune checkpoint inhibitor treatment represents a promising approach toward treating cancer and has been shown to be effective in a subset of melanoma, non-small cell lung cancer (NSCLC), and kidney cancers. Recent studies have suggested that the number of nonsynonymous mutations (NsM) can be used to select melanoma and NSCLC patients most likely to benefit from checkpoint inhibitor treatment. It is hypothesized that a higher burden of NsM generates novel epitopes and gene products, detected by the immune system as foreign. We conducted an assessment of NsM across 7,757 tumor samples drawn from 26 cancers sequenced in the Cancer Genome Atlas (TCGA) Project to estimate the subset of cancers (both types and fractions thereof) that fit the profile suggested for melanoma and NSCLC. An additional independent set of 613 tumors drawn from 5 cancers were analyzed for replication. An analysis of the receiver operating characteristic curves of published data on checkpoint inhibitor response in melanoma and NSCLC data estimates a cutoff of 192 NsM with 74% sensitivity and 59.3% specificity to discriminate potential clinical benefit. Across the 7,757 samples of TCGA, 16.2% displayed an NsM count that exceeded the threshold of 192. It is notable that more than 30% of bladder, colon, gastric, and endometrial cancers have NsM counts above 192, which was also confirmed in melanoma and NSCLC. Our data could inform the prioritization of tumor types (and subtypes) for possible clinical trials to investigate further indications for effective use of immune checkpoint inhibitors, particularly in adult cancers. Cancer Res; 76(13); 3767-72. ©2016 AACR. PMID:27197178

  7. Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model.

    PubMed

    Reardon, David A; Gokhale, Prafulla C; Klein, Sarah R; Ligon, Keith L; Rodig, Scott J; Ramkissoon, Shakti H; Jones, Kristen L; Conway, Amy Saur; Liao, Xiaoyun; Zhou, Jun; Wen, Patrick Y; Van Den Abbeele, Annick D; Hodi, F Stephen; Qin, Lei; Kohl, Nancy E; Sharpe, Arlene H; Dranoff, Glenn; Freeman, Gordon J

    2016-02-01

    Inhibition of immune checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and its ligand PD-L1, has demonstrated exciting and durable remissions across a spectrum of malignancies. Combinatorial regimens blocking complementary immune checkpoints further enhance the therapeutic benefit. The activity of these agents for patients with glioblastoma, a generally lethal primary brain tumor associated with significant systemic and microenvironmental immunosuppression, is not known. We therefore systematically evaluated the antitumor efficacy of murine antibodies targeting a broad panel of immune checkpoint molecules, including CTLA-4, PD-1, PD-L1, and PD-L2 when administered as single-agent therapy and in combinatorial regimens against an orthotopic, immunocompetent murine glioblastoma model. In these experiments, we observed long-term tumor-free survival following single-agent anti-PD-1, anti-PD-L1, or anti-CTLA-4 therapy in 50%, 20%, and 15% of treated animals, respectively. Combination therapy of anti-CTLA-4 plus anti-PD-1 cured 75% of the animals, even against advanced, later-stage tumors. In long-term survivors, tumor growth was not seen upon intracranial tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. Inhibitory immune checkpoint blockade quantitatively increased activated CD8(+) and natural killer cells and decreased suppressive immune cells in the tumor microenvironment and draining cervical lymph nodes. Our results support prioritizing the clinical evaluation of PD-1, PD-L1, and CTLA-4 single-agent targeted therapy as well as combination therapy of CTLA-4 plus PD-1 blockade for patients with glioblastoma. PMID:26546453

  8. CTLA-4 and PD-L1 Checkpoint Blockade Enhances Oncolytic Measles Virus Therapy

    PubMed Central

    Engeland, Christine E; Grossardt, Christian; Veinalde, Rūta; Bossow, Sascha; Lutz, Diana; Kaufmann, Johanna K; Shevchenko, Ivan; Umansky, Viktor; Nettelbeck, Dirk M; Weichert, Wilko; Jäger, Dirk; von Kalle, Christof; Ungerechts, Guy

    2014-01-01

    We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (> 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application. PMID:25156126

  9. Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint.

    PubMed

    Keaton, Mignon A; Lew, Daniel J

    2006-12-01

    The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial. PMID:17055334

  10. MicroRNA-224 Induces G1/S Checkpoint Release in Liver Cancer

    PubMed Central

    An, Fangmei; Olaru, Alexandru V.; Mezey, Esteban; Xie, Qing; Li, Ling; Piontek, Klaus B.; Selaru, Florin M.

    2015-01-01

    Profound changes in microRNA (miR) expression levels are frequently found in liver cancers compared to the normal liver. In this study, we evaluate the expression of miR-224 in human HCC and CCA, as well as its downstream targets and affected pathways. We show that miR-224 is upregulated in a large cohort of human CCA, similar to its upregulation in human HCC. For the purpose of studying the roles of miR-224 in HCC and CCA, we enforced miR-224 expression in cells. mRNA arrays followed by Ingenuity Pathway Analysis (IPA)-identified putative molecules and pathways downstream of miR-224. Phenotypically, we report that enforced expression of miR-224 increases the growth rate of normal cholangiocytes, CCA cell lines, and HCC cell lines. In addition, we identified, in an unbiased fashion, that one of the major biologic processes affected by miR-224 is Gap1 (G1) to Synthesis (S) transition checkpoint release. We next identified p21, p15, and CCNE1 as downstream targets of miR-224 and confirmed the coordinated downregulation results in the increased phosphorylation of Retinoblastoma (Rb) with resulting G1/S checkpoint release. Our data suggest that miR-224 is a master regulator of cell cycle progression, and that its overexpression results in G1/S checkpoint release followed by accelerated cell growth. PMID:26343737

  11. Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function

    PubMed Central

    Murfuni, Ivana; Basile, Giorgia; Subramanyam, Shyamal; Malacaria, Eva; Bignami, Margherita; Spies, Maria; Franchitto, Annapaola; Pichierri, Pietro

    2013-01-01

    In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress. PMID:24204313

  12. Molecular basis of the essential s phase function of the rad53 checkpoint kinase.

    PubMed

    Hoch, Nicolas C; Chen, Eric S-W; Buckland, Robert; Wang, Shun-Chung; Fazio, Alessandro; Hammet, Andrew; Pellicioli, Achille; Chabes, Andrei; Tsai, Ming-Daw; Heierhorst, Jörg

    2013-08-01

    The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed. PMID:23754745

  13. Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

    PubMed Central

    Hoch, Nicolas C.; Chen, Eric S.-W.; Buckland, Robert; Wang, Shun-Chung; Fazio, Alessandro; Hammet, Andrew; Pellicioli, Achille; Chabes, Andrei; Tsai, Ming-Daw

    2013-01-01

    The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed. PMID:23754745

  14. Orchestrating immune check-point blockade for cancer immunotherapy in combinations.

    PubMed

    Perez-Gracia, Jose Luis; Labiano, Sara; Rodriguez-Ruiz, Maria E; Sanmamed, Miguel F; Melero, Ignacio

    2014-04-01

    Inhibitory receptors on immune system cells respond to membrane-bound and soluble ligands to abort or mitigate the intensity of immune responses by raising thresholds of activation, halting proliferation, favoring apoptosis or inhibiting/deviating effector function differentiation. Such evolutionarily selected inhibitory mechanisms are termed check-points and therefore check-point inhibitors empower any ongoing anti-cancer immune response that might have been too weak or exhausted. Monoclonal antibodies (mAb) interfering with CTLA-4-CD80/86, PD-1 - PD-L1, TIM-3-GAL9 and LAG3-MHC-II belong to this category of check-point inhibitors. The anti-CTLA-4 mAb ipilimumab has been approved for metastatic melanoma. Anti-PD-1 and anti-PD-L1 mAbs have shown extremely encouraging clinical activity. The potential of combination strategies with these agents has recently been highlighted by clinical observations on CTLA-4+PD-1 combined blockade in melanoma patients. PMID:24485523

  15. p53 and Translation Attenuation Regulate Distinct Cell Cycle Checkpoints during Endoplasmic Reticulum (ER) Stress*

    PubMed Central

    Thomas, Sally E.; Malzer, Elke; Ordóñez, Adriana; Dalton, Lucy E.; van ′t Wout, Emily F. A.; Liniker, Elizabeth; Crowther, Damian C.; Lomas, David A.; Marciniak, Stefan J.

    2013-01-01

    Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G1 phase, although recent studies have identified a novel ER stress G2 checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G2 delay involving CHK1 and a later induction of G1 arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G1 checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G2 progression prior to ultimate G1 arrest. PMID:23341460

  16. RNF8 Transduces the DNA-Damage Signal Via Histone Ubiquitylation And Checkpoint Protein Assembly

    SciTech Connect

    Huen, M.S.Y.; Grant, R.; Manke, I.; Minn, K.; Yu, X.; Yaffe, M.B.; Chen, J.

