Science.gov

Sample records for 9-1-1 checkpoint clamp

  1. RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling.

    PubMed

    Lindsey-Boltz, Laura A; Kemp, Michael G; Capp, Christopher; Sancar, Aziz

    2015-01-01

    The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9- and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.

  2. Choreography of the 9-1-1 checkpoint complex: DDK puts a check on the checkpoints.

    PubMed

    Paek, Andrew L; Weinert, Ted

    2010-11-24

    Checkpoint proteins respond to DNA damage by halting the cell cycle until the damage is repaired. In this issue of Molecular Cell, Furuya et al. (2010) provide evidence that checkpoint proteins need to be removed from sites of damage in order to properly repair it.

  3. 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

  4. 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.

  5. A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

    PubMed

    Song, Wei; Levin, David S; Varkey, Johnson; Post, Sean; Bermudez, Vladimir P; Hurwitz, Jerard; Tomkinson, Alan E

    2007-08-03

    DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

  6. Replication factor C is a more effective proliferating cell nuclear antigen (PCNA) opener than the checkpoint clamp loader, Rad24-RFC.

    PubMed

    Thompson, Jennifer A; Marzahn, Melissa R; O'Donnell, Mike; Bloom, Linda B

    2012-01-13

    Clamp loaders from all domains of life load clamps onto DNA. The clamp tethers DNA polymerases to DNA to increase the processivity of synthesis as well as the efficiency of replication. Here, we investigated proliferating cell nuclear antigen (PCNA) binding and opening by the Saccharomyces cerevisiae clamp loader, replication factor C (RFC), and the DNA damage checkpoint clamp loader, Rad24-RFC, using two separate fluorescence intensity-based assays. Analysis of PCNA opening by RFC revealed a two-step reaction in which RFC binds PCNA before opening PCNA rather than capturing clamps that have transiently and spontaneously opened in solution. The affinity of RFC for PCNA is about an order of magnitude lower in the absence of ATP than in its presence. The affinity of Rad24-RFC for PCNA in the presence of ATP is about an order magnitude weaker than that of RFC for PCNA, similar to the RFC-PCNA interaction in the absence of ATP. Importantly, fewer open clamp loader-clamp complexes are formed when PCNA is bound by Rad24-RFC than when bound by RFC.

  7. Overproduction and purification of RFC-related clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae.

    PubMed

    Bylund, Göran O; Majka, Jerzy; Burgers, Peter M J

    2006-01-01

    The replication clamp PCNA and its loader RFC (Replication Factor C) are central factors required for processive replication and coordinated DNA repair. Recently, several additional related clamp loaders have been identified. These alternative clamp loaders contain the small Rfc2-5 subunits of RFC, but replace the large Rfc1 subunit by a pathway-specific alternative large subunit, Rad24 for the DNA damage checkpoint, Ctf18 for the establishment of sister chromatid cohesion, and Elg1 for a general function in chromosome stability. In order to define biochemical functions for these loaders, the loaders were overproduced in yeast and purified at a milligram scale. To aid in purification, the large subunit of each clamp loader was fused to a GST-tag that, after purification could be easily removed by a rhinoviral protease. This methodology yielded all clamp loaders in high yield and with high enzymatic activity. The yeast 9-1-1 checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, was overproduced and purified in a similar manner.

  8. 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.

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

    PubMed

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

    2010-06-01

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

  10. The extent of error-prone replication restart by homologous recombination is controlled by Exo1 and checkpoint proteins.

    PubMed

    Tsang, Ellen; Miyabe, Izumi; Iraqui, Ismail; Zheng, Jiping; Lambert, Sarah A E; Carr, Antony M

    2014-07-01

    Genetic instability, a hallmark of cancer, can occur when the replication machinery encounters a barrier. The intra-S-phase checkpoint maintains stalled replication forks in a replication-competent configuration by phosphorylating replisome components and DNA repair proteins to prevent forks from catastrophically collapsing. Here, we report a novel function of the core Schizosaccharomyces pombe checkpoint sensor kinase, Rad3 (an ATR orthologue), that is independent of Chk1 and Cds1 (a CHK2 orthologue); Rad3(ATR) regulates the association of recombination factors with collapsed forks, thus limiting their genetic instability. We further reveal antagonistic roles for Rad3(ATR) and the 9-1-1 clamp - Rad3(ATR) restrains MRN- and Exo1-dependent resection, whereas the 9-1-1 complex promotes Exo1 activity. Interestingly, the MRN complex, but not its nuclease activity, promotes resection and the subsequent association of recombination factors at collapsed forks. The biological significance of this regulation is revealed by the observation that Rad3(ATR) prevents Exo1-dependent genome instability upstream of a collapsed fork without affecting the efficiency of recombination-mediated replication restart. We propose that the interplay between Rad3(ATR) and the 9-1-1 clamp functions to fine-tune the balance between the need for the recovery of replication through recombination and the risk of increased genome instability.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Force-Measuring Clamps

    NASA Technical Reports Server (NTRS)

    Nunnelee, Mark

    2003-01-01

    Force-measuring clamps have been invented to facilitate and simplify the task of measuring the forces or pressures applied to clamped parts. There is a critical need to measure clamping forces or pressures in some applications for example, while bonding sensors to substrates or while clamping any sensitive or delicate parts. Many manufacturers of adhesives and sensors recommend clamping at specific pressures while bonding sensors or during adhesive bonding between parts in general. In the absence of a force-measuring clamp, measurement of clamping force can be cumbersome at best because of the need for additional load sensors and load-indicating equipment. One prior method of measuring clamping force involved the use of load washers or miniature load cells in combination with external power sources and load-indicating equipment. Calibrated spring clamps have also been used. Load washers and miniature load cells constitute additional clamped parts in load paths and can add to the destabilizing effects of loading mechanisms. Spring clamps can lose calibration quickly through weakening of the springs and are limited to the maximum forces that the springs can apply. The basic principle of a force-measuring clamp can be implemented on a clamp of almost any size and can enable measurement of a force of almost any magnitude. No external equipment is needed because the component(s) for transducing the clamping force and the circuitry for supplying power, conditioning the output of the transducers, and displaying the measurement value are all housed on the clamp. In other words, a force-measuring clamp is a complete force-application and force-measurement system all in one package. The advantage of unitary packaging of such a system is that it becomes possible to apply the desired clamping force or pressure with precision and ease.

  16. 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.

  17. Examining the Fundamental Obstructs of Adopting Cloud Computing for 9-1-1 Dispatch Centers in the USA

    ERIC Educational Resources Information Center

    Osman, Abdulaziz

    2016-01-01

    The purpose of this research study was to examine the unknown fears of embracing cloud computing which stretches across measurements like fear of change from leaders and the complexity of the technology in 9-1-1 dispatch centers in USA. The problem that was addressed in the study was that many 9-1-1 dispatch centers in USA are still using old…

  18. Epigenetics meets immune checkpoints.

    PubMed

    Covre, Alessia; Coral, Sandra; Di Giacomo, Anna Maria; Taverna, Pietro; Azab, Mohammad; Maio, Michele

    2015-06-01

    Epigenetic alterations play a pivotal role in cancer development and progression. Pharmacologic reversion of such alterations is feasible, and second generation "epigenetic drugs" are in development and have been demonstrated to possess significant immunomodulatory properties. This knowledge, together with the availability of new and highly effective immunotherapeutic agents including immune checkpoint(s) blocking monoclonal antibodies, allows us to plan for highly innovative proof-of-principle combination studies that will likely open the path to more effective anticancer therapies.

  19. Photovoltaic panel clamp

    SciTech Connect

    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.

  20. Photovoltaic panel clamp

    SciTech Connect

    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.

  1. Clamping in Boltzmann machines.

    PubMed

    Livesey, M

    1991-01-01

    A certain assumption that appears in the proof of correctness of the standard Boltzmann machine learning procedure is investigated. The assumption, called the clamping assumption, concerns the behavior of a Boltzmann machine when some of its units are clamped to a fixed state. It is argued that the clamping assumption is essentially an assertion of the time reversibility of a certain Markov chain underlying the behavior of the Boltzmann machine. As such, the clamping assumption is generally false, though it is certainly true of the Boltzmann machines themselves. The author also considers how the concept of the Boltzmann machine may be generalized while retaining the validity of the clamping assumption.

  2. 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.

  3. Barriers and facilitators to using 9-1-1 and emergency medical services in a limited English proficiency Chinese community.

    PubMed

    Ong, Brandon N; Yip, Mei Po; Feng, Sherry; Calhoun, Rebecca; Meischke, Hendrika W; Tu, Shin-Ping

    2012-04-01

    Effective communication during a medical emergency is crucial for an appropriate emergency medical services (EMS) response. This exploratory qualitative study explored intentions to use 9-1-1 in a Chinese speaking community and the barriers and facilitators to accessing EMS. Focus groups with Chinese adults who self-reported limited English proficiency were conducted. An inductive iterative approach was used to categorize and connect themes identified in the discussions. Language difficulties, negative perceptions of EMS, perceived costs of using emergency services, and no previous experience with 9-1-1 were commonly described as barriers to calling EMS during emergencies. Positive past experiences with EMS and encountering an emergency situation perceived as too great to manage alone are common facilitators for calling 9-1-1. Further exploration is necessary to assess barriers to calling 9-1-1 unique to specific communities, test findings, and tailor interventions to improve EMS communication.

  4. 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.

  5. Laser beam guard clamps

    SciTech Connect

    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.

  6. 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...

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. The PCNA-RFC families of DNA clamps and clamp loaders.

    PubMed

    Majka, Jerzy; Burgers, Peter M J

    2004-01-01

    The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

  12. Targeting checkpoint kinase 1 in cancer therapeutics.

    PubMed

    Tse, Archie N; Carvajal, Richard; Schwartz, Gary K

    2007-04-01

    Progression through the cell cycle is monitored by surveillance mechanisms known as cell cycle checkpoints. Our knowledge of the biochemical nature of checkpoint regulation during an unperturbed cell cycle and following DNA damage has expanded tremendously over the past decade. We now know that dysfunction in cell cycle checkpoints leads to genomic instability and contributes to tumor progression, and most agents used for cancer therapy, such as cytotoxic chemotherapy and ionizing radiation, also activate cell cycle checkpoints. Understanding how checkpoints are regulated is therefore important from the points of view of both tumorigenesis and cancer treatment. In this review, we present an overview of the molecular hierarchy of the checkpoint signaling network and the emerging role of checkpoint targets, especially checkpoint kinase 1, in cancer therapy. Further, we discuss the results of recent clinical trials involving the nonspecific checkpoint kinase 1 inhibitor, UCN-01, and the challenges we face with this new therapeutic approach.

  13. 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.

  14. 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.

  15. Recovery from the DNA Replication Checkpoint

    PubMed Central

    Chaudhury, Indrajit; Koepp, Deanna M.

    2016-01-01

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

  16. Inner Voltage Clamping

    PubMed Central

    Feldberg, Stephen W.; Delgado, Alicia B.

    1978-01-01

    Ketterer, et al. (1971) have suggested that a combination of electrostatic and chemical interactions may cause hydrophobic ions absorbed within a bilayer lipid membrane to reside in two potential wells, each close to a membrane surface. The resulting two planes of charges would define three regions of membrane dielectric: two identical outer regions each between a plane of absorbed charges and the plane of closest approach of ions in the aqueous phase; and the inner region between the two planes of adsorbed charges. The theory describing charge translocation across the inner region is based on a simple three-capacitor model. A significant theoretical conclusion is that the difference between the voltage across the inner region, Vi, and the voltage across the entire membrane, Vm, is directly proportional to the amount of charge that has flowed in a voltage clamp experiment. We demonstrate that we can construct an “inner voltage clamp” that can maintain, with positive feedback, a constant inner voltage, Vi. The manifestation of proper feedback is that the clamp current (after a voltage step) will exhibit pure (i.e., single time-constant) exponential decay, because the voltage dependent rate constants governing translocation will be independent of time. The “pureness” of the exponential is maximized when the standard deviation of the least-square fit of the appropriate exponential equation to the experimental data is minimized. The concomitant feedback is directly related to the capacitances of the inner and outer membrane regions, Ci and Co. Experimental results with tetraphenylborate ion adsorbed in bacterial phosphatidylethanolamine/n-decane bilayers indicate Ci ∼ 5 · 10-7F/cm2 and Co ≈ 5 · 10-5F/cm2. PMID:620078

  17. New checkpoints in cancer immunotherapy.

    PubMed

    Ni, Ling; Dong, Chen

    2017-03-01

    Immune responses must be fine-tuned to allow effective clearance of invading pathogens, while maintain tolerance to self-antigens. T cells are the major effector cells for fighting and killing tumor cells. Immune checkpoints play a pivotal role in T cell activation, and determine the functional outcome of T cell receptor (TCR) signaling. The blockade of immune checkpoints CTLA-4 and PD-1 has already been one of the most successful cancer immunotherapies. In this review, we will focus on three novel inhibitory B7 family checkpoint molecules, B7-H3, B7S1 and VISTA. The aim of this article is to summarize their expressions in tumors as well as their roles in controlling and suppressing T cell immune responses and anti-tumor immunity. These pathways may be explored in future cancer immunotherapy.

  18. 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

  19. Cell cycle regulation by checkpoints.

    PubMed

    Barnum, Kevin J; O'Connell, Matthew J

    2014-01-01

    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.

  20. 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.

  1. Saddle Clamp With Captive Components

    NASA Technical Reports Server (NTRS)

    Belrose, Charles R.

    1993-01-01

    Saddle clamp modified to prevent parts from falling off when installed or removed. Allows easy access for tightening or loosening bolts, and retains alignment with tube mounted in it when opened. All parts are held captive - bolts by retaining washers, floating nuts by pressing and swaging, and upper clamp band by tether. Upper and lower bolt flanges offset from each other to ensure access.

  2. Plastic Clamp Retains Clevis Pin

    NASA Technical Reports Server (NTRS)

    Cortes, R. G.

    1983-01-01

    Plastic clamp requires no special installation or removal tools. Clamp slips easily over end of pin. Once engaged in groove, holds pin securely. Installed and removed easily without special tools - screwdriver or putty knife adequate for prying out of groove. Used to retain bearings, rollers pulleys, other parts that rotate. Applications include slowly and intermittently rotating parts in appliances.

  3. Non-volatile memory for checkpoint storage

    DOEpatents

    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.

  4. 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.

  5. 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.

  6. Phenotypic checkpoints regulate neuronal development.

    PubMed

    Ben-Ari, Yehezkel; Spitzer, Nicholas C

    2010-11-01

    Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and constitute presymptomatic signatures of neurological disorders when they go awry.

  7. 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

  8. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; Hargrove, Paul; Roman, Eric

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core maps and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.

  9. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  10. Surviving the breakup: the DNA damage checkpoint.

    PubMed

    Harrison, Jacob C; Haber, James E

    2006-01-01

    In response to even a single chromosomal double-strand DNA break, cells enact the DNA damage checkpoint. This checkpoint triggers cell cycle arrest, providing time for the cell to repair damaged chromosomes before entering mitosis. This mechanism helps prevent the segregation of damaged or mutated chromosomes and thus promotes genomic stability. Recent work has elucidated the molecular mechanisms underlying several critical steps in checkpoint activation, notably the recruitment of the upstream checkpoint kinases of the ATM and ATR families to different damaged DNA structures and the molecular events through which these kinases activate their effectors. Chromatin modification has emerged as one important component of checkpoint activation and maintenance. Following DNA repair, the checkpoint pathway is inactivated in a process termed recovery. A related but genetically distinct process, adaptation, controls cell cycle re-entry in the face of unrepairable damage.