    2009-06-01

    DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 {angstrom}. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

  17. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints

    PubMed Central

    Koyama, Shohei; Akbay, Esra A.; Li, Yvonne Y.; Herter-Sprie, Grit S.; Buczkowski, Kevin A.; Richards, William G.; Gandhi, Leena; Redig, Amanda J.; Rodig, Scott J.; Asahina, Hajime; Jones, Robert E.; Kulkarni, Meghana M.; Kuraguchi, Mari; Palakurthi, Sangeetha; Fecci, Peter E.; Johnson, Bruce E.; Janne, Pasi A.; Engelman, Jeffrey A.; Gangadharan, Sidharta P.; Costa, Daniel B.; Freeman, Gordon J.; Bueno, Raphael; Hodi, F. Stephen; Dranoff, Glenn; Wong, Kwok-Kin; Hammerman, Peter S.

    2016-01-01

    Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade. PMID:26883990

  18. Autoimmune Bullous Skin Disorders with Immune Checkpoint Inhibitors Targeting PD-1 and PD-L1.

    PubMed

    Naidoo, Jarushka; Schindler, Katja; Querfeld, Christiane; Busam, Klaus; Cunningham, Jane; Page, David B; Postow, Michael A; Weinstein, Alyona; Lucas, Anna Skripnik; Ciccolini, Kathryn T; Quigley, Elizabeth A; Lesokhin, Alexander M; Paik, Paul K; Chaft, Jamie E; Segal, Neil H; D'Angelo, Sandra P; Dickson, Mark A; Wolchok, Jedd D; Lacouture, Mario E

    2016-05-01

    Monoclonal antibodies (mAb) targeting immune checkpoint pathways such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) may confer durable disease control in several malignancies. In some patients, immune checkpoint mAbs cause cutaneous immune-related adverse events. Although the most commonly reported cutaneous toxicities are mild, a subset may persist despite therapy and can lead to severe or life-threatening toxicity. Autoimmune blistering disorders are not commonly associated with immune checkpoint mAb therapy. We report a case series of patients who developed bullous pemphigoid (BP), an autoimmune process classically attributed to pathologic autoantibody formation and complement deposition. Three patients were identified. Two patients developed BP while receiving the anti-PD-1 mAb nivolumab, and one while receiving the anti-PD-L1 mAb durvalumab. The clinicopathologic features of each patient and rash, and corresponding radiologic findings at the development of the rash and after its treatment, are described. Patients receiving an anti-PD-1/PD-L1 mAb may develop immune-related BP. This may be related to both T-cell- and B-cell-mediated responses. Referral to a dermatologist for accurate diagnosis and management is recommended. Cancer Immunol Res; 4(5); 383-9. ©2016 AACR. PMID:26928461

  19. The Mitotic Checkpoint Gene, SIL is Regulated by E2F1

    PubMed Central

    Erez, Ayelet; Chaussepied, Marie; Tina, Colaizzo-Anas; Aplan, Peter; Ginsberg, Doron; Izraeli, Shai

    2009-01-01

    The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in-vivo. Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F. PMID:18649360

  20. The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells.

    PubMed

    Carballo, Jesús A; Pincheira, Juana; de la Torre, Consuelo

    2006-01-01

    Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability. PMID:16874408

  1. A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis

    PubMed Central

    Daley, William P.; Kohn, Joshua M.; Larsen, Melinda

    2011-01-01

    Cleft formation is the initial step of branching morphogenesis in many organs. We previously demonstrated that ROCK 1 regulates a non-muscle myosin II-dependent mechanochemical checkpoint to transition initiated clefts to progressing clefts in developing submandibular salivary glands. Here, we report that ROCK-mediated integrin activation and subsequent formation of focal adhesion complexes comprise this mechanochemical checkpoint. Inhibition of ROCK1 and non-muscle myosin II activity decreased integrin β1 activation in the cleft region and interfered with localization and activation of focal adhesion complex proteins, such as focal adhesion kinase (FAK). Inhibition of FAK activity also prevented cleft progression, by disrupting recruitment of the focal adhesion proteins talin and vinculin and subsequent fibronectin assembly in the cleft region while decreasing ERK1/2 activation. These results demonstrate that inside-out integrin signaling leading to a localized recruitment of active FAK-containing focal adhesion protein complexes generates a mechanochemical checkpoint that facilitates progression of branching morphogenesis. PMID:22016182

  2. Checkpoint kinase 1 inhibitors as targeted molecular agents for clear cell carcinoma of the ovary

    PubMed Central

    KOBAYASHI, HIROSHI; SHIGETOMI, HIROSHI; YOSHIMOTO, CHIHARU

    2015-01-01

    In clear cell carcinoma of the ovary, chemoresistance frequently results in treatment failure. The present study aimed to review the potential association of transcription factor hepatocyte nuclear factor (HNF)-1β with cell cycle checkpoint machinery, as a mechanism for chemoresistance. The English-language literature on the subject was reviewed to identify genomic alterations and aberrant molecular pathways interacting with chemoresistance in clear cell carcinoma. Oxidative stress induced by repeated hemorrhage induces greater susceptibility of endometriotic cells to DNA damage, and subsequent malignant transformation results in endometriosis-associated ovarian cancer. Molecular changes, including those in HNF-1β and checkpoint kinase 1 (Chk1), may be a manifestation of essential alterations in cell cycle regulation, detoxification and chemoresistance in clear cell carcinoma. Chk1 is a critical signal transducer in the cell cycle checkpoint machinery. DNA damage, in turn, increases persistent phosphorylation of Chk1 and induction of G2/M phase cell cycle arrest in cells overexpressing HNF-1β. HNF-1β deletion induces apoptosis, suggesting that enhanced levels of HNF-1β may be associated with chemoresistance. Targeted therapy with Chk1 inhibitors may be explored as a potential treatment modality for patients with clear cell carcinoma. This provides a novel direction for combination therapy, including targeting of Chk1, which may overcome drug resistance and improve treatment efficacy. PMID:26622535

  3. The expression of DNA damage checkpoint proteins and prognostic implication in metastatic brain tumors.

    PubMed

    Seol, Ho Jun; Yoo, Hae Yong; Jin, Juyoun; Joo, Kyeung Min; Kim, Hyeong-Seok; Yoon, Su Jin; Choi, Seung Ho; Kim, Yonghyun; Pyo, Hong Ryull; Lim, Do-Hoon; Kim, Wook; Um, Hong-Duck; Kim, Jong Hyun; Lee, Jung-Ii; Nam, Do-Hyun

    2011-01-01

    The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, DNA damage checkpoint signaling pathway activation after irradiation has received increasing attention. The association between the expression levels and survival outcome was evaluated to find possible therapeutic targets in brain metastasis. Radiosensitivity of human non-small cell lung cancer cell lines was determined by checking their viability after treatment with varying doses of ionizing radiation (IR). The expression of DNA checkpoint proteins was analyzed by Western blots and immunohistochemistry. On the basis of the clinical data for the patients, the association between the expression of the components and patients' survival was investigated. The expression levels of TopBP1 and phosphorylated Chk1 (P-Chk1) protein were higher in radioresistant lung cancer cell lines compared to radiosensitive cell lines. We previously assessed radiation survival of lung cancer cell lines after treating them with Chk1 inhibitor, AZD7762. AZD7762 significantly sensitized both radioresistant and radiosensitive cells to IR. We also observed a strong inverse relationship between progression-free survival (PFS) and expression level of P-Chk1 and TopBP1. This study, which is the first clinical report that connects DNA damage checkpoints and prognosis of brain metastasis, supports these two proteins to be promising targets for overcoming the radioresistance in brain metastasis. PMID:22329197

  4. Efficient checkpointing schemes for depletion perturbation solutions on memory-limited architectures

    SciTech Connect

    Stripling, H. F.; Adams, M. L.; Hawkins, W. D.