  11. 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

  12. 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.

  13. Intellectual property issues of immune checkpoint inhibitors

    PubMed Central

    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

  14. 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.

  15. Molecular modeling-based analysis of interactions in the RFC-dependent clamp-loading process.

    PubMed

    Venclovas, Ceslovas; Colvin, Michael E; Thelen, Michael P

    2002-10-01

    Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli delta-beta interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental

  16. 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.

  17. A structural hinge in eukaryotic MutY homologues mediates catalytic activity and Rad9-Rad1-Hus1 checkpoint complex interactions.

    PubMed

    Luncsford, Paz J; Chang, Dau-Yin; Shi, Guoli; Bernstein, Jade; Madabushi, Amrita; Patterson, Dimeka N; Lu, A-Lien; Toth, Eric A

    2010-10-29

    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.

  18. Checkpoint inhibitors in Hodgkin's lymphoma.

    PubMed

    Jezeršek Novaković, Barbara

    2016-04-01

    Hodgkin's lymphoma is unusual among cancers in that it consists of a small number of malignant Hodgkin/Reed-Sternberg cells in a sea of immune system cells, including T cells. Most of these T cells are reversibly inactivated in different ways and their reactivation may induce a very strong immune response to cancer cells. One way of reactivation of T cells is with antibodies blocking the CTLA-4 and especially with antibodies directed against PD-1 or the PD-L1 ligand thereby reversing the tumor-induced downregulation of T-cell function and augmenting antitumor immune activity at the priming (CTLA-4) or tissue effector (PD-1) phase. Immune checkpoint inhibitors have been evidenced as an additional treatment option with substantial effectiveness and acceptable toxicity in heavily pretreated patients with Hodgkin's lymphoma. Particularly, PD-1 blockade with nivolumab and pembrolizumab has demonstrated significant single-agent activity in this select population.

  19. Immune checkpoint therapy for pancreatic cancer

    PubMed Central

    Johansson, Henrik; Andersson, Roland; Bauden, Monika; Hammes, Sarah; Holdenrieder, Stefan; Ansari, Daniel

    2016-01-01

    Novel treatment modalities are necessary for pancreatic cancer. Immunotherapy with immune checkpoint inhibition has shown effect in other solid tumors, and could have a place in pancreatic cancer treatment. Most available clinical studies on immune checkpoint inhibitors for pancreatic cancer are not yet completed and are still recruiting patients. Among the completed trials, there have been findings of a preliminary nature such as delayed disease progression and enhanced overall survival after treatment with immune checkpoint inhibitors in mono- or combination therapy. However, due to small sample sizes, major results are not yet identifiable. The present article provides a clinical overview of immune checkpoint inhibition in pancreatic cancer. PubMed, ClinicalTrials.gov and American Society of Clinical Oncology’s meeting abstracts were systematically searched for relevant clinical studies. Four articles, five abstracts and 25 clinical trials were identified and analyzed in detail. PMID:27920468

  20. 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.

  1. The RFC clamp loader: structure and function.

    PubMed

    Yao, Nina Y; O'Donnell, Mike

    2012-01-01

    The eukaryotic RFC clamp loader couples the energy of ATP hydrolysis to open and close the circular PCNA sliding clamp onto primed sites for use by DNA polymerases and repair factors. Structural studies reveal clamp loaders to be heteropentamers. Each subunit contains a region of homology to AAA+ proteins that defines two domains. The AAA+ domains form a right-handed spiral upon binding ATP. This spiral arrangement generates a DNA binding site within the center of RFC. DNA enters the central chamber through a gap between the AAA+ domains of two subunits. Specificity for a primed template junction is achieved by a third domain that blocks DNA, forcing it to bend sharply. Thus only DNA with a flexible joint can bind the central chamber. DNA entry also requires a slot in the PCNA clamp, which is opened upon binding the AAA+ domains of the clamp loader. ATP hydrolysis enables clamp closing and ejection of RFC, completing the clamp loading reaction.

  2. Toward an optimal online checkpoint solution under a two-level HPC checkpoint model

    DOE PAGES

    Di, Sheng; Robert, Yves; Vivien, Frederic; ...

    2016-03-29

    The traditional single-level checkpointing method suffers from significant overhead on large-scale platforms. Hence, multilevel checkpointing protocols have been studied extensively in recent years. The multilevel checkpoint approach allows different levels of checkpoints to be set (each with different checkpoint overheads and recovery abilities), in order to further improve the fault tolerance performance of extreme-scale HPC applications. How to optimize the checkpoint intervals for each level, however, is an extremely difficult problem. In this paper, we construct an easy-to-use two-level checkpoint model. Checkpoint level 1 deals with errors with low checkpoint/recovery overheads such as transient memory errors, while checkpoint level 2more » deals with hardware crashes such as node failures. Compared with previous optimization work, our new optimal checkpoint solution offers two improvements: (1) it is an online solution without requiring knowledge of the job length in advance, and (2) it shows that periodic patterns are optimal and determines the best pattern. We evaluate the proposed solution and compare it with the most up-to-date related approaches on an extreme-scale simulation testbed constructed based on a real HPC application execution. Simulation results show that our proposed solution outperforms other optimized solutions and can improve the performance significantly in some cases. Specifically, with the new solution the wall-clock time can be reduced by up to 25.3% over that of other state-of-the-art approaches. Lastly, a brute-force comparison with all possible patterns shows that our solution is always within 1% of the best pattern in the experiments.« less

  3. Toward an optimal online checkpoint solution under a two-level HPC checkpoint model

    SciTech Connect

    Di, Sheng; Robert, Yves; Vivien, Frederic; Cappello, Franck

    2016-03-29

    The traditional single-level checkpointing method suffers from significant overhead on large-scale platforms. Hence, multilevel checkpointing protocols have been studied extensively in recent years. The multilevel checkpoint approach allows different levels of checkpoints to be set (each with different checkpoint overheads and recovery abilities), in order to further improve the fault tolerance performance of extreme-scale HPC applications. How to optimize the checkpoint intervals for each level, however, is an extremely difficult problem. In this paper, we construct an easy-to-use two-level checkpoint model. Checkpoint level 1 deals with errors with low checkpoint/recovery overheads such as transient memory errors, while checkpoint level 2 deals with hardware crashes such as node failures. Compared with previous optimization work, our new optimal checkpoint solution offers two improvements: (1) it is an online solution without requiring knowledge of the job length in advance, and (2) it shows that periodic patterns are optimal and determines the best pattern. We evaluate the proposed solution and compare it with the most up-to-date related approaches on an extreme-scale simulation testbed constructed based on a real HPC application execution. Simulation results show that our proposed solution outperforms other optimized solutions and can improve the performance significantly in some cases. Specifically, with the new solution the wall-clock time can be reduced by up to 25.3% over that of other state-of-the-art approaches. Lastly, a brute-force comparison with all possible patterns shows that our solution is always within 1% of the best pattern in the experiments.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Molecular Mechanisms of DNA Replication Checkpoint Activation

    PubMed Central

    Recolin, Bénédicte; van der Laan, Siem; Tsanov, Nikolay; Maiorano, Domenico

    2014-01-01

    The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability. PMID:24705291

  10. Checkpoint triggering in a computer system

    SciTech Connect

    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.

  11. Immune checkpoint inhibitors for cancer treatment.

    PubMed

    Park, Junsik; Kwon, Minsuk; Shin, Eui-Cheol

    2016-11-01

    During immune responses antigen-specific T cells are regulated by several mechanisms, including through inhibitory receptors and regulatory T cells, to avoid excessive or persistent immune responses. These regulatory mechanisms, which are called 'immune checkpoints', suppress T cell responses, particularly in patients with chronic viral infections and cancer where viral antigens or tumor antigens persist for a long time and contribute to T cell exhaustion. Among these regulatory mechanisms, cytotoxic T lymphocyte associated protein-4 (CTLA-4) and programmed cell death 1 (PD-1) are the most well-known receptors and both have been targeted for drug development. As a result, anti-CTLA-4 and anti-PD-1 (or anti-PD-L1) antibodies were recently developed as immune checkpoint inhibitors for use in cancer treatments. In this review we describe several receptors that function as immunological checkpoints as well as the pharmaceuticals that target them.

  12. 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.

  13. 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…

  14. 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

  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. The monogenean which lost its clamps.

    PubMed

    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 steps in an

  17. Detailed Modeling and Evaluation of a Scalable Multilevel Checkpointing System

    SciTech Connect

    Mohror, Kathryn; Moody, Adam; Bronevetsky, Greg; de Supinski, Bronis R.

    2014-09-01

    High-performance computing (HPC) systems are growing more powerful by utilizing more components. As the system mean time before failure correspondingly drops, applications must checkpoint frequently to make progress. But, at scale, the cost of checkpointing becomes prohibitive. A solution to this problem is multilevel checkpointing, which employs multiple types of checkpoints in a single run. Moreover, lightweight checkpoints can handle the most common failure modes, while more expensive checkpoints can handle severe failures. We designed a multilevel checkpointing library, the Scalable Checkpoint/Restart (SCR) library, that writes lightweight checkpoints to node-local storage in addition to the parallel file system. We present probabilistic Markov models of SCR's performance. We show that on future large-scale systems, SCR can lead to a gain in machine efficiency of up to 35 percent, and reduce the load on the parallel file system by a factor of two. In addition, we predict that checkpoint scavenging, or only writing checkpoints to the parallel file system on application termination, can reduce the load on the parallel file system by 20 × on today's systems and still maintain high application efficiency.

  18. The spindle checkpoint and chromosome segregation in meiosis.

    PubMed

    Gorbsky, Gary J

    2015-07-01

    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 made 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 a 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.

  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. 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.

  1. Dynamic clamp with StdpC software.

    PubMed

    Kemenes, Ildikó; Marra, Vincenzo; Crossley, Michael; Samu, Dávid; Staras, Kevin; Kemenes, György; Nowotny, Thomas

    2011-03-01

    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, for 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 through 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 establishing cell recording.

  2. Diverless pipeline repair clamp: Phase 1

    SciTech Connect

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

    1991-12-01

    Offshore oil and gas developments are underway for water depths beyond which divers can function. The economic lifelines of these projects are the pipelines which will transport the products to shore. In preparation for the day when one of these pipelines will require repair because of a leak, the Pipeline Research Committee of the American Gas Association is funding research directed at developing diverless pipeline repair capabilities. Several types of damage are possible, ranging from latent weld defects on one end of the spectrum to damage resulting in parting of the pipe at the other end. This study is specifically directed toward laying the groundwork for development of a diverless pipeline repair clamp for use in repair of leaks resulting from minor pipe defects. The incentive for a clamp type repair is costs. When compared to replacing a section of pipe, either by welding or by mechanical means, the clamp type repair requires much less disturbance of the pipe, less time, fewer operations and less equipment. This report summarizes (1) capabilities of remotely operated vehicles (ROV's) and associated systems, (2) highlights areas for further research and development, (3) describes the required capabilities of the diverless repairclamp, (4) investigates some alternatives to the diverless clamp, (5) overviews the state of the art in leak repair clamps, and (6) critiques several possible generic clamp concepts.

  3. Sound absorption by clamped poroelastic plates.

    PubMed

    Aygun, H; Attenborough, K

    2008-09-01

    Measurements and predictions have been made of the absorption coefficient and the surface acoustic impedance of poroelastic plates clamped in a large impedance tube and separated from the rigid termination by an air gap. The measured and predicted absorption coefficient and surface impedance spectra exhibit low frequency peaks. The peak frequencies observed in the absorption coefficient are close to those predicted and measured in the deflection spectra of the clamped poroelastic plates. The influences of the rigidity of the clamping conditions and the width of the air gap have been investigated. Both influences are found to be important. Increasing the rigidity of clamping reduces the low frequency absorption peaks compared with those measured for simply supported plates or plates in an intermediate clamping condition. Results for a closed cell foam plate and for two open cell foam plates made from recycled materials are presented. For identical clamping conditions and width of air gap, the results for the different materials differ as a consequence mainly of their different elasticity, thickness, and cell structure.

  4. [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.

  5. Perfect Rainbow Tradeoff with Checkpoints Revisited

    PubMed Central

    2016-01-01

    The rainbow tradeoff is an algorithm for inverting one-way functions that is widely used in practice to recover passwords from unsalted password hashes. An auxiliary technique referred to as checkpoints can be applied to the rainbow tradeoff to reduce the time taken for these inversions. Working out a rigorous theory that can explain and predict the effects of this technique involves delicate manipulations of the random function and is thus a challenging task. In this work, we compare three existing theoretical analyses of the checkpoint technique. We first demonstrate that the claims made by the three works are incompatible with each other. We then carry out experiments designed to highlight these incompatibilities, obtaining experimental evidences that show just one of the three analyses to be correct. Finally, we discuss the obscure theoretical errors made by the two inadequate analyses. PMID:27855190

  6. Targeting immune checkpoints in malignant glioma

    PubMed Central

    Li, Tete; Liu, Yong-Jun; Chen, Wei; Chen, Jingtao

    2017-01-01

    Malignant glioma is the most common and a highly aggressive cancer in the central nervous system (CNS). Cancer immunotherapy, strategies to boost the bodys anti-cancer immune responses instead of directly targeting tumor cells, recently achieved great success in treating several human solid tumors. Although once considered immune privileged and devoid of normal immunological functions, CNS is now considered a promising target for cancer immunotherapy, featuring the recent progresses in neurobiology and neuroimmunology and a highly immunosuppressive state in malignant glioma. In this review, we focus on immune checkpoint inhibitors, specifically, antagonizing monoclonal antibodies for programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and indoleamine 2,3-dioxygenase (IDO). We discuss advances in the working mechanisms of these immune checkpoint molecules, their status in malignant glioma, and current preclinical and clinical trials targeting these molecules in malignant glioma. PMID:27756892

  7. Renal effects of immune checkpoint inhibitors.

    PubMed

    Izzedine, Hassan; Mateus, Christine; Boutros, Céline; Robert, Caroline; Rouvier, Philippe; Amoura, Zahir; Mathian, Alexis

    2016-12-26

    Recent advances in immune checkpoint inhibitor (ICPI) development have led to major improvements in oncology patient outcomes. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are two essential immune checkpoint receptors. Ipilimumab and tremelimumab (anti-CTLA-4-blocking antibodies) and pembrolizumab and nivolumab (antibodies targeting PD-1 receptors) have already been approved by US Food and Drug Administration in several malignancies. Two different forms of ICPI-induced renal damage have been identified, including acute (granulomatous) tubulointerstitial nephritis and immune complex glomerulonephritis. The observed acute renal damage can be reversed upon ICPI drug discontinuation and renal function can recover back to normal following the introduction of systemic corticosteroid treatment. Any delay in treating this complication could result in definitive and irreversible renal injury.

  8. Programmed death 1 immune checkpoint inhibitors.

    PubMed

    Trivedi, Meghna S; Hoffner, Brianna; Winkelmann, Jennifer L; Abbott, Maura E; Hamid, Omid; Carvajal, Richard D

    2015-12-01

    Programmed death 1 (PD-1) is an immune checkpoint that provides inhibitory signals to the immune system in order to modulate the activity of T cells in peripheral tissues and maintain self-tolerance in the setting of infection and inflammation. In cancer, the immune checkpoints are exploited so that the tumor cells are able to evade the immune system. Immune checkpoint inhibitors are a type of cancer immunotherapy that targets pathways such as PD-1 in order to reinvigorate and enhance the immune response against tumor cells. The US Food and Drug Administration (FDA) has approved 2 PD-1 inhibitors, nivolumab and pembrolizumab, and several others are under investigation. Although PD-1 inhibitors have demonstrated activity in many different types of malignancies, FDA approval has been granted only in melanoma and in non-small cell lung cancer (NSCLC). Identifying biomarkers that can predict response to PD-1 inhibitors is critical to maximizing the benefit of these agents. Future directions for PD-1 inhibitors include investigation of combination therapies, use in malignancies other than melanoma and NSCLC, and refinement of biomarkers.

  9. 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.

  10. 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....

  11. 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....

  12. 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....

  13. 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....

  14. 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....

  15. 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.

  16. 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.

  17. 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.

  18. The pathophysiology of aortic cross-clamping.

    PubMed

    Zammert, Martin; Gelman, Simon

    2016-09-01

    During open aortic surgery, interrupting the blood flow through the aorta by applying a cross-clamp is often a key step to allow for surgical repair. As a consequence, ischemia is induced in parts of the body distal to the clamp site. This significant alteration in the blood flow is almost always associated with hemodynamic changes. Upon release of the cross-clamp, the blood flow is restored, triggering an ischemia-reperfusion response, leading to many pathophysiological processes such as inflammation, humoral changes, and metabolite circulation that could lead to injury in many organ systems and may significantly influence the postoperative outcome. It is therefore important to understand these processes and how they can be treated in order to allow for safe surgical aortic repairs while ensuring the best possible outcomes.

  19. 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.

  20. A dnaN Plasmid Shuffle Strain for Rapid In Vivo Analysis of Mutant Escherichia coli β Clamps Provides Insight Into the Role of Clamp in umuDC-Mediated Cold Sensitivity

    PubMed Central

    Babu, Vignesh M. P.; Sutton, Mark D.

    2014-01-01

    The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C. PMID:24896652

  1. A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

    PubMed

    Babu, Vignesh M P; Sutton, Mark D

    2014-01-01

    The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

  2. 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

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

    PubMed

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

    2015-01-01

    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 Saccharomyces 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.

  4. Π-Clamp-mediated cysteine conjugation.

    PubMed

    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.

  5. Patch-clamp amplifiers on a chip.

    PubMed

    Weerakoon, Pujitha; Culurciello, Eugenio; Yang, Youshan; Santos-Sacchi, Joseph; Kindlmann, Peter J; Sigworth, Fred J

    2010-10-15

    We present the first, fully integrated, two-channel implementation of a patch-clamp measurement system. With this "PatchChip" two simultaneous whole-cell recordings can be obtained with rms noise of 8pA in a 10kHz bandwidth. The capacitance and series-resistance of the electrode can be compensated up to 10pF and 100MΩ respectively under computer control. Recordings of hERG and Na(v) 1.7 currents demonstrate the system's capabilities, which are on par with large, commercial patch-clamp instrumentation. By reducing patch-clamp amplifiers to a millimeter size micro-chip, this work paves the way to the realization of massively parallel, high-throughput patch-clamp systems for drug screening and ion-channel research. The PatchChip is implemented in a 0.5μm silicon-on-sapphire process; its size is 3×3mm(2) and the power consumption is 5mW per channel with a 3.3V power supply.

  6. Tactical Checkpoint: Hail/Warn Suppress/Stop (Poster)

    DTIC Science & Technology

    2010-11-15

    distractor , optical suppression , human behavior, checkpoint, ambient light, driver suppression , human experimentation, light, paintball, obscuration...HAIL/WARN AND - SUPPRESS /STOP Poster Presented at the 2010 Directed Energies Professional Society Meeting, 15-19 November 2010. 5a. CONTRACT NUMBER...warning to a driver that is approaching a checkpoint. The laser, MCNC light, and the windshield obscuration were evaluated for their suppression

  7. 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.

  8. [Adverse events of immune checkpoint inhibitors].

    PubMed

    Foller, S; Oppel-Heuchel, H; Fetter, I; Winkler, Y; Grimm, M-O

    2017-04-01

    After immune checkpoint inhibitor therapy was approved for renal cell carcinoma last year, this new immune therapy has spread to urology. Further approvals (atezolizumab, nivolumab, pembrolizumab) are expected in 2017 for metastatic urothelial carcinoma that has progressed following treatment with platinum-based chemotherapy. With expanding use of immune checkpoint inhibitors, experience in diagnosing and managing immune-mediated adverse events increases. Although of low incidence, grade 3/4 toxicities play a central role. Organs most common for immune-mediated adverse events are skin, liver (hepatitis), kidneys (nephritis), gastrointestinal tract (diarrhea and colitis), lungs (pneumonitis), and endocrine organs (hyper-, hypothyroidism and hypophysitis). Diagnostic workup includes routine laboratory tests (including liver function tests) and may be supplemented with hormone values. In cases of pneumonitis or hypophysitis, imaging (high-resolution CT, MRI) can confirm diagnoses. Immune-mediated toxicities are treated with therapy interruption and administration of corticosteroids (and in individual cases additional immunosuppression). Dose modification is not intended!

  9. Mammalian oocyte development: checkpoints for competence.

    PubMed

    Fair, Trudee

    2010-01-01

    During the lifespan of the female, biochemical changes occur in the ovarian environment. These changes are brought about by numerous endogenous and exogenous factors, including husbandry practices, production demands and disease, and can have a profound effect on ovarian oocyte quality and subsequent embryo development. Despite many investigations, there is no consensus regarding the time or period of follicular oocyte development that is particularly sensitive to insult. Here, the key molecular and morphological events that occur during oocyte and follicle growth are reviewed, with a specific focus on identifying critical checkpoints in oocyte development. The secondary follicle stage appears to be a key phase in follicular oocyte development because major events such as activation of the oocyte transcriptome, sequestration of the zona pellucida, establishment of bidirectional communication between the granulosa cells and the oocyte and cortical granule synthesis occur during this period of development. Several months later, the periovulatory period is also characterised by the occurrence of critical events, including appropriate degradation or polyadenylation of mRNA transcripts, resumption of meiosis, spindle formation, chromosome alignment and segregation, and so should also be considered as a potential checkpoint of oocyte development.

  10. Dynamics and Stability of Pinned-Clamped and Clamped-Pinned Cylindrical Shells Conveying Fluid

    NASA Astrophysics Data System (ADS)

    Misra, A. K.; Wong, S. S. T.; Païdoussis, M. P.

    2001-11-01

    The paper examines the dynamics and stability of fluid-conveying cylindrical shells having pinned-clamped or clamped-pinned boundary conditions, where ``pinned'' is an abbreviation for ``simply supported''. Flügge's equations are used to describe the shell motion, while the fluid-dynamic perturbation pressure is obtained utilizing the linearized potential flow theory. The solution is obtained using two methods - the travelling wave method and the Fourier-transform approach. The results obtained by both methods suggest that the negative damping of the clamped-pinned systems and positive damping of the pinned-clamped systems, observed by previous investigators for any arbitrarily small flow velocity, are simply numerical artefacts; this is reinforced by energy considerations, in which the work done by the fluid on the shell is shown to be zero. Hence, it is concluded that both systems are conservative.

  11. Mechanical properties of a lap joint under uniform clamping pressure

    NASA Technical Reports Server (NTRS)

    Diller, S. V.; Metherell, A. F.

    1969-01-01

    Equations were derived for the load deflection relations, the energy dissipation per cycle, and the instantaneous rate of dissipation for a lap joint idealized as two overlapping plates clamped together under a uniform clamping pressure.

  12. Towards a Dynamic Clamp for Neurochemical Modalities

    PubMed Central

    Rivera, Catalina Maria; Kwon, Hyuck-Jin; Hashmi, Ali; Yu, Gan; Zhao, Jiheng; Gao, Jianlong; Xu, Jie; Xue, Wei; Dimitrov, Alexander G.

    2015-01-01

    The classic dynamic clamp technique uses a real-time electrical interface between living cells and neural simulations in order to investigate hypotheses about neural function and structure. One of the acknowledged drawbacks of that technique is the limited control of the cells' chemical microenvironment. In this manuscript, we use a novel combination of nanosensor and microfluidic technology and microfluidic and neural simulations to add sensing and control of chemical concentrations to the dynamic clamp technique. Specifically, we use a microfluidic lab-on-a-chip to generate distinct chemical concentration gradients (ions or neuromodulators), to register the concentrations with embedded nanosensors and use the processed signals as an input to simulations of a neural cell. The ultimate goal of this project is to close the loop and provide sensor signals to the microfluidic lab-on-a-chip to mimic the interaction of the simulated cell with other cells in its chemical environment. PMID:25946635

  13. Clamp on ultrasonic instruments in subsea applications

    SciTech Connect

    Haugen, S.; Hodgson, S.; Upchurch, J.; McMahan, J.; Hazelrigg, K.; Mundorff, J.

    1995-12-01

    Monitoring of solids flow in pipelines has until recently required pipework intervention in order to insert probes and switches. Both sand particles and cleaning pigs are by nature destructive to these directly exposed detectors due to erosion and inertia. The instruments presented in this paper provide a superior alternative in ultrasonic clamp-on technology requiring only superficial pipework installation. Wells can now be operated efficiently in a way that minimizes the risk of erosion and collapse. Pigging operations can be monitored both by on-line pig detection and debris monitoring thereby allowing a safe and optimized survey to take place. The non-intrusive clamp-on concept raises standards in instrument reliability, durability and measurement performance.

  14. A tissue checkpoint regulates type 2 immunity

    PubMed Central

    Van Dyken, Steven J.; Nussbaum, Jesse C.; Lee, Jinwoo; Molofsky, Ari B.; Liang, Hong-Erh; Pollack, Joshua L.; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Marson, Alexander; Erle, David J.; Locksley, Richard M.

    2017-01-01

    Group 2 innate lymphoid cells (ILC2s) and CD4+ T helper type 2 (Th2) cells are defined by their similar effector cytokines, which together mediate the features of allergic immunity. Here, we show that tissue ILC2s and Th2 cells differentiate independently but share overlapping effector function programs mediated by exposure to the tissue-derived cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Loss of these three tissue signals does not affect lymph node priming but abrogates terminal differentiation of effector Th2 cells and adaptive lung inflammation in a T cell-intrinsic manner. Our findings suggest how diverse perturbations activate type 2 immunity, uncovering a shared local tissue-elicited checkpoint that can be exploited to control both innate and adaptive allergic inflammation. PMID:27749840

  15. 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.

  16. The Intra-S Checkpoint Responses to DNA Damage

    PubMed Central

    Iyer, Divya Ramalingam; Rhind, Nicholas

    2017-01-01

    Faithful duplication of the genome is a challenge because DNA is susceptible to damage by a number of intrinsic and extrinsic genotoxins, such as free radicals and UV light. Cells activate the intra-S checkpoint in response to damage during S phase to protect genomic integrity and ensure replication fidelity. The checkpoint prevents genomic instability mainly by regulating origin firing, fork progression, and transcription of G1/S genes in response to DNA damage. Several studies hint that regulation of forks is perhaps the most critical function of the intra-S checkpoint. However, the exact role of the checkpoint at replication forks has remained elusive and controversial. Is the checkpoint required for fork stability, or fork restart, or to prevent fork reversal or fork collapse, or activate repair at replication forks? What are the factors that the checkpoint targets at stalled replication forks? In this review, we will discuss the various pathways activated by the intra-S checkpoint in response to damage to prevent genomic instability. PMID:28218681

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

    PubMed Central

    Najor, Nicole A.; Weatherford, Layne; Brush, George S.

    2016-01-01

    In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control. PMID:27678521

  4. 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.

  5. 33 CFR 183.532 - Clips, straps, and hose clamps.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Clips, straps, and hose clamps... (CONTINUED) BOATING SAFETY BOATS AND ASSOCIATED EQUIPMENT Fuel Systems Equipment Standards § 183.532 Clips, straps, and hose clamps. (a) Each clip, strap, and hose clamp must: (1) Be made from a...

  6. 33 CFR 183.532 - Clips, straps, and hose clamps.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Clips, straps, and hose clamps... (CONTINUED) BOATING SAFETY BOATS AND ASSOCIATED EQUIPMENT Fuel Systems Equipment Standards § 183.532 Clips, straps, and hose clamps. (a) Each clip, strap, and hose clamp must: (1) Be made from a...

  7. Damping mechanisms of single-clamped and prestressed double-clamped resonant polymer microbeams

    NASA Astrophysics Data System (ADS)

    Schmid, S.; Hierold, C.

    2008-11-01

    In this article, an investigation of the damping mechanisms of resonant single- and double-clamped polymer microbeams for a frequency range from 10 kHz to 5 MHz is presented. The suspended structures are made of SU-8, an epoxy-type photoresist, by means of a sacrificial layer technique. The vibration was measured with a laser-Doppler vibrometer in high vacuum at different temperatures and at atmospheric pressure. The influence of air damping in rarefied air was investigated and the intrinsic damping mechanisms were determined in high vacuum (p <0.05 Pa). After excluding a variety of possible damping factors, the dominant intrinsic dissipation mechanism of the single-clamped microbeams was understood to be the material damping with maximum quality factors (Q) of around 70 at 20 °C. Quality factors of up to 720 at 20 °C were measured for stringlike double-clamped microbeams, which suggest a different intrinsic damping mechanism than material loss. It is shown that internal damping mechanisms due to flexure and elongation have a small impact on the damping of stretched strings. Modeling the clamping loss based on the wave transmission into the suspended anchor plates indicates that it is the dominant intrinsic dissipation in the prestressed double-clamped microresonators. At atmospheric pressure it was shown that at low frequencies the quality factors of single-clamped and stringlike double-clamped microbeams are limited by the squeeze-film air damping. At high frequencies the quality factors are limited by the specific intrinsic damping. In between the two particular regions with a specific dominant damping mechanism the quality factors show a maximum.

  8. A divergent role of the SIRT1-TopBP1 axis in regulating metabolic checkpoint and DNA damage checkpoint.

    PubMed

    Liu, Tongzheng; Lin, Yi-Hui; Leng, Wenchuan; Jung, Sung Yun; Zhang, Haoxing; Deng, Min; Evans, Debra; Li, Yunhui; Luo, Kuntian; Qin, Bo; Qin, Jun; Yuan, Jian; Lou, Zhenkun

    2014-12-04

    DNA replication is executed only when cells have sufficient metabolic resources and undamaged DNA. Nutrient limitation and DNA damage cause a metabolic checkpoint and DNA damage checkpoint, respectively. Although SIRT1 activity is regulated by metabolic stress and DNA damage, its function in these stress-mediated checkpoints remains elusive. Here we report that the SIRT1-TopBP1 axis functions as a switch for both checkpoints. With glucose deprivation, SIRT1 is activated and deacetylates TopBP1, resulting in TopBP1-Treslin disassociation and DNA replication inhibition. Conversely, SIRT1 activity is inhibited under genotoxic stress, resulting in increased TopBP1 acetylation that is important for the TopBP1-Rad9 interaction and activation of the ATR-Chk1 pathway. Mechanistically, we showed that acetylation of TopBP1 changes the conformation of TopBP1, thereby facilitating its interaction with distinct partners in DNA replication and checkpoint activation. Taken together, our studies identify the SIRT1-TopBP1 axis as a key signaling mode in the regulation of the metabolic checkpoint and the DNA damage checkpoint.

  9. Immunogenic chemotherapy sensitizes tumors to checkpoint blockade therapy

    PubMed Central

    Pfirschke, Christina; Engblom, Camilla; Rickelt, Steffen; Cortez-Retamozo, Virna; Garris, Christopher; Pucci, Ferdinando; Yamazaki, Takahiro; Colame, Vichnou Poirier; Newton, Andita; Redouane, Younes; Lin, Yi-Jang; Wojtkiewicz, Gregory; Iwamoto, Yoshiko; Mino-Kenudson, Mari; Huynh, Tiffany G.; Hynes, Richard O.; Freeman, Gordon J.; Kroemer, Guido; Zitvogel, Laurence; Weissleder, Ralph; Pittet, Mikael J.