    2013-07-01

    We describe a methodology for decreasing the memory footprint and machine I/O load associated with the need to access a forward solution during an adjoint solve. Specifically, we are interested in the depletion perturbation equations, where terms in the adjoint Bateman and transport equations depend on the forward flux solution. Checkpointing is the procedure of storing snapshots of the forward solution to disk and using these snapshots to recompute the parts of the forward solution that are necessary for the adjoint solve. For large problems, however, the storage cost of just a few copies of an angular flux vector can exceed the available RAM on the host machine. We propose a methodology that does not checkpoint the angular flux vector; instead, we write and store converged source moments, which are typically of a much lower dimension than the angular flux solution. This reduces the memory footprint and I/O load of the problem, but requires that we perform single sweeps to reconstruct flux vectors on demand. We argue that this trade-off is exactly the kind of algorithm that will scale on advanced, memory-limited architectures. We analyze the cost, in terms of FLOPS and memory footprint, of five checkpointing schemes. We also provide computational results that support the analysis and show that the memory-for-work trade off does improve time to solution. (authors)

  5. A novel UGGT1 and p97-dependent checkpoint for native ectodomains with ionizable intramembrane residue

    PubMed Central

    Merulla, Jessica; Soldà, Tatiana; Molinari, Maurizio

    2015-01-01

    Only native polypeptides are released from the endoplasmic reticulum (ER) to be transported at the site of activity. Persistently misfolded proteins are retained and eventually selected for ER-associated degradation (ERAD). The paradox of a structure-based protein quality control is that functional polypeptides may be destroyed if they are architecturally unfit. This has health-threatening implications, as shown by the numerous “loss-of-function” proteopathies, but also offers chances to intervene pharmacologically to promote bypassing of the quality control inspection and export of the mutant, yet functional protein. Here we challenged the ER of human cells with four modular glycopolypeptides designed to alert luminal and membrane protein quality checkpoints. Our analysis reveals the unexpected collaboration of the cytosolic AAA-ATPase p97 and the luminal quality control factor UDP-glucose:glycoprotein glucosyltransferase (UGGT1) in a novel, BiP- and CNX-independent checkpoint. This prevents Golgi transport of a chimera with a native ectodomain that passes the luminal quality control scrutiny but displays an intramembrane defect. Given that human proteopathies may result from impaired transport of functional polypeptides with minor structural defects, identification of quality checkpoints and treatments to bypass them as shown here upon silencing or pharmacologic inhibition of UGGT1 or p97 may have important clinical implications. PMID:25694454

  6. Trail without Catheter after Transurethral Resection of Prostate: Clamp It or Not?

    PubMed Central

    Talreja, Vikash; Saeed, Summaya; Rani, Kiran; Farid, Farah Naz; Haider, Mumtazuddin

    2016-01-01

    Background. There has been argument between clinical practitioners about clamping catheter or not prior to its removal after transurethral resection of prostate (TURP). We conducted a clinical trial to assess whether clamping has any role in early bladder tone recovery particularly in patients who undergo TURP. Methods. Randomized clinical trial was conducted at a tertiary care hospital, Karachi from January 2014 to July 2015. Eighty-six study participants who underwent TURP were randomly allocated into two groups of 43 participants each. In Group I, patient's Foley catheter was not clamped prior to its removal and in Group II Foley catheter was clamped. Data of all subjects were analyzed using SPSS version 20. Results. There was no significant difference in age and weight of resected tissues between two groups. Among 4 patients in Group I who required recatheterization, 1 patient was discharged with catheter as compared to Group II in which 2 patients were discharged with catheter (P = 0.99). Only 1 patient (2.3%) in Group II had bleeding which required recatheterization. Length of stay was significantly affected by early and free removal of Foley catheter (P < 0.001). Conclusion. The results of current study identified that clamping whether done or not had no significant impact on urinary retention. PMID:27034894

  7. Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure.

    PubMed

    Yao, N; Hurwitz, J; O'Donnell, M

    2000-01-14

    Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed. PMID:10625694

  8. Nonlinear Bending Stiffness of Plates Clamped by Bolted Joints under Bending Moment

    NASA Astrophysics Data System (ADS)

    Naruse, Tomohiro; Shibutani, Yoji

    Equivalent stiffness of plates clamped by bolted joints for designing should be evaluated according to not only the strength of bolted joints but also the deformation and vibration characteristics of the structures. When the applied external axial load or the bending moment is sufficiently small, the contact surfaces of the bolted joint are stuck together, and thus both the bolt and the clamped plates deform linearly. Although the sophisticated VDI 2230 code gives the appropriate stiffness of clamped plates for the infinitesimal deformation, the stiffness may vary nonlinearly with increasing the loading because of changing the contact state. Therefore, the present paper focuses on the nonlinear behaviour of the bending stiffness of clamped plates by using Finite Element (FE) analyses, taking the contact condition on bearing surfaces and between the plates into account. The FE models of the plates with thicknesses of 3.2, 4.5, 6.0 and 9.0 mm tightened with M8, 10, 12 and 16 bolts were constructed. The relation between bending moment and bending compliance of clamped plates is found to be categorized into three regions, namely, (i) constant compliance with fully stuck contact surfaces, (ii) transition showing the nonlinear compliance, and (iii) constant compliance with one-side contact surfaces. The mechanical models for these three regions are proposed and compared with FEM solutions. The prediction on the bounds of three regions is in a fairly good agreement except the case with smaller bolts and thicker plates.

  9. A bacterial toxin inhibits DNA replication elongation through a direct interaction with the β sliding clamp.

    PubMed

    Aakre, Christopher D; Phung, Tuyen N; Huang, David; Laub, Michael T

    2013-12-12

    Toxin-antitoxin (TA) systems are ubiquitous on bacterial chromosomes, yet the mechanisms regulating their activity and the molecular targets of toxins remain incompletely defined. Here, we identify SocAB, an atypical TA system in Caulobacter crescentus. Unlike canonical TA systems, the toxin SocB is unstable and constitutively degraded by the protease ClpXP; this degradation requires the antitoxin, SocA, as a proteolytic adaptor. We find that the toxin, SocB, blocks replication elongation through an interaction with the sliding clamp, driving replication fork collapse. Mutations that suppress SocB toxicity map to either the hydrophobic cleft on the clamp that binds DNA polymerase III or a clamp-binding motif in SocB. Our findings suggest that SocB disrupts replication by outcompeting other clamp-binding proteins. Collectively, our results expand the diversity of mechanisms employed by TA systems to regulate toxin activity and inhibit bacterial growth, and they suggest that inhibiting clamp function may be a generalizable antibacterial strategy. PMID:24239291

  10. The effects of thoracic aortic cross-clamping and declamping on visceral organ blood flow.

    PubMed Central

    Oyama, M; McNamara, J J; Suehiro, G T; Suehiro, A; Sue-Ako, K

    1983-01-01

    Blood flow was measured using radioactive microspheres in 11 macaque monkeys 1) before hemorrhage shock, 2) after onset of shock, 3) after aortic cross-clamping and resuscitation, and 4) after release of the cross-clamp and stabilization. Hemodynamic parameters (cardiac output, arterial, right atrial and left atrial pressure) and blood gases were also monitored. Total abdominal organ flow fell with hemorrhage and fell further with aortic clamping. Reinfusion of shed volume did not restore abdominal organ flow (4.7% baselines) but increased LAP and cardiac output to the upper body. Release of the cross-clamp produced profound acidosis that was treated effectively with NcHCO3. After stabilization of blood, flow to kidney remained low (49% baseline) although intestinal flow was increased threefold (320% of baseline). It is clear that thoracic aortic cross-clamping in shock further compromises already reduced visceral blood flow and may contribute to the problem of ischemic multiple organ failure after resuscitation from hemorrhagic shock. PMID:6830352

  11. The Role of the C-Clamp in Wnt-Related Colorectal Cancers

    PubMed Central

    Ravindranath, Aditi J.; Cadigan, Ken M.

    2016-01-01

    T-cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factors are major regulators of Wnt targets, and the products of the TCF7 and TCF7L2 genes have both been implicated in the progression of colorectal cancer in animal models and humans. TCFs recognize specific DNA sequences through their high mobility group (HMG) domains, but invertebrate TCFs and some isoforms of vertebrate TCF7 and TCF7L2 contain a second DNA binding domain known as the C-clamp. This review will cover the basic properties of C-clamps and their importance in Wnt signaling, using data from Drosophila, C. elegans, and mammalian cell culture. The connection between C-clamp containing TCFs and colorectal cancer will also be discussed. PMID:27527215

  12. The Anion Paradox in Sodium Taste Reception: Resolution by Voltage-Clamp Studies

    NASA Astrophysics Data System (ADS)

    Ye, Qing; Heck, Gerard L.; Desimone, John A.

    1991-11-01

    Sodium salts are potent taste stimuli, but their effectiveness is markedly dependent on the anion, with chloride yielding the greatest response. The cellular mechanisms that mediate this phenomenon are not known. This "anion paradox" has been resolved by considering the field potential that is generated by restricted electrodiffusion of the anion through paracellular shunts between taste-bud cells. Neural responses to sodium chloride, sodium acetate, and sodium gluconate were studied while the field potential was voltage-clamped. Clamping at electronegative values eliminated the anion effect, whereas clamping at electropositive potentials exaggerated it. Thus, field potentials across the lingual epithelium modulate taste reception, indicating that the functional unit of taste reception includes the taste cell and its paracellular microenvironment.