    2016-01-01

    SUMMARY Checkpoint blockade immunotherapies can be extraordinarily effective, but may benefit only the minority of patients whose tumors are pre-infiltrated by T cells. Here, using lung adenocarcinoma mouse models, including genetic models, we show that autochthonous tumors that lacked T cell infiltration and resisted current treatment options could be successfully sensitized to host antitumor T cell immunity when using appropriately selected immunogenic drugs (e.g. oxaliplatin combined with cyclophosphamide for treatment against tumors expressing oncogenic Kras and lacking Trp53). The antitumor response was triggered by direct drug actions on tumor cells, relied on innate immune sensing through toll-like receptor 4 signaling, and ultimately depended on CD8+ T cell antitumor immunity. Furthermore, instigating tumor infiltration by T cells sensitized tumors to checkpoint inhibition and controlled cancer durably. These findings indicate that the proportion of cancers responding to checkpoint therapy can be feasibly and substantially expanded by combining checkpoint blockade with immunogenic drugs. PMID:26872698

  10. Radiotherapy and immune checkpoint blockades: a snapshot in 2016

    PubMed Central

    Koo, Taeryool; Kim, In Ah

    2016-01-01

    Immune checkpoint blockades including monoclonal antibodies (mAbs) of cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1) have been emerged as a promising anticancer therapy. Several immune checkpoint blockades have been approved by US Food and Drug Administration (FDA), and have shown notable success in clinical trials for patients with advanced melanoma and non-small cell lung cancer. Radiotherapy is a promising combination partner of immune checkpoint blockades due to its potent pro-immune effect. This review will cover the current issue and the future perspectives for combined with radiotherapy and immune checkpoint blockades based upon the available preclinical and clinical data. PMID:28030901

  11. Testing and evaluation of stretching strain in clamped-clamped beams for energy harvesting

    NASA Astrophysics Data System (ADS)

    Emad, Ahmed; Mahmoud, Mohamed A. E.; Ghoneima, Maged; Dessouky, Mohamed

    2016-11-01

    In this paper, evaluation of stretching strain capabilities to harvest energy from a piezoelectric clamped-clamped beam is theoretically modeled and experimentally tested. The utilization of stretching strain has many advantages as: elimination of any substrate material, and the simple electrode configuration. The doubly clamped structure exhibits a highly nonlinear frequency response (Hardening Duffing) that widens the bandwidth during the frequency up-ward sweep. The wide bandwidth makes it suitable for practical applications. A design of 53.5 {{mm}}3 (29.7 {{mm}}3 piezoelectric material + 23.8 {{mm}}3 proof mass) energy harvester was tested using PVDF (polyvinylidene fluoride) that can generate up to 15 μW from vibrations of 0.5{g} at 128 {Hz} and 2 MΩ load. The design can also generate up to 41 μ {{W}} from vibrations of 1{g} at 140 {Hz} and 2 MΩ load.

  12. Timing of umbilical cord clamping of term infants.

    PubMed

    Ceriani Cernadas, José María

    2017-04-01

    For at least over 200 years, multiple controversies have arisen around the timing of umbilical cord clamping. In the past decades, early cord clamping (within the first 15 seconds) had markedly prevailed. Only in the 21st century, randomized controlled trials have demonstrated the benefits of delayed cord clamping (at 2-3 minutes). Delayed cord clamping has been practiced in obstetrics based on the recommendations made by scientific societies and in systematic reviews, which have provided solid evidence to support this practice in term infants. This review describes the most relevant factors supporting the use of delayed cord clamping in term infants. It points out the essential role played by physiological mechanisms which, undoubtedly, allow us to understand the benefits of delayed cord clamping and advise us to wait for what nature has established. Other relevant aspects supporting delayed cord clamping are also described here.

  13. Tactical Checkpoint: Hail/Warn Suppress/Stop (Briefing Charts)

    DTIC Science & Technology

    2010-11-15

    U.S. Army Research, Development and Engineering Command Tactical Checkpoint: Hail/Warn Suppress /Stop Target Behavioral Response Laboratory... SUPPRESS /STOP Presented at the 2010 Directed Energies Professional Society Meeting, 15-19 November 2010. 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c...driver that is approaching a checkpoint. The laser, MCNC light, and the windshield obscuration were evaluated for their suppression capabilities (ability

  14. Whole-GUV patch-clamping.

    PubMed

    Garten, Matthias; Mosgaard, Lars D; Bornschlögl, Thomas; Dieudonné, Stéphane; Bassereau, Patricia; Toombes, Gilman E S

    2017-01-10

    Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

  15. HORMAD1-dependent checkpoint/surveillance mechanism eliminates asynaptic oocytes.

    PubMed

    Kogo, Hiroshi; Tsutsumi, Makiko; Ohye, Tamae; Inagaki, Hidehito; Abe, Takaya; Kurahashi, Hiroki

    2012-06-01

    Meiotic pachytene checkpoints monitor the failure of homologous recombination and synapsis to ensure faithful chromosome segregation during gamete formation. To date, the molecular basis of the mammalian pachytene checkpoints has remained largely unknown. We here report that mouse HORMAD1 is required for a meiotic prophase checkpoint that eliminates asynaptic oocytes. Hormad1-deficient mice are infertile and show an extensive failure of homologous pairing and synapsis, consistent with the evolutionarily conserved function of meiotic HORMA domain proteins. Unexpectedly, Hormad1-deficient ovaries contain a normal number of oocytes despite asynapsis and consequently produce aneuploid oocytes, indicating a checkpoint failure. By the analysis of Hormad1/Spo11 double mutants, the Hormad1 deficiency was found to abrogate the massive oocyte loss in the Spo11-deficient ovary. The Hormad1 deficiency also causes the eventual loss of pseudo sex body in the Spo11-deficient ovary and testis. These results suggest the involvement of HORMAD1 in the repressive chromatin domain formation that is proposed to be important in the meiotic prophase checkpoints. We also show the extensive phosphorylation of HORMAD1 in the Spo11-deficient testis and ovary, suggesting an involvement of novel DNA damage-independent phosphorylation signaling in the surveillance mechanism. Our present results provide clues to HORMAD1-dependent checkpoint in response to asynapsis in mammalian meiosis.

  16. Patch Clamping Protoplasts from Vascular Plants 1

    PubMed Central

    Elzenga, J. Theo M.; Keller, Christopher P.; Van Volkenburgh, Elizabeth

    1991-01-01

    A method is described for the isolation of protoplasts (Pisum sativum, Phaseolus vulgaris, Avena sativa, Arabidopsis thaliana) in preparation for ion flux studies using patch clamp electrophysiology. Protoplasts that have been exposed to hydrolytic, cell wall degrading, enzymes for as little as 5 minutes form gigaseals (seal resistance higher than 10 giga Ohm) with the patch pipette with success rates greater than 40%. Sealing of these protoplasts is fast, averaging less than 2 minutes. This method yields high rates of gigaseal formation in a variety of tissues from both monocots and dicots and will enhance data collection in ion flux studies of plasma membranes of vascular plants. PMID:16668586

  17. The Clamp Loader Assembles the β Clamp onto Either a 3′ or 5′ Primer Terminus

    PubMed Central

    Park, Mee Sook; O'Donnell, Mike

    2009-01-01

    Clamp loaders assemble sliding clamps onto 3′ primed sites for DNA polymerases. Clamp loaders are thought to be specific for a 3′ primed site, and unable to bind a 5′ site. We demonstrate here that the Escherichia coli γ complex clamp loader can load the β clamp onto a 5′ primed site, although with at least 20-fold reduced efficiency relative to loading at a 3′ primed site. Preferential clamp loading at a 3′ site does not appear to be due to DNA binding, as the clamp loader forms an avid complex with β at a 5′ site. Preferential loading at a 3′ versus a 5′ site occurs at the ATP hydrolysis step, needed to close the ring around DNA. We also address DNA structural features that are recognized for preferential loading at a 3′ site. Although the single-stranded template strand extends in opposite directions from 3′ and 5′ primed sites, thus making it a favorite candidate for distinguishing between 3′ and 5′ sites, the single-strand polarity at a primed template junction does not determine 3′ site selection for clamp loading. Instead, we find that clamp loader recognition of a 3′ site lies in the duplex portion of the primed site, not the single-strand portion. We present evidence that the β clamp facilitates its own loading specificity for a 3′ primed site. Implications to eukaryotic clamp loader complexes are proposed. PMID:19759020

  18. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4

    PubMed Central

    Chan, Leon Y.; Amon, Angelika

    2009-01-01

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function. PMID:19605686

  19. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4.

    PubMed

    Chan, Leon Y; Amon, Angelika

    2009-07-15

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.

  20. 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.

  1. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    PubMed

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

  2. 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

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

    PubMed Central

    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. DOI: http://dx.doi.org/10.7554/eLife.04463.001 PMID:25831964

  4. 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.

  5. 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.

  6. Nanoindentation characteristics of clamped freestanding Cu membranes.

    PubMed

    Wang, Tong Hong; Fang, Te-Hua; Kang, Shao-Hui; Lin, Yu-Cheng

    2007-04-04

    This research employed instrumented nanoindentation to address the issue of bending to stretching-induced deformation of clamped freestanding Cu membranes. The experimental results show that indentation-induced plastic deformation only comes into effect at the centre and the indented edge of the Cu membrane when the indenter is applied, while the other locations remain undamaged. A step-by-step evolution was presumed for the time histories of the bending to stretching-induced deformation and for the timing of the significant change in slope of the load-deflection curve. Deformation was deliberately introduced at the transition from the single-point bending indentation to the surface stretching indentation at the impact location touched with the indenter. Good elastic recovery was found at locations away from the indenter. A similar finding can be arrived at by means of finite element analysis.

  7. Pipe lifting hook having clamp assembly

    SciTech Connect

    Codner, J.A.

    1984-06-12

    A pipe lifting hook is provided having a generally ''C'' shaped hook member having an elongated lower portion being insertable within the end of a joint of pipe and having an upper portion positionable above the pipe and provided with lifting connection means. The hook member is frictionally clamped to the pipe by grip shoe means that is movably supported by the upper portion of the hook member and is selectably movable from a released position out of contact with said pipe to a locked position in frictional locking engagement with the outer surface of the pipe. A ratchet mechanism couples said grip shoe means to the upper portion of the hook member and is manually positionable to lock said grip shoe means at said locked position or release said grip shoe means for movement toward said released position thereof.

  8. WWOX modulates the ATR-mediated DNA damage checkpoint response.

    PubMed

    Abu-Odeh, Mohammad; Hereema, Nyla A; Aqeilan, Rami I

    2016-01-26

    For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability.

  9. WWOX modulates the ATR-mediated DNA damage checkpoint response

    PubMed Central

    Abu-Odeh, Mohammad; Hereema, Nyla A.; Aqeilan, Rami I.

    2016-01-01

    For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability. PMID:26675548

  10. PKCɛ switches Aurora B specificity to exit the abscission checkpoint

    PubMed Central

    Pike, Tanya; Brownlow, Nicola; Kjaer, Svend; Carlton, Jeremy; Parker, Peter J.

    2016-01-01

    The ‘NoCut', or Aurora B abscission checkpoint can be activated if DNA is retained in the cleavage furrow after completion of anaphase. Checkpoint failure leads to incomplete abscission and a binucleate outcome. These phenotypes are also observed after loss of PKCɛ in transformed cell models. Here we show that PKCɛ directly modulates the Aurora B-dependent abscission checkpoint by phosphorylating Aurora B at S227. This phosphorylation invokes a switch in Aurora B specificity, with increased phosphorylation of a subset of target substrates, including the CPC subunit Borealin. This switch is essential for abscission checkpoint exit. Preventing the phosphorylation of Borealin leads to abscission failure, as does expression of a non-phosphorylatable Aurora B S227A mutant. Further, depletion of the ESCRT-III component and Aurora B substrate CHMP4C enables abscission, bypassing the PKCɛ–Aurora B exit pathway. Thus, we demonstrate that PKCɛ signals through Aurora B to exit the abscission checkpoint and complete cell division. PMID:28004745

  11. 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

  12. Kinetochore localization of spindle checkpoint proteins: who controls whom?

    PubMed

    Vigneron, Suzanne; Prieto, Susana; Bernis, Cyril; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2004-10-01

    The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.

  13. PKCɛ switches Aurora B specificity to exit the abscission checkpoint.

    PubMed

    Pike, Tanya; Brownlow, Nicola; Kjaer, Svend; Carlton, Jeremy; Parker, Peter J

    2016-12-22

    The 'NoCut', or Aurora B abscission checkpoint can be activated if DNA is retained in the cleavage furrow after completion of anaphase. Checkpoint failure leads to incomplete abscission and a binucleate outcome. These phenotypes are also observed after loss of PKCɛ in transformed cell models. Here we show that PKCɛ directly modulates the Aurora B-dependent abscission checkpoint by phosphorylating Aurora B at S227. This phosphorylation invokes a switch in Aurora B specificity, with increased phosphorylation of a subset of target substrates, including the CPC subunit Borealin. This switch is essential for abscission checkpoint exit. Preventing the phosphorylation of Borealin leads to abscission failure, as does expression of a non-phosphorylatable Aurora B S227A mutant. Further, depletion of the ESCRT-III component and Aurora B substrate CHMP4C enables abscission, bypassing the PKCɛ-Aurora B exit pathway. Thus, we demonstrate that PKCɛ signals through Aurora B to exit the abscission checkpoint and complete cell division.

  14. The emerging role of immune checkpoint inhibition in malignant lymphoma

    PubMed Central

    Hude, Ida; Sasse, Stephanie; Engert, Andreas; Bröckelmann, Paul J.

    2017-01-01

    To evade elimination by the host immune system, tumor cells commonly exploit physiological immune checkpoint pathways, restraining efficient anti-tumor immune cell function. Growing understanding of the complex dialog between tumor cells and their microenvironment contributed to the development of immune checkpoint inhibitors. This innovative strategy has demonstrated paradigm-shifting clinical activity in various malignancies. Antibodies targeting programmed death 1 and cytotoxic T-lymphocyte-associated protein-4 are also being investigated in lymphoid malignancies with varying levels of activity and a favorable toxicity profile. To date, evaluated only in the setting of relapsed or refractory disease, anti-programmed death 1 antibodies such as nivolumab and pembrolizumab show encouraging response rates particularly in classical Hodgkin lymphoma but also in follicular lymphoma and diffuse-large B-cell lymphoma. As the first immune checkpoint inhibitor in lymphoma, nivolumab was approved for the treatment of relapsed or refractory classical Hodgkin lymphoma by the Food and Drug Administration in May 2016. In this review, we assess the role of the pathways involved and potential rationale of checkpoint inhibition in various lymphoid malignancies. In addition to data from current clinical trials, immune-related side effects, potential limitations and future perspectives including promising combinatory approaches with immune checkpoint inhibition are discussed. PMID:27884973

  15. 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.