  13. The Role of the C-Clamp in Wnt-Related Colorectal Cancers.

    PubMed

    Ravindranath, Aditi J; Cadigan, Ken M

    2016-01-01

    T-cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factors are major regulators of Wnt targets, and the products of the TCF7 and TCF7L2 genes have both been implicated in the progression of colorectal cancer in animal models and humans. TCFs recognize specific DNA sequences through their high mobility group (HMG) domains, but invertebrate TCFs and some isoforms of vertebrate TCF7 and TCF7L2 contain a second DNA binding domain known as the C-clamp. This review will cover the basic properties of C-clamps and their importance in Wnt signaling, using data from Drosophila, C. elegans, and mammalian cell culture. The connection between C-clamp containing TCFs and colorectal cancer will also be discussed. PMID:27527215

  14. Delayed cord clamping in red blood cell alloimmunization: safe, effective, and free?

    PubMed Central

    2016-01-01

    Hemolytic disease of the newborn (HDN), an alloimmune disorder due to maternal and fetal blood type incompatibility, is associated with fetal and neonatal complications related to red blood cell (RBC) hemolysis. After delivery, without placental clearance, neonatal hyperbilirubinemia may develop from ongoing maternal antibody-mediated RBC hemolysis. In cases refractory to intensive phototherapy treatment, exchange transfusions (ET) may be performed to prevent central nervous system damage by reducing circulating bilirubin levels and to replace antibody-coated red blood cells with antigen-negative RBCs. The risks and costs of treating HDN are significant, but appear to be decreased by delayed umbilical cord clamping at birth, a strategy that promotes placental transfusion to the newborn. Compared to immediate cord clamping (ICC), safe and beneficial short-term outcomes have been demonstrated in preterm and term neonates receiving delayed cord clamping (DCC), a practice that may potentially be effective in cases RBC alloimmunization. PMID:27186530

  15. Temporary clamping of branch pulmonary artery for pulmonary hemorrhage after endarterectomy.

    PubMed

    Reddy, Srinivasa; Rajanbabu, Balram Babu; Kumar, Nalkunda Kyathaplar Sunil; Rajani, Indira

    2013-10-01

    A 49-year-old man underwent pulmonary thromboendarterectomy for chronic thromboembolic pulmonary hypertension. A massive pulmonary hemorrhage developed, which was identified to be from the right lower lobe, when weaning off cardiopulmonary bypass was attempted. He was managed by temporary overnight clamping of the right pulmonary artery, after the upper lobe branch. The next morning the clamp was removed, the bleeding had stopped completely, and his chest was closed. The patient was discharged on the 21st day. At 14 months' follow-up, he is in New York Heart Association functional class I. In suitable patients, temporary clamping of branch pulmonary artery can be a useful salvage measure, as in this patient. PMID:24088460

  16. Delayed cord clamping in red blood cell alloimmunization: safe, effective, and free?

    PubMed

    McAdams, Ryan M

    2016-04-01

    Hemolytic disease of the newborn (HDN), an alloimmune disorder due to maternal and fetal blood type incompatibility, is associated with fetal and neonatal complications related to red blood cell (RBC) hemolysis. After delivery, without placental clearance, neonatal hyperbilirubinemia may develop from ongoing maternal antibody-mediated RBC hemolysis. In cases refractory to intensive phototherapy treatment, exchange transfusions (ET) may be performed to prevent central nervous system damage by reducing circulating bilirubin levels and to replace antibody-coated red blood cells with antigen-negative RBCs. The risks and costs of treating HDN are significant, but appear to be decreased by delayed umbilical cord clamping at birth, a strategy that promotes placental transfusion to the newborn. Compared to immediate cord clamping (ICC), safe and beneficial short-term outcomes have been demonstrated in preterm and term neonates receiving delayed cord clamping (DCC), a practice that may potentially be effective in cases RBC alloimmunization. PMID:27186530

  17. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

    PubMed Central

    Hayner, Jaclyn N.; Douma, Lauren G.; Bloom, Linda B.

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. PMID:25159615

  18. The Effects of G2-Phase Enrichment and Checkpoint Abrogation on Low-Dose Hyper-Radiosensitivity

    SciTech Connect

    Krueger, Sarah A.; Wilson, George D.; Piasentin, Evano; Joiner, Michael C.; Marples, Brian

    2010-08-01

    Purpose: An association between low-dose hyper-radiosensitivity (HRS) and the 'early' G2/M checkpoint has been established. An improved molecular understanding of the temporal dynamics of this relationship is needed before clinical translation can be considered. This study was conducted to characterize the dose response of the early G2/M checkpoint and then determine whether low-dose radiation sensitivity could be increased by synchronization or chemical inhibition of the cell cycle. Methods and Materials: Two related cell lines with disparate HRS status were used (MR4 and 3.7 cells). A double-thymidine block technique was developed to enrich the G2-phase population. Clonogenic cell survival, radiation-induced G2-phase cell cycle arrest, and deoxyribonucleic acid double-strand break repair were measured in the presence and absence of inhibitors to G2-phase checkpoint proteins. Results: For MR4 cells, the dose required to overcome the HRS response (approximately 0.2 Gy) corresponded with that needed for the activation of the early G2/M checkpoint. As hypothesized, enriching the number of G2-phase cells in the population resulted in an enhanced HRS response, because a greater proportion of radiation-damaged cells evaded the early G2/M checkpoint and entered mitosis with unrepaired deoxyribonucleic acid double-strand breaks. Likewise, abrogation of the checkpoint by inhibition of Chk1 and Chk2 also increased low-dose radiosensitivity. These effects were not evident in 3.7 cells. Conclusions: The data confirm that HRS is linked to the early G2/M checkpoint through the damage response of G2-phase cells. Low-dose radiosensitivity could be increased by manipulating the transition of radiation-damaged G2-phase cells into mitosis. This provides a rationale for combining low-dose radiation therapy with chemical synchronization techniques to improve increased radiosensitivity.

  19. Phosphorylation-Independent Inhibition of Cdc28p by the Tyrosine Kinase Swe1p in the Morphogenesis Checkpoint

    PubMed Central

    McMillan, John N.; Sia, Rey A. L.; Bardes, Elaine S. G.; Lew, Daniel J.

    1999-01-01

    The morphogenesis checkpoint in budding yeast delays cell cycle progression in G2 when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G2 arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p. Results of experiments using a nonphosphorylatable CDC28Y19F allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation. We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28E12K allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28Y19F allele suggested that Swe1p is still able to inhibit CDC28Y19F in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p. Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy. The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms. PMID:10454545

  20. Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

    PubMed Central

    Kaisari, Sharon; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Hershko, Avram

    2016-01-01

    The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system. PMID:26755599

  1. Measurement of transmembrane potential and current in cardiac muscle: a new voltage clamp method.

    PubMed Central

    Goldman, Y; Morad, M

    1977-01-01

    1. A single sucrose gap voltage clamp technique was developed to correct for artifacts of 'leakage' corrent and extracellular resistance making possible improved measurement of membrane current and membrane potential in cardiac muscle. 2. A fourth compartment termed 'guard gap' was added to the sucrose gap. The guard gap is maintained at the same potential as the Reinger pool, so that no extracellular leakage current can flow into the Ringer pool. Comparison of experimental results with the predictions of an idealized cable model indicates that the guard gap is effective in trapping leakage current. 3. The slow charging of membrane capacitance due to extracellular series resistance was accelerated by applying a 'pre-pulse' of the command potential past the final voltage clamp value. 4. A second technique, termed 'chopped current pulse clamp', was used to compensate for the extracellular resistance throughout the voltage clamp step. The applied current was turned on and off at a frequency of 0-5-2 kHz. The membrane potential sampled during the zero current phase was fed back through the clamp loop. 5. With either of these compensation techniques, the voltage and current traces settle to effectively constant values within 2-4 msec after initiation of a hyperpolarizing voltage clamp step from rest. 6. The membrane conductance measured by the prepulse and chopped current-pulse technique are equal and confirm a higher conductance at rest than during the plateau of the action potential. 7. The 'instantaneous' current-voltage relation of the membrane is linear during the plateau of the frog ventricular action potential. PMID:301933

  2. Cracking associated with micrometeoroid impact craters in anodized aluminum alloy clamps on LDEF

    NASA Technical Reports Server (NTRS)

    Murr, Lawrence E.; Niou, Chorng S.; Quinones, Stella; Murr, Kyle S.

    1992-01-01

    The Long Duration Exposure Facility (LDEF) is a reusable hollow-cylindrical satellite sustaining a total of 57 different experiments. The 130 sq m of spacecraft surface area included anodized 6061-T6 Al alloy bay frames and clamps for holding experiment trays in the bay areas. Attention is presently given to the micrometeoroid impact crater features observed on two tray clamps recovered from the LDEF leading-edge locations. It is found that even very subtle surface modifications in structural alloy anodizing can influence micrometeoroid impact crater cracking, notable radial cracking due to the ejecta-rim of the impact craters.

  3. High pressure clamp for electrical measurements up to 8 GPa and temperature down to 77 K

    NASA Astrophysics Data System (ADS)

    Bandyopadhyay, A. K.; Nalini, A. V.; Gopal, E. S. R.; Subramanyam, S. V.