  16. Immune checkpoint and inflammation as therapeutic targets in pancreatic carcinoma

    PubMed Central

    Kimbara, Shiro; Kondo, Shunsuke

    2016-01-01

    Pancreatic adenocarcinoma (PAC) is one of the most deadly malignant neoplasms, and the efficacy of conventional cytotoxic chemotherapy is far from satisfactory. Recent research studies have revealed that immunosuppression and inflammation are associated with oncogenesis, as well as tumor development, invasion, and metastasis in PAC. Thus, immunosuppression-related signaling, especially that involving immune checkpoint and inflammation, has emerged as novel treatment targets for PAC. However, PAC is an immune-resistant tumor, and it is still unclear whether immune checkpoint or anti-inflammation therapies would be an ideal strategy. In this article, we will review immune checkpoint and inflammation as potential targets, as well as clinical trials and the prospects for immunotherapy in PAC. PMID:27672267

  17. The Abscission Checkpoint: Making It to the Final Cut.

    PubMed

    Nähse, Viola; Christ, Liliane; Stenmark, Harald; Campsteijn, Coen

    2017-01-01

    Cytokinesis is the final stage of cell division and is concluded by abscission of the intercellular bridge to physically separate the daughter cells. Timing of cytokinetic abscission is monitored by a molecular machinery termed the abscission checkpoint. This machinery delays abscission in cells with persistent chromatin in the intercellular bridge. Recent work has also uncovered its response to high membrane tension, nuclear pore defects, and DNA replication stress. Although it is known that the abscission checkpoint depends on persistent activity of the Aurora B protein kinase, we have only recently begun to understand its molecular basis. We propose here a molecular framework for abscission checkpoint signaling and we discuss outstanding questions relating to its function and physiological relevance.

  18. Combination Approaches with Immune-Checkpoint Blockade in Cancer Therapy.

    PubMed

    Swart, Maarten; Verbrugge, Inge; Beltman, Joost B

    2016-01-01

    In healthy individuals, immune-checkpoint molecules prevent autoimmune responses and limit immune cell-mediated tissue damage. Tumors frequently exploit these molecules to evade eradication by the immune system. Over the past years, immune-checkpoint blockade of cytotoxic T lymphocyte antigen-4 and programed death-1 emerged as promising strategies to activate antitumor cytotoxic T cell responses. Although complete regression and long-term survival is achieved in some patients, not all patients respond. This review describes promising, novel combination approaches involving immune-checkpoint blockade in the context of the cancer-immunity cycle, aimed at increasing response rates to the single treatments. Specifically, we discuss combinations that promote antigen release and presentation, that further amplify T cell activation, that inhibit trafficking of regulatory T cells or MSDCs, that stimulate intratumoral T cell infiltration, that increase cancer recognition by T cells, and that stimulate tumor killing.

  19. Analysis of the Functionality of the Mitotic Checkpoints.

    PubMed

    Fraschini, Roberta

    2017-01-01

    During cell division the main goal of the cell is to produce two daughter cells with the same genome as the mother, i.e., maintain its genetic stability. Since this issue is essential to preserve the cell ability to proliferate properly, all eukaryotic cells have developed several pathways, called mitotic checkpoints, that regulate mitotic entry, progression, and exit in response to different cellular signals. Given the evolutive conservation of mechanisms and proteins involved in the cell cycle control from yeast to humans, the budding yeast S. cerevisiae has been very helpful to gain insight in these complex regulations. Here, we describe how the checkpoint can be activated and which cellular phenotypes can be used as markers of checkpoint activation.

  20. Combination Approaches with Immune-Checkpoint Blockade in Cancer Therapy

    PubMed Central

    Swart, Maarten; Verbrugge, Inge; Beltman, Joost B.

    2016-01-01

    In healthy individuals, immune-checkpoint molecules prevent autoimmune responses and limit immune cell-mediated tissue damage. Tumors frequently exploit these molecules to evade eradication by the immune system. Over the past years, immune-checkpoint blockade of cytotoxic T lymphocyte antigen-4 and programed death-1 emerged as promising strategies to activate antitumor cytotoxic T cell responses. Although complete regression and long-term survival is achieved in some patients, not all patients respond. This review describes promising, novel combination approaches involving immune-checkpoint blockade in the context of the cancer-immunity cycle, aimed at increasing response rates to the single treatments. Specifically, we discuss combinations that promote antigen release and presentation, that further amplify T cell activation, that inhibit trafficking of regulatory T cells or MSDCs, that stimulate intratumoral T cell infiltration, that increase cancer recognition by T cells, and that stimulate tumor killing. PMID:27847783

  1. Epigenetic regulation of immune checkpoints: another target for cancer immunotherapy?

    PubMed

    Ali, Mahmoud A; Matboli, Marwa; Tarek, Marwa; Reda, Maged; Kamal, Kamal M; Nouh, Mahmoud; Ashry, Ahmed M; El-Bab, Ahmed Fath; Mesalam, Hend A; Shafei, Ayman El-Sayed; Abdel-Rahman, Omar

    2017-01-01

    Epigenetic changes in oncogenes and tumor-suppressor genes contribute to carcinogenesis. Understanding the epigenetic and genetic components of tumor immune evasion is crucial. Few cancer genetic mutations have been linked to direct correlations with immune evasion. Studies on the epigenetic modulation of the immune checkpoints have revealed a critical interaction between epigenetic and immune modulation. Epigenetic modifiers can activate many silenced genes. Some of them are immune checkpoints regulators that turn on immune responses and others turn them off resulting in immune evasion. Many forms of epigenetic inheritance mechanisms may play a role in regulation of immune checkpoints including: covalent modifications, noncoding RNA and histone modifications. In this review, we will show how the potential interaction between epigenetic and immune modulation may lead to new approaches for specific epigenome/immunome-targeted therapies for cancer.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. A quantitative systems view of the spindle assembly checkpoint

    PubMed Central

    Ciliberto, Andrea; Shah, Jagesh V

    2009-01-01

    The idle assembly checkpoint acts to delay chromosome segregation until all duplicated sister chromatids are captured by the mitotic spindle. This pathway ensures that each daughter cell receives a complete copy of the genome. The high fidelity and robustness of this process have made it a subject of intense study in both the experimental and computational realms. A significant number of checkpoint proteins have been identified but how they orchestrate the communication between local spindle attachment and global cytoplasmic signalling to delay segregation is not yet understood. Here, we propose a systems view of the spindle assembly checkpoint to focus attention on the key regulators of the dynamics of this pathway. These regulators in turn have been the subject of detailed cellular measurements and computational modelling to connect molecular function to the dynamics of spindle assembly checkpoint signalling. A review of these efforts reveals the insights provided by such approaches and underscores the need for further interdisciplinary studies to reveal in full the quantitative underpinnings of this cellular control pathway. PMID:19629044

  8. Immunomodulatory Drugs: Immune Checkpoint Agents in Acute Leukemia

    PubMed Central

    Knaus, Hanna A.; Kanakry, Christopher G.; Luznik, Leo; Gojo, Ivana

    2016-01-01

    Intrinsic immune responses to acute leukemia are inhibited by a variety of mechanisms, such as aberrant antigen expression by leukemia cells, secretion of immunosuppressive cytokines and expression of inhibitory enzymes in the tumor microenvironment, expansion of immunoregulatory cells, and activation of immune checkpoint pathways, all leading to T cell dysfunction and/or exhaustion. Leukemic cells, similar to other tumor cells, hijack these inhibitory pathways to evade immune recognition and destruction by cytotoxic T lymphocytes. Thus, blockade of immune checkpoints has emerged as a highly promising approach to augment innate anti-tumor immunity in order to treat malignancies. Most evidence for the clinical efficacy of this immunotherapeutic strategy has been seen in patients with metastatic melanoma, where anti-CTLA-4 and anti-PD-1 antibodies have recently revolutionized treatment of this lethal disease with otherwise limited treatment options. To meet the high demand for new treatment strategies in acute leukemia, clinical testing of these promising therapies is commencing. Herein, we review the biology of multiple inhibitory checkpoints (including CTLA-4, PD-1, TIM-3, LAG-3, BTLA, and CD200R) and their contribution to immune evasion by acute leukemias. In addition, we discuss the current state of preclinical and clinical studies of immune checkpoint inhibition in acute leukemia, which seek to harness the body’s own immune system to fight leukemic cells. PMID:25981611

  9. [Immune Checkpoint for a Kidney Cancer and Other Cancers].

    PubMed

    Kageyama, Shunichiro; Yamaguchi, Shigeo; Miura, Kayo; Kato, Shunsuke

    2016-06-01

    Immune checkpoint inhibitors have been getting increasing attention in the field of cancer treatment, resulting in the investigation of numerous drugs and target cancers. Clinical trials of immune checkpoint inhibitors have focused on malignant melanomas and non-small cell lung cancer;however, recently, clinical trials have been carried out for other cancers. To date, 31 phase III clinical trials have been conducted for 13 types of cancer. Recently, the results of the CheckMate025 kidney cancer and CheckMate141 head and neck cancer trials have been reported. These reports showed that nivolumab significantly enhanced overall survival in comparison to that associated with an existing second-line treatment drug. Based on these results, the approval of nivolumab for use in renal cell cancer and head and neck cancer is expected in the near future. Furthermore, the results of 20 phase III clinical trials will be submitted from 2017 to 2019, expanding the approval of immune checkpoint inhibitors. However, many issues such as biomarker searches, the evaluation of antitumor effects, and the impact on medical economy remain to be resolved. In this report, we outline clinical trial trends and the future prospects for immune checkpoint inhibitors.

  10. Immunomodulatory Drugs: Immune Checkpoint Agents in Acute Leukemia.

    PubMed

    Knaus, Hanna A; Kanakry, Christopher G; Luznik, Leo; Gojo, Ivana

    2017-01-01

    Intrinsic immune responses to acute leukemia are inhibited by a variety of mechanisms, such as aberrant antigen expression by leukemia cells, secretion of immunosuppressive cytokines and expression of inhibitory enzymes in the tumor microenvironment, expansion of immunoregulatory cells, and activation of immune checkpoint pathways, all leading to T cell dysfunction and/or exhaustion. Leukemic cells, similar to other tumor cells, hijack these inhibitory pathways to evade immune recognition and destruction by cytotoxic T lymphocytes. Thus, blockade of immune checkpoints has emerged as a highly promising approach to augment innate anti-tumor immunity in order to treat malignancies. Most evidence for the clinical efficacy of this immunotherapeutic strategy has been seen in patients with metastatic melanoma, where anti-CTLA-4 and anti-PD-1 antibodies have recently revolutionized treatment of this lethal disease with otherwise limited treatment options. To meet the high demand for new treatment strategies in acute leukemia, clinical testing of these promising therapies is commencing. Herein, we review the biology of multiple inhibitory checkpoints (including CTLA-4, PD-1, TIM-3, LAG-3, BTLA, and CD200R) and their contribution to immune evasion by acute leukemias. In addition, we discuss the current state of preclinical and clinical studies of immune checkpoint inhibition in acute leukemia, which seek to harness the body's own immune system to fight leukemic cells.

  11. Trends in Aortic Clamp Use During Coronary Artery Bypass Surgery: The Effect of Aortic Clamping Strategies on Neurologic Outcomes

    PubMed Central

    Daniel, William T.; Kilgo, Patrick; Puskas, John D.; Thourani, Vinod H.; Lattouf, Omar M.; Guyton, Robert A.; Halkos, Michael E.

    2013-01-01

    Objective The purpose of this study was to determine the impact of different clamping strategies during CABG on the incidence of postoperative stroke. Methods In this case-control study, all patients at Emory hospitals from 2002–2009 with postoperative stroke after isolated CABG (N=141) were matched 1:4 to a contemporaneous cohort of patients without postoperative stroke (N=565). Patients were matched according to the Society of Thoracic Surgeons (STS) Predicted Risk of Postoperative Stroke (PROPS), which is based on 26 variables. On- (ONCAB) and off-pump (OPCAB) patients were matched separately. Multiple logistic regression analysis with adjusted odds ratios (OR) was performed to identify operative variables associated with postoperative stroke. Results Among the ONCAB cohort, the single cross-clamp technique was associated with a decreased risk of stroke compared to the double clamp (cross clamp + partial clamp) technique (OR=0.385, p=0.044). Within the OPCAB cohort, there was no significant difference in stroke according to clamp use. Epiaortic ultrasound of the ascending aorta increased from 45.3% in 2002 to 89.4% in 2009. From 2002–2009, clamp use decreased from 97.7% of cases to 72.7%. Conclusions During ONCAB, the use of a single cross-clamp compared to the double clamp technique decreases the risk of postoperative stroke. The use of any aortic clamp has decreased and epiaortic ultrasound use has increased from 2002–2009, indicating a change in operative technique and surgeon awareness of the potential complications associated with manipulation of the aorta. PMID:23477689

  12. Single electrode dynamic clamp with StdpC

    PubMed Central

    Samu, David; Marra, Vincenzo; Kemenes, Ildiko; Crossley, Michael; Kemenes, György; Staras, Kevin; Nowotny, Thomas

    2012-01-01

    Dynamic clamp is a powerful approach for electrophysiological investigations allowing researchers to introduce artificial electrical components into target neurons to simulate ionic conductances, chemical or electrotonic inputs or connections to other cells. Due to the rapidly changing and potentially large current injections during dynamic clamp, problematic voltage artifacts appear on the electrode used to inject dynamic clamp currents into a target neuron. Dynamic clamp experiments, therefore, typically use two separate electrodes in the same cell, one for recording membrane potential and one for injecting currents. The requirement for two independent electrodes has been a limiting factor for the use of dynamic clamp in applications where dual recordings of this kind are difficult or impossible to achieve. The recent development of an active electrode compensation (AEC) method has overcome some of these prior limitations, permitting artifact-free dynamic clamp experimentation with a single electrode. Here we describe an AEC method for the free dynamic clamp software StdpC. The AEC component of StdpC is the first such system implemented for the use of non-expert users and comes with a set of semi-automated configuration and calibration procedures that facilitate its use. We briefly introduce the AEC method and its implementation in StdpC and then validate it with an electronic model cell and in two different biological preparations. PMID:22898473

  13. Single electrode dynamic clamp with StdpC.

    PubMed

    Samu, David; Marra, Vincenzo; Kemenes, Ildiko; Crossley, Michael; Kemenes, György; Staras, Kevin; Nowotny, Thomas

    2012-10-15

    Dynamic clamp is a powerful approach for electrophysiological investigations allowing researchers to introduce artificial electrical components into target neurons to simulate ionic conductances, chemical or electrotonic inputs or connections to other cells. Due to the rapidly changing and potentially large current injections during dynamic clamp, problematic voltage artifacts appear on the electrode used to inject dynamic clamp currents into a target neuron. Dynamic clamp experiments, therefore, typically use two separate electrodes in the same cell, one for recording membrane potential and one for injecting currents. The requirement for two independent electrodes has been a limiting factor for the use of dynamic clamp in applications where dual recordings of this kind are difficult or impossible to achieve. The recent development of an active electrode compensation (AEC) method has overcome some of these prior limitations, permitting artifact-free dynamic clamp experimentation with a single electrode. Here we describe an AEC method for the free dynamic clamp software StdpC. The AEC component of StdpC is the first such system implemented for the use of non-expert users and comes with a set of semi-automated configuration and calibration procedures that facilitate its use. We briefly introduce the AEC method and its implementation in StdpC and then validate it with an electronic model cell and in two different biological preparations.

  14. Avoiding aortic clamping during CABG reduces postoperative stroke

    PubMed Central

    Moss, Emmanuel; Puskas, John D; Thourani, Vinod H; Kilgo, Patrick; Chen, Edward P; Leshnower, Bradley G; Lattouf, Omar M; Guyton, Robert A.; Glas, Kathryn E; Halkos, Michael E.