    1980-01-01

    A compact clamp-type high pressure cell for carrying out electrical conductivity measurements on small solid samples of size 1 mm or less at pressures upto 8 GPa (i.e., 80 kbar) and for use down to 77 K has been designed and fabricated. The pressure generated in the sample region has been calibrated at room temperature against the polymorphic phase transitions of Bismuth and Ytterbium. The pressure relaxation of the clamp at low temperatures has been estimated by monitoring the electrical conductivity behavior of lead.

  4. Clamping of RNA with PNA enables targeting of microRNA.

    PubMed

    Ghidini, Alice; Bergquist, Helen; Murtola, Merita; Punga, Tanel; Zain, Rula; Strömberg, Roger

    2016-06-21

    To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have studied BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs. PMID:27203783

  5. Ultrafast spectroscopy of super high frequency mechanical modes of doubly clamped beams

    NASA Astrophysics Data System (ADS)

    Ristow, Oliver; Merklein, Moritz; Grossmann, Martin; Hettich, Mike; Schubert, Martin; Bruchhausen, Axel; Grebing, Jochen; Erbe, Artur; Mounier, Denis; Gusev, Vitalyi; Scheer, Elke; Dekorsy, Thomas; Barretto, Elaine C. S.

    2013-12-01

    We use ultrafast pump-probe spectroscopy to study the mechanical vibrations in the time domain of doubly clamped silicon nitride beams. Beams with two different clamping conditions are investigated. Finite element method calculations are performed to analyse the mode spectra of both structures. By calculating the strain integral on the surface of the resonators, we are able to reproduce the effect of the detection mechanism and identify all the measured modes. We show that our spectroscopy technique combined with our modelling tools allow the investigation of several different modes in the super high frequency range (3-30 GHz) and above, bringing more information about the vibration modes of nanomechanical resonators.

  6. Role of phosphorylation of Cdc20 in p31comet-stimulated disassembly of the mitotic checkpoint complex

    PubMed Central

    Miniowitz-Shemtov, Shirly; Eytan, Esther; Ganoth, Dvora; Sitry-Shevah, Danielle; Dumin, Elena; Hershko, Avram

    2012-01-01

    The mitotic checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is turned on, it promotes the formation of the mitotic checkpoint complex (MCC), which inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 bound to the APC/C activator Cdc20. When the checkpoint is satisfied, MCC is disassembled and APC/C becomes active. Previous studies have shown that the Mad2-binding protein p31comet promotes the dissociation of Cdc20 from BubR1 in MCC in a process that requires ATP. We now show that a part of MCC dissociation is blocked by inhibitors of cyclin-dependent kinases (Cdks) and that purified Cdk1–cyclin B stimulates this process. The mutation of all eight potential Cdk phosphorylation sites of Cdc20 partially prevented its release from BubR1. Furthermore, p31comet stimulated Cdk-catalyzed phosphorylation of Cdc20 in MCC. It is suggested that the binding of p31comet to Mad2 in MCC may trigger a conformational change in Cdc20 that facilitates its phosphorylation by Cdk, and that the latter process may promote its dissociation from BubR1. PMID:22566641

  7. Cell cycle execution point analysis of ORC function and characterization of the checkpoint response to ORC inactivation in Saccharomyces cerevisiae.

    PubMed

    Gibson, Daniel G; Bell, Stephen P; Aparicio, Oscar M

    2006-06-01

    Chromosomal replication initiates through the assembly of a prereplicative complex (pre-RC) at individual replication origins in the G1-phase, followed by activation of these complexes in the S-phase. In Saccharomyces cerevisiae, the origin recognition complex (ORC) binds replication origins throughout the cell cycle and participates in pre-RC assembly. Whether the ORC plays an additional role subsequent to pre-RC assembly in replication initiation or any other essential cell cycle process is not clear. To study the function of the ORC during defined cell cycle periods, we performed cell cycle execution point analyses with strains containing a conditional mutation in the ORC1, ORC2 or ORC5 subunit of ORC. We found that the ORC is essential for replication initiation, but is dispensable for replication elongation or later cell cycle events. Defective initiation in ORC mutant cells results in incomplete replication and mitotic arrest enforced by the DNA damage and spindle assembly checkpoint pathways. The involvement of the spindle assembly checkpoint implies a defect in kinetochore-spindle attachment or sister chromatid cohesion due to incomplete replication and/or DNA damage. Remarkably, under semipermissive conditions for ORC1 function, the spindle checkpoint alone suffices to block proliferation, suggesting this checkpoint is highly sensitive to replication initiation defects. We discuss the potential significance of these overlapping checkpoints and the impact of our findings on previously postulated role(s) of ORCs in other cell cycle functions. PMID:16716188

  8. The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint

    PubMed Central

    Marquitz, Aron R.; Harrison, Jacob C.; Bose, Indrani; Zyla, Trevin R.; McMillan, John N.; Lew, Daniel J.

    2002-01-01

    The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (GAP) previously shown to act on Rho1p, and we now show that it also acts on Cdc42p, the GTPase primarily responsible for establishment of cell polarity in yeast. Whereas the morphogenesis role of Bem2p required GAP activity, the checkpoint role of Bem2p did not. Instead, this function required an N-terminal Bem2p domain. Thus, this single protein has a GAP-dependent role in promoting cell polarity and a GAP-independent role in responding to defects in cell polarity by enacting the checkpoint. Surprisingly, Swe1p accumulation occurred normally in bem2 cells, but they were nevertheless unable to promote Cdc28p phosphorylation. Therefore, Bem2p defines a novel pathway in the morphogenesis checkpoint. PMID:12145202

  9. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

    PubMed

    Pike, Brietta L; Tenis, Nora; Heierhorst, Jörg

    2004-09-17

    Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways. PMID:15271990

  10. Genetic Control of the Trigger for the G2/M Checkpoint

    SciTech Connect

    Hall, Eric J.; Smilenov, Lubomir B.; Young, Erik F.

    2013-10-01

    The work undertaken in this project addressed two seminal areas of low dose radiation biology that are poorly understood and controversial. These areas are the challenge to the linear-no-threshold (LNT) paradigm at low doses of radiation and, the fundamental elements of radiation bystander effect biology Genetic contributions to low dose checkpoint engagement: The LNT paradigm is an extrapolation of known, measured cancer induction endpoints. Importantly, data for lower doses is often not available. Debatably, radiation protection standards have been introduced which are prudently contingent on the adherence of cancer risk to the established trend seen at higher doses. Intriguing findings from other labs have hinted at separate DNA damage response programs that engage at low or high levels of radiation. Individual radiation sensitivity commensurate with hemizygosity for a radiation sensitivity gene has been estimated at 1-2% in the U.S.. Careful interrogation of the DNA damage response at low doses of radiation became important and served as the basis for this grant. Several genes were tested in combinations to determine if combined haploinsufficiency for multiple radiosensitizing genes could render a cell more sensitive to lower levels of acute radiation exposure. We measured a classical radiation response endpoint, cell cycle arrest prior to mitosis. Mouse embryo fibroblasts were used and provided a uniform, rapidly dividing and genetically manipulable population of study. Our system did not report checkpoint engagement at acute doses of gamma rays below 100 mGy. The system did report checkpoint engagement reproducibly at 500 mGy establishing a threshold for activation between 100 and 500 mGy. Engagement of the checkpoint was ablated in cells nullizygous for ATM but was otherwise unperturbed in cells combinatorially haploinsufficient for ATM and Rad9, ATM and PTEN or PTEN and Rad9. Taken together, these experiments tell us that, in a sensitive fibroblast culture

  11. Requirement for PLK1 kinase activity in the maintenance of a robust spindle assembly checkpoint

    PubMed Central

    O'Connor, Aisling; Maffini, Stefano; Rainey, Michael D.; Kaczmarczyk, Agnieszka; Gaboriau, David; Musacchio, Andrea; Santocanale, Corrado

    2016-01-01

    ABSTRACT During mitotic arrest induced by microtubule targeting drugs, the weakening of the spindle assembly checkpoint (SAC) allows cells to progress through the cell cycle without chromosome segregation occurring. PLK1 kinase plays a major role in mitosis and emerging evidence indicates that PLK1 is also involved in establishing the checkpoint and maintaining SAC signalling. However, mechanistically, the role of PLK1 in the SAC is not fully understood, with several recent reports indicating that it can cooperate with either one of the major checkpoint kinases, Aurora B or MPS1. In this study, we assess the role of PLK1 in SAC maintenance. We find that in nocodazole-arrested U2OS cells, PLK1 activity is continuously required for maintaining Aurora B protein localisation and activity at kinetochores. Consistent with published data we find that upon PLK1 inhibition, phosphoThr3-H3, a marker of Haspin activity, is reduced. Intriguingly, Aurora B inhibition causes PLK1 to relocalise from kinetochores into fewer and much larger foci, possibly due to incomplete recruitment of outer kinetochore proteins. Importantly, PLK1 inhibition, together with partial inhibition of Aurora B, allows efficient SAC override to occur. This phenotype is more pronounced than the phenotype observed by combining the same PLK1 inhibitors with partial MPS1 inhibition. We also find that PLK1 inhibition does not obviously cooperate with Haspin inhibition to promote SAC override. These results indicate that PLK1 is directly involved in maintaining efficient SAC signalling, possibly by cooperating in a positive feedback loop with Aurora B, and that partially redundant mechanisms exist which reinforce the SAC. PMID:26685311