    2014-01-01

    Objective The purpose of this study was to determine whether the incidence of postoperative stroke (PS) could be reduced by eliminating aortic clamping during CABG. Methods From 2002–2013, 12,079 patients underwent primary, isolated CABG at a single US academic institution. Aortic manipulation was completely avoided by using in-situ internal mammary arteries for inflow in 1,552 (12.9%) patients (no-touch), a clampless facilitating device (CFD) was used for proximal anastomoses in 1,548 (12.8%) patients, and aortic clamping was used in 8,979 (74.3%) patients. These strategies were assessed in a logistic regression model controlling for relevant variables. Results The overall incidence of PS was 1.4% (n=165), with an unadjusted incidence of 0.6% (n=10) in the no-touch group, 1.2% (n=18) in the CFD group, and 1.5% (n=137) in the clamp group (p<0.01 for no-touch vs clamp). The ratio of observed to expected stroke rate increased as the degree of aortic manipulation increased, from 0.48 in the no-touch group, to 0.61 in the CFD group, and 0.95 in the clamp group. Aortic clamping was independently associated with an increase in PS compared to a no-touch technique (AOR 2.50, p<0.01). When separated by CPB utilization, both the off-pump partial clamp and on-pump cross-clamp techniques increased the risk of PS compared to no-touch (AOR 2.52, p<0.01 and AOR 4.25, p<0.001, respectively). Conclusion A no-aortic touch technique has the lowest risk for postoperative stroke for patients undergoing CABG. Clamping the aorta during CABG increases the risk of PS, regardless of the severity of aortic disease. PMID:25293356

  15. Reciprocating clamp apparatus for thermoforming plastic containers

    SciTech Connect

    Beck, M.H.; Harry, I.L.; Krishnakumar, S.M.

    1984-03-06

    This relates to the forming of containers and like hollow articles from sheets or webs of thermoplastic material. Two webs or sheets are simultaneously acted upon by way of a forming apparatus which includes a reciprocating clamp first cooperable with one outer platen and then the other in sequence wherein, while a first web or sheet is being formed within a plurality of mold cavities to define a plurality of hollow articles such as containers, the other sheet or web may be stripped from its respective mold set and a new sheet or a new portion of a sheet or web may be advanced into position for molding. The forming apparatus may be constructed in a manner wherein the web portions which are to be formed may be billowed away from the mold cavities as an initial step in the stretching and orientation of the thermoplastic material. The thermoplastic material may be heated to the desired forming temperature using separate sets of rf electrodes so that only those portions of the thermoplastic material which are to be formed need be heated. Two sets of containers of different sizes may be formed from the webs or sheets, and then internested to form double wall containers wherein the walls may be formed of different materials and wherein the outer material may be a barrier material, or wherein the walls of the containers are spaced apart to form an insulated container. This abstract forms no part of the specification of this application and is not to be construed as limiting the claims of the application.

  16. 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

  17. The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis.

    PubMed

    Venkei, Zsolt G; Yamashita, Yukiko M

    2015-01-01

    Asymmetric cell division is utilized by a broad range of cell types to generate two daughter cells with distinct cell fates. In stem cell populations asymmetric cell division is believed to be crucial for maintaining tissue homeostasis, failure of which can lead to tissue degeneration or hyperplasia/tumorigenesis. Asymmetric cell divisions also underlie cell fate diversification during development. Accordingly, the mechanisms by which asymmetric cell division is achieved have been extensively studied, although the check points that are in place to protect against potential perturbation of the process are poorly understood. Drosophila melanogaster male germline stem cells (GSCs) possess a checkpoint, termed the centrosome orientation checkpoint (COC), that monitors correct centrosome orientation with respect to the component cells of the niche to ensure asymmetric stem cell division. To our knowledge, the COC is the only checkpoint mechanism identified to date that specializes in monitoring the orientation of cell division in multicellular organisms. Here, by establishing colcemid-induced microtubule depolymerization as a sensitive assay, we examined the characteristics of COC activity and find that it functions uniquely in GSCs but not in their differentiating progeny. We show that the COC operates in the G2 phase of the cell cycle, independently of the spindle assembly checkpoint. This study may provide a framework for identifying and understanding similar mechanisms that might be in place in other asymmetrically dividing cell types.

  18. 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.

  19. Studying mechanosensitive ion channels with an automated patch clamp.

    PubMed

    Barthmes, Maria; Jose, Mac Donald F; Birkner, Jan Peter; Brüggemann, Andrea; Wahl-Schott, Christian; Koçer, Armağan

    2014-03-01

    Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.

  20. Laser Transmission Welding of Thermoplastics With Dual Clamping Devices

    NASA Astrophysics Data System (ADS)

    Devrient, M.; Knoll, B.; Geiger, R.

    Beside well chosen process parameters a geometrical joining partner design suitable for laser transmission welding and adequate clamping pressure appliance is necessary to form high quality welds. Amongst other clamping techniques Dual Clamping Devices (DCD) are a promising approach to fulfill the process needs and to get to a robust and also fail safe clamping. When using DCD the laser beam has to pass thin non transmitting bars while a weld seam is formed. So the laser beam is partial refracted, reflected and absorbed. Here the influences of the bars onto the laser beam intensity distribution behind the bars rather in the interaction zone of the laser beam and the two joining partners are investigated. Welding experiments with DCD are carried out and discussed. Based on the experimental results thermal process simulations are performed, to get a deeper knowledge about the effect of the bars onto the spatially and temporally changing temperature field within the joining partners.

  1. The Application of Mechanical Clamps to Portsmouth Connectors.

    DTIC Science & Technology

    1981-11-23

    Resistance Readings ..... .............. .64 TABLE 12 - ALT SUMM!ARY - POLYURETHANE CONNECTORS Resis- ance Readings ..... .............. .65 v7 ft...34 FIGURE 17 Pressure Tank Pressure Fittings . ........ . 37 vi I THE APPLICATION OF MECHANICAL CLAMPS TO PORTSMOUTH CONNECTORS BACKGROUN )D This

  2. Spatial dynamics, thermalization, and gain clamping in a photon condensate

    NASA Astrophysics Data System (ADS)

    Keeling, Jonathan; Kirton, Peter

    2016-01-01

    We study theoretically the effects of pump-spot size and location on photon condensates. By exploring the inhomogeneous molecular excitation fraction, we make clear the relation between spatial equilibration, gain clamping, and thermalization in a photon condensate. This provides a simple understanding of several recent experimental results. We find that as thermalization breaks down, gain clamping is imperfect, leading to "transverse spatial hole burning" and multimode condensation. This opens the possibility of engineering the gain profile to control the condensate structure.

  3. Nonlinear response of a clamped-clamped beam with internal resonance under sinusoidal excitation

    NASA Astrophysics Data System (ADS)

    Afaneh, Abdul-Hafiz Ahmed

    1992-01-01

    The nonlinear response characteristics of a clamped-clamped beam is investigated analytically, numerically, and experimentally. The beam is under an initial static axial load and subjected to a harmonic excitation of its support. Two ranges of the axial load are considered. These are below (the beam is initially straight) and above Euler buckling load (the beam is initially buckled). Hamilton's principle is used to derive a fourth order partial differential equation of motion which is descritized and reduced to a set of second order ordinary differential equations by applying Galerkin's method. Under certain values of the static load, the normal modes are nonlinearly coupled and this coupling results in a fourth order internal resonance condition between the first three modes when the beam is initially straight. Second and third order internal resonance conditions occur between the first two modes for the case of initially buckled beam. The multiple scales method showed the significant effects of these internal resonance conditions on the system behavior. In the straight beam case, the third mode which is externally excited transfers energy to the first two modes within a small range of internal detuning. Outside this region, the response is governed by a unimodal response of the third mode. In the neighborhood of 1:1 internal resonance, it is found that within the region of two mode interaction, the solution is either stationary or nonstationary depending on the excitation level and system parameters. Saturation and jump phenomena are found to take place in the case of two mode interaction with 2:1 internal resonance. Numerical simulation and experimental testing confirmed these predictions and revealed the occurrence of multifurcation, snap-through (escaping from one well to the other in an irregular manner), and chaotic motion.

  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. Replication, checkpoint suppression and structure of centromeric DNA.

    PubMed

    Romeo, Francesco; Falbo, Lucia; Costanzo, Vincenzo

    2016-11-01

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

  6. Checkpoint inhibitors in lung cancer: latest developments and clinical potential

    PubMed Central

    Schvartsman, Gustavo; Ferrarotto, Renata; Massarelli, Erminia

    2016-01-01

    Lung cancer is the leading cause of cancer death in the United States. The vast majority of patients are diagnosed with metastatic disease with a 5-year survival rate of less than 5%. After first-line chemotherapy or biomarker-matched targeted therapy, only suitable for a small group of patients, further systemic therapy options rendered very limited, if any, benefit until recently. Checkpoint inhibitors have significantly improved outcomes in patients with metastatic non-small cell lung cancer (NSCLC) and are currently an established second-line therapeutic option. In this manuscript, we review the mechanism of action of checkpoint inhibitors, present the available data with approved and experimental agents, discuss the progress that has already been made in the field, as well as toxicity awareness, and future perspectives. PMID:27800034

  7. 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.

  8. Update on immune checkpoint inhibitors in gynecological cancers

    PubMed Central

    2017-01-01

    In recent years, progress in our understanding of immune-modulatory signaling pathways in immune cells and the tumor microenvironment (TME) has led to rejuvenated interest in cancer immunotherapy. In particular, immunotherapy targeting the immune checkpoint receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell-death 1 (PD-1), and programmed cell-death ligand 1 (PD-L1) have demonstrated clinical activity in a wide variety of tumors, including gynecological cancers. This review will focus on the emerging clinical data on the therapeutic role of immune checkpoint inhibitors, and potential strategies to enhance the efficacy of this class of compounds, in the context of gynecological cancers. It is anticipated that future biomarker-directed clinical trials will provide further insights into the mechanisms underlying response and resistance to immunotherapy, and help guide our approach to designing therapeutic combinations that have the potential to enhance the benefit of immunotherapy in patients with gynecologic cancers. PMID:28028993

  9. Abscission checkpoint control: stuck in the middle with Aurora B.

    PubMed

    Carmena, Mar

    2012-07-01

    At the end of cell division, the cytoplasmic bridge joining the daughter cells is severed through a process that involves scission of the plasma membrane. The presence of chromatin bridges 'stuck' in the division plane is sensed by the chromosomal passenger complex (CPC) component Aurora B kinase, triggering a checkpoint that delays abscission until the chromatin bridges have been resolved. Recent work has started to shed some light on the molecular mechanism by which the CPC controls the timing of abscission.

  10. Central Tolerance Blockade to Augment Checkpoint Immunotherapy in Melanoma

    DTIC Science & Technology

    2016-09-01

    needed to bring anti-RANKL antibody to the clinic for treating advanced melanoma patients. To date, our findings include: RANKL is expressed at high...promising therapy for enhancing checkpoint inhibitor efficacy in advanced melanoma. This observation has immediate clinical relevance given the FDA...What was the impact on the development of the principal discipline(s) of the project? Based on our findings, we are planning a Phase 2 clinical

  11. 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.

  12. 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

  13. Managing immune checkpoint-blocking antibody side effects.

    PubMed

    Postow, Michael A

    2015-01-01

    Immune checkpoint-blocking antibodies that enhance the immune system's ability to fight cancer are becoming important components of treatment for patients with a variety of malignancies. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) was the first immune checkpoint to be clinically targeted, and ipilimumab, an inhibitor of CTLA-4, was approved by the U.S. Food and Drug Administration (FDA) for patients with advanced melanoma. The programmed cell death-1 (PD-1) receptor and one of its ligands, PD-L1, more recently have shown great promise as therapeutic targets in a variety of malignancies. Nivolumab and pembrolizumab recently have been FDA- approved for patients with melanoma and additional approvals within this therapeutic class are expected. The use of anti-CTLA-4 and anti-PD-1/PD-L1 antibodies is associated with side effects known as immune-related adverse events (irAEs). Immune-related adverse events affect the dermatologic, gastrointestinal, hepatic, endocrine, and other organ systems. Temporary immunosuppression with corticosteroids, tumor necrosis factor-alpha antagonists, mycophenolate mofetil, or other agents can be effective treatment. This article describes the side-effect profile of the checkpoint-blocking antibodies that target CTLA-4 and PD-1/PD-L1 and provides suggestions on how to manage specific irAEs.

  14. Immune checkpoint modulation for non-small cell lung cancer.

    PubMed

    Soria, Jean-Charles; Marabelle, Aurélien; Brahmer, Julie R; Gettinger, Scott

    2015-05-15

    Therapies targeting immune checkpoints have recently shown encouraging activity in patients with heavily pretreated advanced non-small cell lung cancer (NSCLC), independently of NSCLC histology or mutational status, with low toxicity profiles when used as monotherapy. Objective response rates of approximately 20% have been reported in patients with advanced NSCLC treated with antagonist antibodies targeting the immune checkpoint, programmed death 1 (PD-1) on activated T cells, or its primary ligand, programmed death ligand 1 (PD-L1) expressed within the tumor microenvironment. Response rates appear to be higher in patients with tumor PD-L1 expression documented by immunohistochemistry, although responses have been appreciated in patients with reportedly PD-L1-negative tumor specimens. Antibodies directed against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), another immunosuppressive T-cell signaling molecule, are also being evaluated in clinical trials, with one randomized phase II trial demonstrating improved immune-related progression-free survival in lung cancer patients when added to standard chemotherapy. Additional clinical trials are combining anti-CTLA-4 antibodies with either anti-PD-1 or anti-PD-L1 antibodies. Combinations of other immune checkpoint antagonists or agonist antibodies with anti-PD-1 or anti-PD-L1 antibodies are also being pursued.

  15. Emerging role of checkpoint blockade therapy in lymphoma

    PubMed Central

    Galanina, Natalie; Kline, Justin; Bishop, Michael R.

    2017-01-01

    Following the successful application of immune checkpoint blockade therapy (CBT) in refractory solid tumors, it has recently gained momentum as a promising modality in the treatment of relapsed lymphoma. This significant therapeutic advance stems from decades of research that elucidated the role of immune regulation pathways and the mechanisms by which tumors can engage these critical pathways to escape immune detection. To date, two main pathways, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1), have emerged as key targets of CBT demonstrating unprecedented activity particularly in heavily pretreated relapsed/refractory Hodgkin lymphoma and some forms of non-Hodgkin disease. Herein we provide a brief discussion of checkpoint blockade in the context of lymphoma biology with a specific focus on novel checkpoint inhibitors and their therapeutic activity. We discuss current clinical trials and the landscape of CBT to underscore both the remarkable progress and foreseeable limitations of this novel treatment strategy. In particular, we build upon state-of-the-art knowledge and clinical insights gained from the early trials to review potential approaches to how CBT may be integrated with other treatment modalities, including chemoimmunotherapy to improve patient outcomes in the future. Finally, as the role of CBT evolves to potentially become a cornerstone of therapy in refractory/relapsed lymphoma, we briefly emphasize the importance of predictive biomarkers in an effort to select appropriate patients who are most likely to derive benefit from CBT. PMID:28203344

  16. Immune Checkpoint Blockade and Hematopoietic Stem Cell Transplant.

    PubMed

    Merryman, Reid W; Armand, Philippe

    2017-02-01

    Allogeneic hematopoietic stem cell transplant (HSCT) relies primarily upon graft-versus-tumor activity for cancer eradication. Relapse remains the principal cause of treatment failure after HSCT, implying frequent immune escape, which in at least some cases, appears to be mediated by increased expression of inhibitory immune checkpoints. In an attempt to restore anti-tumor immunity, checkpoint blockade therapy (CBT) targeting PD-1 and CLTA-4 has been used in conjunction with both allogeneic and autologous HSCT. Clinical experience in this setting is limited to several small clinical trials and case series, but together they suggest that treatment with CBT can effectively amplify anti-tumor immune responses. However, intrinsic to its mechanism is also the risk that CBT in the HSCT setting may also cause significant immune toxicity. Fatal immune-related adverse events and graft-versus-host disease have been observed, but in most cases, immune side effects appear to be reversible with steroids and CBT discontinuation. As clinical investigation continues, improved understanding of immune checkpoint biology will be critical to optimize safe and efficacious treatment strategies.