  12. Human SAD1 kinase is involved in UV-induced DNA damage checkpoint function.

    PubMed

    Lu, Rui; Niida, Hiroyuki; Nakanishi, Makoto

    2004-07-23

    Checkpoint activation by DNA damage during G(2) prevents activation of cyclin B/Cdc2 complexes, and as a consequence, mitotic entry is blocked. Although initiation and maintenance of G(2) arrest are known to be regulated by at least two distinct signaling pathways, including those of p38MAPK and ataxia-telangiectasia-mutated (ATM)- and Rad3-related (ATR)-Chk1 in higher eukaryotes, the actual number of signaling pathways involved in this regulation is still elusive. In the present study, we identified human SAD1 (hsSAD1) by searching a sequence data base. The predicted hsSAD1 protein comprises 778 amino acids and shares significant homology with the fission yeast Cdr2, a mitosis-regulatory kinase, and Caenorhabditis elegans SAD1, a neuronal cell polarity regulator. HsSAD1 transcript was expressed ubiquitously with the highest levels of expression in brain and testis. HsSAD1 specifically phosphorylated Wee1A, Cdc25-C, and -B on Ser-642, Ser-216, and Ser-361 in vitro, respectively. Overexpression of hsSAD1 resulted in an increased phosphorylation of Cdc25C on Ser-216 in vivo. DNA damage induced by UV or methyl methane sulfonate but not by IR enhanced endogenous hsSAD1 kinase activity in a caffeine-sensitive manner and caused translocation of its protein from cytoplasm to nucleus. Overexpression of wild-type hsSAD1 induced G(2)/M arrest in HeLa S2 cells. Furthermore, UV-induced G(2)/M arrest was partially abrogated by the reduced expression of hsSAD1 using small interfering RNA. These results suggest that hsSAD1 acts as checkpoint kinase upon DNA damage induced by UV or methyl methane sulfonate. The identification of this new kinase suggests the existence of an alternative checkpoint pathway other than those of ATR-Chk1 and p38MAPK. PMID:15150265

  13. Transcriptional regulation of mitotic checkpoint gene MAD1 by p53.

    PubMed

    Chun, Abel C S; Jin, Dong-Yan

    2003-09-26

    p53 regulates a number of genes through transcriptional activation and repression. p53-dependent mitotic checkpoint has been described, but the underlying mechanism is still obscure. Here we examined the effect of p53 on the expression of a human mitotic checkpoint protein, Mitosis Arrest Deficiency 1 (MAD1), in cultured human cells. The expression of MAD1 was reduced when the cells were overexpressing exogenously introduced wild-type p53. The same reduction was also observed when the cells were treated with anticancer agents 5-fluorouracil and cisplatin or were irradiated with UV. Consistently, MAD1 promoter activity diminished in a dose-dependent manner when induced by p53, indicating that p53 repressed MAD1 at a transcriptional level. Intriguingly, several tumor hot spot mutations in p53 (V143A, R175H, R248W, and R273H) did not abolish the ability of p53 to repress MAD1 expression. By serial truncation of the MAD1 promoter, we confined the p53-responsive element to a 38-bp region that represents a novel sequence distinct from the known p53 consensus binding site. Trichostatin A, a histone deacetylase inhibitor, relieved the p53 transrepression activity on MAD1. Chromatin immunoprecipitation assay revealed that p53, histone deacetylase 1, and co-repressor mSin3a associated with the MAD1 promoter in vivo. Taken together, our findings suggest a regulatory mechanism for the mitotic checkpoint in which MAD1 is inhibited by p53. PMID:12876282

  14. A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology

    PubMed Central

    Ander, Marcel; Subramaniam, Sivaraman; Fahmy, Karim; Stewart, A. Francis; Schäffer, Erik

    2015-01-01

    Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction. PMID:26271032

  15. Development of a reusable, low-shock clamp band separation system for small spacecraft release applications

    NASA Astrophysics Data System (ADS)

    Dowen, David; Christiansen, Scott; Arulf, Orjan

    2001-09-01

    In small spacecraft, the proximity of sensitive components to release systems has led to the need for low-shock spacecraft release systems. Marmon band systems are often desirable for their flight history, structural capability, and reliability. Until recently, only pyrotechnically released clamp bands were readily available. The clamp band system described in ths paper reduces shock in two ways: it eliminates shock typically associated with pyrotechnic release devices as well as utilizing a release device that reduces the shock associated with the rapid release of the preload strain energy. Patented Fast Acting Shockless Separation Nut (FASSN) technology is utilized to convert strain energy stored in the system into rotational energy of a flywheel. Early FASSN devices were designed for discrete point applications and were somewhat large and massive. Additional development of the FASSN device has reduced the size and weight to enable the use of the technology in a medium sized (23 to 60 cm diameter) clamp band system. This paper describes the overall design, performance, and initial test results for the FASSN-based, non-pyrotechnic, low-shock clamp band release system.

  16. ELECTROCARDIOGRAPHIC RESPONSES OF RAT FETUSES WITH CLAMPED OR INTACT UMBILICAL CORDS TO ACUTE MATERNAL UTERINE ISCHEMIA

    EPA Science Inventory

    Uterine ischemia results in severe cardiac disturbances in the fetus. It has been postulated that these effects are due to interaction with the ischemic uterus or placenta and not due to hypoxia or build up of metabolites in the fetus. The fetal cardiac responses to uterine clamp...

  17. Construction, Calibration, and Validation of a Simple Patch-Clamp Amplifier for Physiology Education

    ERIC Educational Resources Information Center

    Rouzrokh, Ali; Ebrahimi, Soltan Ahmed; Mahmoudian, Massoud

    2009-01-01

    A modular patch-clamp amplifier was constructed based on the Strickholm design, which was initially published in 1995. Various parts of the amplifier such as the power supply, input circuit, headstage, feedback circuit, output and nulling circuits were redesigned to use recent software advances and fabricated using the common lithographic printed…

  18. A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology.

    PubMed

    Ander, Marcel; Subramaniam, Sivaraman; Fahmy, Karim; Stewart, A Francis; Schäffer, Erik

    2015-08-01

    Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA-protein interaction. PMID:26271032

  19. A Single Subunit Directs the Assembly of the Escherichia coli DNA Sliding Clamp Loader

    PubMed Central

    Park, Ah Young; Jergic, Slobodan; Politis, Argyris; Ruotolo, Brandon T.; Hirshberg, Daniel; Jessop, Linda L.; Beck, Jennifer L.; Barsky, Daniel; O’Donnell, Mike; Dixon, Nicholas E.; Robinson, Carol V.

    2016-01-01

    SUMMARY Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (τ/γ) proteins, δ, and δ′, which together form an asymmetric pentameric ring that also interacts with ψχ. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that γ exists exclusively as a stable homotetramer, while τ is in a monomer-dimer-trimer-tetramer equilibrium. δ′ plays a direct role in the assembly as a τ/γ oligomer breaker, thereby facilitating incorporation of lower oligomers. With δ′, both δ and ψχ stabilize the trimeric form of DnaX, thus completing the assembly. When τ and γ are present simultaneously, mimicking the situation in vivo, subunit exchange between τ and γ tetramers occurs rapidly to form heterocomplexes but is retarded when δδ′ is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed. PMID:20223211

  20. Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus

    PubMed Central

    Vass, Robert H.; Chien, Peter

    2013-01-01

    Chromosome replication relies on sliding clamps that are loaded by energy-dependent complexes. In Escherichia coli, the ATP-binding clamp loader subunit DnaX exists as both long (τ) and short (γ) forms generated through programmed translational frameshifting, but the need for both forms is unclear. Here, we show that in Caulobacter crescentus, DnaX isoforms are unexpectedly generated through partial proteolysis by the AAA+ protease casein lytic proteinase (Clp) XP. We find that the normally processive ClpXP protease partially degrades DnaX to produce stable fragments upon encountering a glycine-rich region adjacent to a structured domain. Increasing the sequence complexity of this region prevents partial proteolysis and generates a τ-only form of DnaX in vivo that is unable to support viability on its own. Growth is restored when γ is provided in trans, but these strains are more sensitive to DNA damage compared with strains that can generate γ through proteolysis. Our work reveals an unexpected mode of partial processing by the ClpXP protease to generate DnaX isoforms, demonstrates that both τ and γ forms of DnaX are required for Caulobacter viability, and identifies a role for clamp loader diversity in responding to DNA damage. The conservation of distinct DnaX isoforms throughout bacteria despite fundamentally different mechanisms for producing them suggests there may be a conserved need for alternate clamp loader complexes during DNA damaging conditions. PMID:24145408

  1. Experimental and numerical analysis of clamped joints in front motorbike suspensions

    NASA Astrophysics Data System (ADS)

    Croccolo, D.; de Agostinis, M.; Vincenzi, N.