  17. Checkpoint inhibition: new treatment options in urologic cancer.

    PubMed

    De Maeseneer, Daan Joost; Delafontaine, Brant; Rottey, Sylvie

    2017-02-01

    Both urothelial (UC) and renal cell cancer (RCC) are highly immunogenic tumours. Recent advances in cellular immunity understanding have resulted in a successful new class of therapeutic agents. Interaction between the programmed cell death 1 (PD1) on regulatory T-cells (Treg) and programmed cell death 1 ligand (PDL1) on cancer cells inhibits an effective immune response and is an important mechanism for cancer cells to evade the immune system. Monoclonal anti-PD1 and anti-PDL1 antibodies inhibit this interaction and are called checkpoint inhibitors. As in non-small lung cancer and melanoma, these agents have shown to be highly active agents in UC and RCC. In metastatic or inoperable UC, no good second line therapy exists and checkpoint inhibitors have shown to be active. Multiple Randomized Clinical Trials (RCT) are underway both in second line and adjuvant settings which could change daily practice. In RCC, an anti-PD1 antibody, Nivolumab, has already proven to be effective in prolonging overall survival, and is included in current guidelines in metastatic RCC in second and third line. RCTs with other anti-PD1 and anti-PDL1 antibodies are ongoing both in metastatic and adjuvant setting. Checkpoint inhibitors have breathed new life into immunotherapy in urologic cancers and are rapidly being included in practice guidelines.

  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. Sobriety checkpoints in Thailand: a review of effectiveness and developments over time.

    PubMed

    Ditsuwan, Vallop; Veerman, J Lennert; Bertram, Melanie; Vos, Theo

    2015-03-01

    This review describes the legal basis for and implementation of sobriety checkpoints in Thailand and identifies factors that influenced their historical development and effectiveness. The first alcohol and traffic injury control law in Thailand was implemented in 1934. The 0.05 g/100 mL blood alcohol concentration limit was set in 1994. Currently, 3 types of sobriety checkpoints are used: general police checkpoints, selective breath testing, and special event sobriety checkpoints. The authors found few reports on the strategies, frequencies, and outcomes for any of these types of checkpoints, despite Thailand having devoted many resources to their implementation. In Thailand and other low-middle income countries, it is necessary to address the country-specific barriers to successful enforcement (including political and logistical issues, lack of equipment, and absence of other supportive alcohol harm reduction measures) before sobriety checkpoints can be expected to be as effective as reported in high-income countries.

  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. Axon voltage-clamp simulations. I. Methods and tests.

    PubMed Central

    Moore, J W; Ramón, F; Joyner, R W

    1975-01-01

    This is the first in a series of four papers in which we present the numerical simulation of the application of the voltage clamp technique to excitable cells. In this paper we describe the application of the Crank-Nicolson (1947) method for the solution of the parabolic partial differential equations that describe a cylindrical cell in which the ionic conductances are functions of voltage and time (Hodgkin and Huxley, 1952). This method is compared with other methods in terms of accuracy and speed of solution for a propagated action potential. In addition, differential equations representing a simple voltage-clamp electronic circuit are presented. Using the voltage clamp circuit equations, we simulate the voltage clamp of a single isopotential membrane patch and show how the parameters of the circuit affect the transient response of the patch to a step change in the control potential.The stimulation methods presented in this series of papers allow the evaluation of voltage clamp control of an excitable cell or a syncytium of excitable cells. To the extent that membrane parameters and geometrical factors can be determined, the methods presented here provide solutions for the voltage profile as a function of time. PMID:1174640

  3. Two-Level Incremental Checkpoint Recovery Scheme for Reducing System Total Overheads

    PubMed Central

    Li, Huixian; Pang, Liaojun; Wang, Zhangquan

    2014-01-01

    Long-running applications are often subject to failures. Once failures occur, it will lead to unacceptable system overheads. The checkpoint technology is used to reduce the losses in the event of a failure. For the two-level checkpoint recovery scheme used in the long-running tasks, it is unavoidable for the system to periodically transfer huge memory context to a remote stable storage. Therefore, the overheads of setting checkpoints and the re-computing time become a critical issue which directly impacts the system total overheads. Motivated by these concerns, this paper presents a new model by introducing i-checkpoints into the existing two-level checkpoint recovery scheme to deal with the more probable failures with the smaller cost and the faster speed. The proposed scheme is independent of the specific failure distribution type and can be applied to different failure distribution types. We respectively make analyses between the two-level incremental and two-level checkpoint recovery schemes with the Weibull distribution and exponential distribution, both of which fit with the actual failure distribution best. The comparison results show that the total overheads of setting checkpoints, the total re-computing time and the system total overheads in the two-level incremental checkpoint recovery scheme are all significantly smaller than those in the two-level checkpoint recovery scheme. At last, limitations of our study are discussed, and at the same time, open questions and possible future work are given. PMID:25111048

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

    DOE PAGES

    Islam, Tanzima Zerin; Mohror, Kathryn; Bagchi, Saurabh; ...

    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

  5. Umbilical cord clamping. An analysis of a usual neonatological conduct.

    PubMed

    Papagno, L

    1998-01-01

    Here we described a critical analysis of the neonatological procedure of early cord clamping, meaning this, within 40 seconds after birth. Fifty three cases are here analysed, in which this practice was not performed, but instead a late umbilical cord clamping was done after birth or after the cord had stopped beating. Variations in hematocrito values within 24 to 36 hours after birth were studied. A transitory polycithemia, with a maximum peak 12 hours post-delivery was observed. These values returned to normal levels between 24 and 36 hours after birth. K vitamin was not administered to any of the newborns. No pathology appeared related to this transitory polycithemia. In can be concluded that the late umbilical cord clamping represents no risk to the new-born and that the pathological phenomena described under these circumstances may be attributed to the increase in K vitamin dependent coagulation factors that are induced by the routinary administration of phitonadione to all normal newborns.

  6. [Advantage of delayed umbilical cord clamping in the newborn infant].

    PubMed

    Menget, A; Mougey, C; Thiriez, G; Riethmuller, D

    2013-09-01

    The timing of umbilical cord clamping remains controversial. Although most maternity wards use the early clamping (5-15s), randomized studies and meta-analyses have demonstrated the benefit of delayed clamping for term and preterm newborn infants over the past 10 years. Indeed, placentofetal transfusion of 20-30 ml/kg in 2-3 min improves the iron status of term infants and prevents infant hypochromic anemia. Infant anemia is a public health problem in many developing countries. For preterm newborns, placental transfusion for 45 s or milking the cord for 15 s improves cardiovascular adaptation, with better hemodynamic stability, as well as decreased intraventricular hemorrhages, need for transfusion, and late-onset sepsis. A new look at this symbolic act is needed and professionals need to be persuaded of the importance of the "wait a minute" policy for a better physiological delivery.

  7. Clamping compared to cauterization for subcutaneous hemostasis in Pfannenstiel incision.

    PubMed

    Ozkaya, Enis; Korkmaz, Vakkas; Kucukozkan, Tuncay

    2011-04-01

    We compared subcutaneous clamping and cauterization for hemostasis at laparotomy with Pfannenstiel incision with reference to surgical site infection, postoperative fever and time taken for incision. A total of 214 patients with consecutive hysterectomies were alternately assigned to incisional hemostasis by clamping (n= 107) or cauterization (n= 107). The groups were similar in terms of age, gravidity, parity, body mass index, uterine size and mean hemoglobin drop. Rates of surgical site infection, postoperative fever and time from skin incision to peritoneal cavity entry were significantly higher in the group with cauterization (p < 0.05).

  8. 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.

  9. Novel Checkpoints and Cosignaling Molecules in Cancer Immunotherapy.

    PubMed

    Giuroiu, Iulia; Weber, Jeffrey

    The recent demonstration of the antitumor efficacy of checkpoint protein inhibition has resulted in the approval of blocking antibodies against the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway in multiple different histologic findings. Therapeutic successes with PD-1/PD-L1 antibodies in melanoma and lung cancer have been followed by approvals in bladder, renal, and head and neck cancers and Hodgkin lymphoma, with others undoubtedly to come. However, PD-1 is only one of many checkpoints and agonistic regulatory molecules expressed on T cells by which maintenance of the balance between costimulatory and coinhibitory signaling pathways is perturbed in cancer. The manipulation of many of these molecules in cancer patients might be associated with clinical benefit. The majority of the T-cell cosignaling receptors belong to either the immunoglobulin superfamily or the tumor necrosis factor receptor superfamily. A total of 29 immunoglobulin superfamily and 26 tumor necrosis factor receptor superfamily cosignaling receptors have been identified that are expressed on T cells, providing fertile ground for development of inhibitory or agonistic antibodies and small molecules as cancer therapeutics. In the current work, we focus on some of the most promising new checkpoints and agonistic or cosignaling molecules that are in early clinical development as single agents or in combinations with PD-1/PD-L1, cytotoxic T-lymphocyte-associated protein 4 blockade, or chemotherapy with an emphasis on those that have reached the clinic and on important targets that are in late preclinical development.

  10. Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy

    PubMed Central

    Rojas, Juan J; Sampath, Padma; Hou, Weizhou; Thorne, Steve H

    2015-01-01

    Purpose Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in pre-clinical models. Perhaps the greatest advance with oncolytic virotherapy has been the appreciation of the importance of activation of the immune response in therapeutic activity. Meanwhile, the understanding that blockade of immune checkpoints (with antibodies that block the binding of PD1 to PDL1 or CTLA4 to B7-2) is critical for an effective anti-tumor immune response has revitalized the field of immunotherapy. The combination of immune activation using an oncolytic virus and blockade of immune checkpoints is therefore a logical next step. Experimental Design Here we explore such combinations and demonstrate their potential to produce enhanced responses in mouse tumor models. Different combinations and regimens were explored in immunocompetent mouse models of renal and colorectal cancer. Bioluminescence imaging and immune assays were used to determine the mechanisms mediating synergistic or antagonistic combinations. Results Interaction between immune checkpoint inhibitors and oncolytic virotherapy was found to be complex, with correct selection of viral strain, antibody and timing of the combination being critical for synergistic effects. Indeed, some combinations produced antagonistic effects and loss of therapeutic activity. A period of oncolytic viral replication and directed targeting of the immune response against the tumor were required for the most beneficial effects, with CD8+ and NK, but not CD4+ cells mediating the effects. Conclusions These considerations will be critical in the design of the inevitable clinical translation of these combination approaches. PMID:26187615

  11. Greatwall and Polo-like Kinase 1 Coordinate to Promote Checkpoint Recovery*

    PubMed Central

    Peng, Aimin; Wang, Ling; Fisher, Laura A.

    2011-01-01

    Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G2/M DNA damage checkpoint and efficient checkpoint recovery. PMID:21708943

  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. Do telomeres ask checkpoint proteins: "gimme shelter-in"?

    PubMed

    Weinert, Ted

    2005-12-01

    Telomeres are complicated structures designed to allow one thing and avoid another. They allow replication of chromosome ends, an issue mostly about telomerase, which we seem to understand (though details of its regulation are works in progress). Telomeres must also avoid being detected as DNA breaks. This is important for two reasons: DNA breaks activate checkpoints that cause arrest of cell division, and DNA breaks engage repair machinery. Clearly, normal telomeres neither activate cell cycle arrest nor allow themselves to be repaired; arrest blocks cell division, and repair fuses chromosomes.

  14. Monitoring spindle orientation: Spindle position checkpoint in charge.

    PubMed

    Caydasi, Ayse K; Ibrahim, Bashar; Pereira, Gislene

    2010-12-11

    Every cell division in budding yeast is inherently asymmetric and counts on the correct positioning of the mitotic spindle along the mother-daughter polarity axis for faithful chromosome segregation. A surveillance mechanism named the spindle position checkpoint (SPOC), monitors the orientation of the mitotic spindle and prevents cells from exiting mitosis when the spindle fails to align along the mother-daughter axis. SPOC is essential for maintenance of ploidy in budding yeast and similar mechanisms might exist in higher eukaryotes to ensure faithful asymmetric cell division. Here, we review the current model of SPOC activation and highlight the importance of protein localization and phosphorylation for SPOC function.

  15. Immune-Related Adverse Events Associated with Immune Checkpoint Inhibitors.

    PubMed

    Day, Daphne; Hansen, Aaron R

    2016-12-01

    Immune checkpoint inhibitors (ICIs), including antibodies targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein-1 (PD-1), have shown durable treatment responses in multiple tumor types by enhancing antitumor immunity. However, removal of self-tolerance can induce autoimmunity and produce a unique immune-driven toxicity profile, termed immune-related adverse events (irAEs). As ICIs gain approval for a growing number of indications, it is imperative clinicians increase their knowledge of and ability to manage irAEs. This review examines the etiology, presentation, kinetics, and treatment of irAEs and aims to provide practical guidance for clinicians.

  16. Cdh1 is an antagonist of the spindle assembly checkpoint.

    PubMed

    Nagai, Masayoshi; Ushimaru, Takashi

    2014-10-01

    The spindle assembly checkpoint (SAC) monitors unsatisfied connections of microtubules to kinetochores and prevents anaphase onset by inhibition of the ubiquitin ligase E3 anaphase-promoting complex or cyclosome (APC/C) in association with the activator Cdc20. Another APC/C activator, Cdh1, exists permanently throughout the cell cycle but it becomes active from telophase to G1. Here, we show that Cdh1 is partially active and mediates securin degradation even in SAC-active metaphase cells. Additionally, Cdh1 mediates Cdc20 degradation in metaphase, promoting formation of the APC/C-Cdh1. These results indicate that Cdh1 opposes the SAC and promotes anaphase transition.

  17. 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...

  18. 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...

  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, 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...

  2. 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.

  3. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Cable clamps and grips. 18.40 Section 18.40 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR TESTING, EVALUATION, AND APPROVAL OF MINING PRODUCTS ELECTRIC MOTOR-DRIVEN MINE EQUIPMENT AND ACCESSORIES Construction and...

  4. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Cable clamps and grips. 18.40 Section 18.40 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR TESTING, EVALUATION, AND APPROVAL OF MINING PRODUCTS ELECTRIC MOTOR-DRIVEN MINE EQUIPMENT AND ACCESSORIES Construction and...

  5. An improved vaseline gap voltage clamp for skeletal muscle fibers

    PubMed Central

    1976-01-01

    A Vaseline gap potentiometric recording and voltage clamp method is developed for frog skeletal muscle fibers. The method is based on the Frankenhaeuser-Dodge voltage clamp for myelinated nerve with modifications to improve the frequency response, to compensate for external series resistance, and to compensate for the complex impedance of the current-passing pathway. Fragments of single muscle fibers are plucked from the semitendinosus muscle and mounted while depolarized by a solution like CsF. After Vaseline seals are formed between fluid pools, the fiber ends are cut once again, the central region is rinsed with Ringer solution, and the feedback amplifiers are turned on. Errors in the potential and current records are assessed by direct measurements with microelectrodes. The passive properties of the preparation are simulated by the "disk" equivalent circuit for the transverse tubular system and the derived parameters are similar to previous measurements with microelectrodes. Action potentials at 5 degrees C are long because of the absence of delayed rectification. Their shape is approximately simulated by solving the disk model with sodium permeability in the surface and tubular membranes. Voltage clamp currents consist primarily of capacity currents and sodium currents. The peak inward sodium current density at 5 degrees C is 3.7 mA/cm2. At 5 degrees C the sodium currents are smoothly graded with increasing depolarization and free of notches suggesting good control of the surface membrane. At higher temperatures a small, late extra inward current appears for small depolarizations that has the properties expected for excitation in the transverse tubular system. Comparison of recorded currents with simulations shows that while the transverse tubular system has regenerative sodium currents, they are too small to make important errors in the total current recorded at the surface under voltage clamp at low temperature. The tubules are definitely not under voltage

  6. Polarization states and dielectric responses of elastically clamped ferroelectric nanocrystals

    NASA Astrophysics Data System (ADS)

    Azovtsev, A. V.; Pertsev, N. A.