    2010-06-01

    Clamped joints are shaft-hub connections used, as an instance, in front motorbike suspensions to lock the steering plates with the legs and the legs with the wheel pin, by means of one or two bolts. The preloading force, produced during the tightening process, should be evaluated accurately, since it must lock safely the shaft, without overcoming the yielding point of the hub. Firstly, friction coefficients have been evaluated on “ad-hoc designed” specimens, by applying the Design of Experiment approach: the applied tightening torque has been precisely related to the imposed preloading force. Then, the tensile state of clamps have been evaluated both via FEM and by leveraging some design formulae proposed by the Authors as function of the preloading force and of the clamp geometry. Finally, the results have been compared to those given by some strain gauges applied on the tested clamps: the discrepancies between numerical analyses, the design formulae and the experimental results remains under a threshold of 10%.

  2. Acute renal allograft rejection after immune checkpoint inhibitor therapy for metastatic melanoma.

    PubMed

    Spain, L; Higgins, R; Gopalakrishnan, K; Turajlic, S; Gore, M; Larkin, J

    2016-06-01

    Immune checkpoint inhibitors such as ipilimumab and nivolumab improve survival in patients with advanced melanoma and are increasingly available to clinicians for use in the clinic. Their safety in organ transplant recipients is not well defined but published case reports describing treatment with ipilimumab have not been complicated by graft rejection. No cases of anti-programmed cell death protein 1 administration are reported in this group. We describe a case of acute graft rejection in a kidney transplant recipient after treatment with nivolumab, after progression on ipilimumab. Potential factors increasing the risk of graft rejection in this case are discussed, in particular the contribution of nivolumab. PMID:26951628

  3. Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

    PubMed Central

    Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F.; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L.; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B.; Murray, Brion W.

    2015-01-01

    Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment

  4. Comparative theoretical study of the binding of potential cancer-treatment drugs to Checkpoint kinase 1

    NASA Astrophysics Data System (ADS)

    Araújo, Pedro M. M.; Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

    2014-01-01

    This Letter focuses the binding between Checkpoint kinase 1 and two molecules with known inhibition potential, C39 and C40. In order to find the most relevant residues the structures were submitted to an optimization process. As expected C39 presented the highest inhibitory power towards Chk1, being this inhibition mode highly dependent on the interactions with Lys38 and Glu91. Glu55 and Asp148 exhibit unfavorable interactions to C39. Glu91 was the most important residues in the binding of C40 to Chk1, while interaction with Lys38, Glu55 and Gly90 resulted in repulsion.

  5. MATLAB implementation of a dynamic clamp with bandwidth >125 KHz capable of generating INa at 37°C

    PubMed Central

    Clausen, Chris; Valiunas, Virginijus; Brink, Peter R.; Cohen, Ira S.

    2012-01-01

    We describe the construction of a dynamic clamp with bandwidth >125 KHz that utilizes a high performance, yet low cost, standard home/office PC interfaced with a high-speed (16 bit) data acquisition module. High bandwidth is achieved by exploiting recently available software advances (code-generation technology, optimized real-time kernel). Dynamic-clamp programs are constructed using Simulink, a visual programming language. Blocks for computation of membrane currents are written in the high-level matlab language; no programming in C is required. The instrument can be used in single- or dual-cell configurations, with the capability to modify programs while experiments are in progress. We describe an algorithm for computing the fast transient Na+ current (INa) in real time, and test its accuracy and stability using rate constants appropriate for 37°C. We then construct a program capable of supplying three currents to a cell preparation: INa, the hyperpolarizing-activated inward pacemaker current (If), and an inward-rectifier K+ current (IK1). The program corrects for the IR drop due to electrode current flow, and also records all voltages and currents. We tested this program on dual patch-clamped HEK293 cells where the dynamic clamp controls a current-clamp amplifier and a voltage-clamp amplifier controls membrane potential, and current-clamped HEK293 cells where the dynamic clamp produces spontaneous pacing behavior exhibiting Na+ spikes in otherwise passive cells. PMID:23224681

  6. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age

    PubMed Central

    Marangos, Petros; Stevense, Michelle; Niaka, Konstantina; Lagoudaki, Michaela; Nabti, Ibtissem; Jessberger, Rolf; Carroll, John

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes from aged mice. These data lead us to propose that the SAC is a major gatekeeper preventing the progression of oocytes harbouring DNA damage. The SAC therefore acts to integrate protection against both aneuploidy and DNA damage by preventing production of abnormal mature oocytes and subsequent embryos. Finally, we suggest escaping this DNA damage checkpoint in maternal ageing may be one of the causes of increased chromosome anomalies in oocytes and embryos from older mothers. PMID:26522734

  7. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age.

    PubMed

    Marangos, Petros; Stevense, Michelle; Niaka, Konstantina; Lagoudaki, Michaela; Nabti, Ibtissem; Jessberger, Rolf; Carroll, John

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes from aged mice. These data lead us to propose that the SAC is a major gatekeeper preventing the progression of oocytes harbouring DNA damage. The SAC therefore acts to integrate protection against both aneuploidy and DNA damage by preventing production of abnormal mature oocytes and subsequent embryos. Finally, we suggest escaping this DNA damage checkpoint in maternal ageing may be one of the causes of increased chromosome anomalies in oocytes and embryos from older mothers. PMID:26522734

  8. Scanning electron microscope/energy dispersive x ray analysis of impact residues on LDEF tray clamps

    NASA Technical Reports Server (NTRS)

    Bernhard, Ronald P.; Durin, Christian; Zolensky, Michael E.

    1992-01-01

    To better understand the nature of particulates in low-Earth orbit (LEO), and their effects on spacecraft hardware, we are analyzing residues found in impacts on the Long Duration Exposure Facility (LDEF) tray clamps. LDEF experiment trays were held in place by 6 to 8 chromic-anodized aluminum (6061-T6) clamps that were fastened to the spacecraft frame using three stainless steel hex bolts. Each clamp exposed an area of approximately 58 sq cm (4.8 cm x 12.7 cm x .45 cm, minus the bolt coverage). Some 337 out of 774 LDEF tray clamps were archived at JSC and are available through the Meteoroid & Debris Special Investigation Group (M&D SIG). Optical scanning of clamps, starting with Bay/Row A01 and working toward H25, is being conducted at JSC to locate and document impacts as small as 40 microns. These impacts are then inspected by Scanning Electron Microscopy/Energy Dispersive X-ray Analysis (SEM/EDXA) to select those features which contain appreciable impact residue material. Based upon the composition of projectile remnants, and using criteria developed at JSC, we have made a preliminary discrimination between micrometeoroid and space debris residue-containing impact features. Presently, 13 impacts containing significant amounts of unmelted and semi-melted micrometeoritic residues were forwarded to Centre National d'Etudes Spatiales (CNES) in France. At the CNES facilities, the upgraded impacts were analyzed using a JEOL T330A SEM equipped with a NORAN Instruments, Voyager X-ray Analyzer. All residues were quantitatively characterized by composition (including oxygen and carbon) to help understand interplanetary dust as possibly being derived from comets and asteroids.

  9. Evaluation of the Safe Ischemic Time of Clamping During Intermittent Pringles Maneuver in Rabbits

    PubMed Central

    Kolahdoozan, Mohsen; Behdad, Akbar; Hosseinpour, Mehrdad; Behdad, Samin; Rezaei, Mohammad Taghi

    2015-01-01

    Background: The liver is the most commonly injured organ in blunt abdominal trauma. Although major hepatic bleeding may be partially controlled with portal triade clamping (the Pringle’s maneuver), continuous prolonged clamping results in liver ischemia. Objectives: The purpose of this study was to determine the safe time of Pringle maneuver based on pathologic changes of liver in rabbit models. Materials and Methods: In an experimental study, 20 New-Zealand white rabbits were selected. In laparotomy, a blunt dissector was passed through the foramen of Winslow and the hepato-duodenal ligament encircled with an umbilical tape. En masse Pringle maneuver was performed using atraumatic flexible clamps. Rabbits were divided into four groups based on Pringle maneuver time (30 minutes, 45 minutes, 60 minutes, and 75 minutes). A hepatic biopsy was performed at the beginning of operation. The degree of tissue injury was evaluated using blood markers. Results: There were five rabbits in each group. At the end of 60 minutes ischemia, only minor alterations were observed in pathological specimens. At the end of 75 minutes, hepatocyte damage and necrosis were observed. The serum levels of alanine aminotransferase (Group A: P = 0.02; Group B: P = 0.01; Group C: P = 0.0002; Group D: P = 0.01) and Aspartate aminotransferase (Group A: P = 0.03; Group B: P = 0.002; Group C: P = 0.0004; Group D: P = 0.0003) were significantly increased post-operatively. The maximum level was in the first day after operation. Conclusions: Continuous portal triade clamping (the Pringle maneuver) during liver ischemia (30 and 45 minutes) in rabbits resulted in no ischemic change. Increasing time of clamping to 30 minutes was safe in intermittent Pringle maneuver. PMID:26848477

  10. Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells

    PubMed Central

    Moolman, M. Charl; Krishnan, Sriram Tiruvadi; Kerssemakers, Jacob W. J.; van den Berg, Aafke; Tulinski, Pawel; Depken, Martin; Reyes-Lamothe, Rodrigo; Sherratt, David J.; Dekker, Nynke H.

    2014-01-01

    The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli β2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both β2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of β2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous β2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism. PMID:25520215

  11. Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells

    NASA Astrophysics Data System (ADS)

    Moolman, M. Charl; Krishnan, Sriram Tiruvadi; Kerssemakers, Jacob W. J.; van den Berg, Aafke; Tulinski, Pawel; Depken, Martin; Reyes-Lamothe, Rodrigo; Sherratt, David J.; Dekker, Nynke H.

    2014-12-01

    The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli β2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both β2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of β2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous β2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism.

  12. ATP is required for the release of the anaphase-promoting complex/cyclosome from inhibition by the mitotic checkpoint

    PubMed Central

    Miniowitz-Shemtov, Shirly; Teichner, Adar; Sitry-Shevah, Danielle; Hershko, Avram

    2010-01-01

    The mitotic (or spindle assembly) checkpoint system ensures accurate segregation of chromosomes by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. This system acts by inhibiting the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase to target securin for degradation. APC/C is inhibited by a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Mad2, and Cdc20. The molecular mechanisms of the inactivation of the mitotic checkpoint, including the release of APC/C from inhibition, remain obscure. It has been reported that polyubiquitylation by the APC/C is required for the inactivation of the mitotic checkpoint [Reddy SK, Rape M, Margansky WA, Kirschner MW (2007) Nature, 446:921–924]. We confirmed the involvement of polyubiquitylation, but found that another process, which requires ATP cleavage at the β–γ position (as opposed to α–β bond scission involved in ubiquitylation), is essential for the release of APC/C from checkpoint inhibition. ATP (β–γ) cleavage is required both for the dissociation of MCC components from APC/C and for the disassembly of free MCC, whereas polyubiquitylation is involved only in the former process. We find that the requirement for ATP (β–γ) cleavage is not due to the involvement of the 26S proteasome and that the phenomena observed are not due to sustained activity of protein kinase Cdk1/cyclin B, caused by inhibition of the degradation of cyclin B. Thus, some other energy-consuming process is needed for the inactivation of the mitotic checkpoint. PMID:20212161

  13. The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity.

    PubMed

    McCleland, Mark L; Gardner, Richard D; Kallio, Marko J; Daum, John R; Gorbsky, Gary J; Burke, Daniel J; Stukenberg, P Todd

    2003-01-01

    We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling. PMID:12514103

  14. The Geography of Deterrence: Exploring the Small Area Effects of Sobriety Checkpoints on Alcohol-Impaired Collision Rates within a City

    ERIC Educational Resources Information Center

    Nunn, Samuel; Newby, William

    2011-01-01

    This article examines alcohol-impaired collision metrics around nine sobriety checkpoint locations in Indianapolis, Indiana, before and after implementation of 22 checkpoints, using a pre/post examination, a pre/post nonequivalent comparison group analysis, and an interrupted time series approach. Traffic safety officials used geographical…

  15. Sum1, a highly conserved WD-repeat protein, suppresses S-M checkpoint mutants and inhibits the osmotic stress cell cycle response in fission yeast.

    PubMed Central

    Humphrey, T; Enoch, T

    1998-01-01

    The S-M checkpoint ensures that entry into mitosis is dependent on completion of DNA replication. In the fission yeast Schizosaccharomyces pombe, the SM checkpoint mutant cdc2-3w is thought to be defective in receiving the checkpoint signal. To isolate genes that function in the checkpoint pathway, we screened an S. pombe cDNA library for genes that, when overexpressed, could suppress the checkpoint defect of cdc2-3w. Using this approach, we have identified a novel gene, sum1+ (suppressor of uncontrolled mitosis). sum1+ encodes a highly conserved WD-transducin repeat protein with striking sequence similarity to the human transforming growth factor (TGF)-beta-receptor interacting protein TRIP-1 and to the translation initiation factor 3 subunit eIF3-p39, encoded by the TIF34 gene in Saccharomyces cerevisiae. S. pombe sum1+ is an essential gene, required for normal cell growth and division. In addition to restoring checkpoint control, overexpression of sum1+ inhibits the normal cell cycle response to osmotic stress. Furthermore, we demonstrate that inactivation of the stress-activated MAP kinase pathway, required for cell cycle stress response, restores the S-M checkpoint in cdc2-3w cells. These results suggest that Suml interacts with the stress-activated MAP kinase pathway and raise the possibility that environmental conditions may influence the checkpoint response in fission yeast. PMID:9560390

  16. A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores

    PubMed Central

    Moudgil, Devinderjit K.; Westcott, Nathan; Famulski, Jakub K.; Patel, Kinjal; Macdonald, Dawn; Hang, Howard

    2015-01-01

    Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein–dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein–protein interaction. PMID:25825516

  17. Spindle assembly checkpoint is sufficient for complete Cdc20 sequestering in mitotic control.

    PubMed

    Ibrahim, Bashar

    2015-01-01

    The spindle checkpoint assembly (SAC) ensures genome fidelity by temporarily delaying anaphase onset, until all chromosomes are properly attached to the mitotic spindle. The SAC delays mitotic progression by preventing activation of the ubiquitin ligase anaphase-promoting complex (APC/C) or cyclosome; whose activation by Cdc20 is required for sister-chromatid separation marking the transition into anaphase. The mitotic checkpoint complex (MCC), which contains Cdc20 as a subunit, binds stably to the APC/C. Compelling evidence by Izawa and Pines (Nature 2014; 10.1038/nature13911) indicates that the MCC can inhibit a second Cdc20 that has already bound and activated the APC/C. Whether or not MCC per se is sufficient to fully sequester Cdc20 and inhibit APC/C remains unclear. Here, a dynamic model for SAC regulation in which the MCC binds a second Cdc20 was constructed. This model is compared to the MCC, and the MCC-and-BubR1 (dual inhibition of APC) core model variants and subsequently validated with experimental data from the literature. By using ordinary nonlinear differential equations and spatial simulations, it is shown that the SAC works sufficiently to fully sequester Cdc20 and completely inhibit APC/C activity. This study highlights the principle that a systems biology approach is vital for molecular biology and could also be used for creating hypotheses to design future experiments. PMID:25977749

  18. RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints.

    PubMed

    Mamidipudi, V; Dhillon, N K; Parman, T; Miller, L D; Lee, K C; Cartwright, C A

    2007-05-01

    Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G(1) and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer. PMID:17072338

  19. ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

    PubMed Central

    Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S.; Peters, Jan-Michael

    2016-01-01

    To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. PMID:26953350

  20. Immune checkpoint inhibitors: the new frontier in non-small-cell lung cancer treatment.

    PubMed

    El-Osta, Hazem; Shahid, Kamran; Mills, Glenn M; Peddi, Prakash

    2016-01-01

    Lung cancer is the major cause for cancer-related death in the US. Although advances in chemotherapy and targeted therapy have improved the outcome of metastatic non-small-cell lung cancer, its prognosis remains dismal. A deeper understanding of the complex interaction between the immune system and tumor microenvironment has identified immune checkpoint inhibitors as new avenue of immunotherapy. Rather than acting directly on the tumor, these therapies work by removing the inhibition exerted by tumor cell or other immune cells on the immune system, promoting antitumoral immune response. To date, two programmed death-1 inhibitors, namely nivolumab and pembrolizumab, have received the US Food and Drug Administration approval for the treatment of advanced non-small-cell lung cancer that failed platinum-based chemotherapy. This manuscript provides a brief overview of the pathophysiology of cancer immune evasion, summarizes pertinent data on completed and ongoing clinical trials involving checkpoint inhibitors, discusses the different strategies to optimize their function, and outlines various challenges that are faced in this promising yet evolving field. PMID:27574451