    2016-12-01

    Polarization states and physical properties of ferroelectrics depend on the mechanical boundary conditions due to electrostrictive coupling between electric polarization and lattice strains. Here, we describe theoretically both equilibrium thermodynamic states and electric permittivities of ferroelectric nanocrystals subjected to the elastic three-dimensional (3D) clamping by a surrounding dielectric material. The problem is solved by the minimization of a special thermodynamic potential that describes the case of an ellipsoidal ferroelectric inclusion embedded into a linear elastic matrix. Numerical calculations are performed for BaTiO3, PbTiO3, and Pb(Zr0.5Ti0.5)O3 nanoparticles surrounded by silica glass. It is shown that, in the case of BaTiO3 and PbTiO3, elastic 3D clamping may change the order of a ferroelectric phase transition from first to second. Furthermore, the mechanical inclusion-matrix interaction shifts the temperatures of structural transitions between different ferroelectric states and even eliminates some ferroelectric phases existing in stress-free BaTiO3 and Pb(Zr0.5Ti0.5)O3 crystals. Another important effect of elastic clamping is the lowering of the symmetry of ferroelectric states in ellipsoidal inclusions, where orthorhombic and monoclinic phases may form instead of the tetragonal and rhombohedral bulk counterparts. Finally, our thermodynamic calculations show that the dielectric responses of studied perovskite ferroelectrics are sensitive to matrix-induced clamping as well. For instance, dielectric peaks occurring at structural transitions between different ferroelectric phases in BaTiO3 appear to be much higher in spherical inclusions than in the freestanding crystal. Predicted clamping-induced enhancement of certain dielectric responses at room temperature indicates that composite materials comprising nanocrystals of perovskite ferroelectrics are promising for device applications requiring the use of high-permittivity dielectrics.

  7. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  8. A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

    PubMed Central

    Ji, Zhejian; Gao, Haishan; Jia, Luying; Li, Bing; Yu, Hongtao

    2017-01-01

    The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment. DOI: http://dx.doi.org/10.7554/eLife.22513.001 PMID:28072388

  9. 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.

  10. 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…

  11. Molecular Pathways: Immune Checkpoint Antibodies and their Toxicities.

    PubMed

    Cousin, Sophie; Italiano, Antoine

    2016-09-15

    The emergence of immune checkpoint inhibitors for solid tumor treatments represents a major oncologic advance. Since the approval of ipilimumab, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibody, for the treatment of metastatic melanoma, many drugs, especially those targeting PD-1/PD-L1, have demonstrated promising antitumor effects in many types of cancer. By reactivating the immune system, these immunotherapies have led to the development of new toxicity profiles, also called immune-related adverse events (irAE). IrAEs can involve many organ systems, and their management is radically different from that of cytotoxic drugs; irAEs require immunosuppressive treatments, such as corticoids or TNFα antibody. In addition, the occurrence of irAEs has raised significant questions. Here, we summarize progress that has been made toward answering these questions, focusing on (i) the impact of immunotherapy dose on irAE occurrence, (ii) the correlation between irAE and patient outcome, (iii) the safety of immune checkpoint inhibitors in patients already treated for autoimmune disease, and (iv) the suspected effect on tumor growth of steroids used for the management of irAEs. Clin Cancer Res; 22(18); 4550-5. ©2016 AACR.

  12. A Splicing-Dependent Transcriptional Checkpoint Associated with Prespliceosome Formation

    PubMed Central

    Chathoth, Keerthi T.; Barrass, J. David; Webb, Shaun; Beggs, Jean D.

    2014-01-01

    Summary There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 protein (Prp5p) is an RNA-stimulated adenosine triphosphatase (ATPase) required for prespliceosome formation in yeast. We demonstrate through in vivo RNA labeling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at Ser5 of the largest subunit’s heptad repeat accumulates over introns and that this defect requires Cus2 protein. A similar accumulation of polymerase was observed when prespliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with prespliceosome formation during cotranscriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription. PMID:24560925

  13. Spindle assembly checkpoint and its regulators in meiosis.

    PubMed

    Sun, Shao-Chen; Kim, Nam-Hyung

    2012-01-01

    BACKGROUND Meiosis is a unique form of cell division in which cells divide twice but DNA is duplicated only once. Errors in chromosome segregation during meiosis will result in aneuploidy, followed by loss of the conceptus during pregnancy or birth defects. During mitosis, cells utilize a mechanism called the spindle assembly checkpoint (SAC) to ensure faithful chromosome segregation. A similar mechanism has been uncovered for meiosis in the last decade, especially in the past several years. METHODS For this review, we included data and relevant information obtained through a PubMed database search for all articles published in English from 1991 through 2011 which included the term 'meiosis', 'spindle assembly checkpoint', or 'SAC'. RESULTS There are 91 studies included. Evidence for the existence of SAC functions in meiosis is provided by studies on the SAC proteins mitotic-arrest deficient-1 (Mad1), Mad2, budding uninhibited by benzimidazole-1 (Bub1), Bub3, BubR1 and Mps1; microtubule-kinetochore attachment regulators Ndc80 complex, chromosomal passenger complex, mitotic centromere-associated kinesin (MCAK), kinetochore null 1 (KNL1) and Mis12 complex and spindle stability regulators. CONCLUSIONS SAC and its regulators exist and function in meiosis, and their malfunctions may cause germ cell aneuploidy. However, species and sexual differences exist. Moreover, interaction of SAC components with other regulators is still poorly understood, which needs further study.

  14. Immunotherapy comes of age: Immune aging & checkpoint inhibitors.

    PubMed

    Elias, Rawad; Karantanos, Theodoros; Sira, Elizabeth; Hartshorn, Kevan L

    2017-02-17

    Immune checkpoint inhibitors (ICIs) are based on the understanding that there are multilayered checks and balances which can be manipulated to unleash already existing, but paralyzed, immune responses to cancer. These agents are safer and more efficacious than classic cytotoxic drugs making them a very attractive therapeutic option, especially in older adults. Current available data do not suggest significant age-associated differences in the clinical profile of ICIs. It must be noted, however, that there is still relatively little information on the use of ICIs in adults over 75years of age and aging is associated with a decline in the immune system or "immunosenescence" which theoretically can reduce the efficacy of these immune based therapies. In this paper, we review the mechanism of action of ICIs, current clinical data on their use in older adults, and age-associated immune changes that might have a direct impact on their activity in this population. We chose to focus on mainly adaptive cellular immunity, and especially on components of the immune system that are implicated directly in the immune checkpoint process.

  15. 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.

  16. Transcription factors of M-phase cyclin CLB2 in the yeast cell wall integrity checkpoint.

    PubMed

    Sekiya, Mizuho; Nogami, Satoru; Ohya, Yoshikazu

    2009-08-01

    The cell wall integrity checkpoint coordinates cell wall synthesis and mitosis in the budding yeast, Saccharomyces cerevisiae. It has been reported that this checkpoint arrests the cell cycle at G2/M phase with repression of the M phase cyclin Clb2p at the transcriptional level, under perturbation of cell wall synthesis. We demonstrate that an override of this checkpoint with accumulation of CLB2 mRNA is induced when negative CLB2 transcription factors are deleted or when positive CLB2 transcription factors are overproduced in cell wall-defective cells. Our data imply that transcription factors for CLB2 are involved in the cell wall integrity checkpoint system and suggest that there are multiple regulation pathways of the checkpoint.

  17. Fhit loss in lung preneoplasia: relation to DNA damage response checkpoint activation

    PubMed Central

    Cirombella, Roberto; Montrone, Giuseppe; Stoppacciaro, Antonella; Giglio, Simona; Volinia, Stefano; Graziano, Paolo; Huebner, Kay; Vecchione, Andrea

    2009-01-01

    Loss of heterozygosity at the FHIT locus is coincident with activation of DNA damage response checkpoint proteins; thus damage at fragile loci may trigger checkpoint activation. We examined preneoplastic lesions adjacent to non-small cell lung carcinomas for alterations to expression of Fhit and activated checkpoint proteins. Expression scores were analyzed for pair-wise associations and correlations among proteins and type of lesion. Hyperplastic and dysplastic lesions were positive for nuclear H2AX expression; 12/20 dysplastic lesions were negative for Fhit expression. Fhit positive lesions showed expression of most checkpoint proteins examined, while Fhit negative lesions showed absence of expression of Chk1 and phosphoChk1. The results show that loss of expression of Fhit is significantly directly correlated with absence of activated Chk1 in dysplasia, and suggest a connection between loss of Fhit and modulation of checkpoint activity. PMID:19931269

  18. Blocking immune checkpoints in prostate, kidney, and urothelial cancer: An overview.

    PubMed

    Alme, Angela K B; Karir, Beerinder S; Faltas, Bishoy M; Drake, Charles G

    2016-04-01

    Despite a long history of immunotherapeutic approaches to treatment, most genitourinary malignancies are not cured by existing immunotherapy regimens. More recently, cell surface molecules known as immune checkpoints have become the focus of efforts to develop more effective immunotherapies. Interactions between these molecules and their ligands inhibit the proliferation and function of tumor-specific lymphocytes. A monoclonal antibody blocking 1 of these checkpoints was approved for the treatment of metastatic melanoma and is now being tested in other malignancies. The objective responses seen in these early trials of checkpoint blockade are driving renewed enthusiasm for cancer immunotherapy. There are several ongoing and planned trials in genitourinary malignancies of single-agent inhibitors, as well as combinations targeting multiple checkpoints or adding other types of therapies to checkpoint blockade.

  19. Blocking Immune Checkpoints in Prostate, Kidney and Urothelial cancer: An Overview

    PubMed Central

    Alme, Angela K.B.; Karir, Beerinder S.; Faltas, Bishoy M.; Drake, Charles G.

    2016-01-01

    Despite a long history of immunotherapeutic approaches to treatment, most genitourinary malignancies are not cured by existing immunotherapy regimens. More recently, cell-surface molecules known as immune checkpoints have become the focus of efforts to develop more effective immunotherapies. Interactions between these molecules and their ligands inhibit the proliferation and function of tumor-specific lymphocytes. A monoclonal antibody blocking one of these checkpoints was approved for the treatment of metastatic melanoma and is now being tested in other malignancies. The objective responses seen in these early trials of checkpoint blockade are driving renewed enthusiasm for cancer immunotherapy. There are several ongoing and planned trials in genitourinary malignancies of single-agent inhibitors, as well as combinations targeting multiple checkpoints or adding other types of therapies to checkpoint blockade. PMID:26923782

  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. 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

  2. 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

  3. Patch-clamp experiments under micro-gravity.

    PubMed

    Meissner, Klaus; Hanke, Wolfgang

    2002-07-01

    For human based space research it is of high importance to understand the influence of gravity on the properties of single ion channels in biological membranes, as these are involved in about all biological processes. The patch clamp technique is the best established method to investigate electrophysiological properties of single ion channels in detail. Consequently, a patch clamp set-up was designed for the drop tower in Bremen, Germany. Using this set-up among others, successfully leech neurons have been patched under micro-gravity, delivering data about ion channel behaviour, which were compared to results from bilayer experiments in the drop tower and to results from lab controls under 1 g and under higher gravity.

  4. 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.

  5. A mathematical model of the euglycemic hyperinsulinemic clamp

    PubMed Central

    Picchini, Umberto; De Gaetano, Andrea; Panunzi, Simona; Ditlevsen, Susanne; Mingrone, Geltrude

    2005-01-01

    Background The Euglycemic Hyperinsulinemic Clamp (EHC) is the most widely used experimental procedure for the determination of insulin sensitivity, and in its usual form the patient is followed under insulinization for two hours. In the present study, sixteen subjects with BMI between 18.5 and 63.6 kg/m2 were studied by long-duration (five hours) EHC. Results From the results of this series and from similar reports in the literature it is clear that, in obese subjects, glucose uptake rates continue to increase if the clamp procedure is prolonged beyond the customary 2 hours. A mathematical model of the EHC, incorporating delays, was fitted to the recorded data, and the insulin resistance behaviour of obese subjects was assessed analytically. Obese subjects had significantly less effective suppression of hepatic glucose output and higher pancreatic insulin secretion than lean subjects. Tissue insulin resistance appeared to be higher in the obese group, but this difference did not reach statistical significance. Conclusion The use of a mathematical model allows a greater amount of information to be recovered from clamp data, making it easier to understand the components of insulin resistance in obese vs. normal subjects. PMID:16269082

  6. Cleaning patch-clamp pipettes for immediate reuse

    PubMed Central

    Kolb, I.; Stoy, W. A.; Rousseau, E. B.; Moody, O. A.; Jenkins, A.; Forest, C. R.

    2016-01-01

    Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator. PMID:27725751

  7. Cleaning patch-clamp pipettes for immediate reuse.

    PubMed

    Kolb, I; Stoy, W A; Rousseau, E B; Moody, O A; Jenkins, A; Forest, C R

    2016-10-11

    Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator.

  8. Selective killing of cancer cells by β-lapachone: Direct checkpoint activation as a strategy against cancer

    PubMed Central

    Li, Youzhi; Sun, Xiangao; LaMont, J. Thomas; Pardee, Arthur B.; Li, Chiang J.

    2003-01-01

    Most chemotherapeutic drugs kill cancer cells by indirectly activating checkpoint-mediated apoptosis after creating nonselective damage to DNA or microtubules, which accounts for their toxicity toward normal cells. We seek to target cancer cells by directly activating checkpoint regulators without creating such damage. Here, we show that β-lapachone selectively induces apoptosis in cancer cells without causing the death of nontransformed cells in culture. This unusual selectivity against cancer cells is preceded by activation of S-phase checkpoint and selective induction of E2F1, a regulator of checkpoint-mediated apoptosis. This study suggests direct checkpoint activation as a strategy against cancer. PMID:12598645

  9. Immune checkpoint inhibitors renal side effects and management.

    PubMed

    Rassy, Elie El; Kourie, Hampig R; Rizkallah, Jamale; Karak, Fadi El; Hanna, Colette; Chelala, Dania N; Ghosn, Marwan

    2016-12-01

    The choice of immunotherapy in the treatment of cancer has improved the prognosis of many patients affected by various malignancies. The high expectations foreseen with immunotherapy have led to fast approvals despite the incomplete understanding of the toxicity profiles in the different organs, including the kidneys. The high prevalence of chronic kidney disease in cancer patients complicates the issue further and requires a better knowledge of the renal safety profile to ensure an optimal safe treatment. This review summarizes the present knowledge of renal adverse events secondary to immune checkpoint inhibitors and discusses their pathophysiology, clinical presentation and adequate management. We also advocate the need for a multidisciplinary approach in patients with immune-related toxic adverse events.

  10. 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.

  11. Xenopus oocyte meiosis lacks spindle assembly checkpoint control

    PubMed Central

    Shao, Hua; Ma, Chunqi; Chen, Eric

    2013-01-01

    The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC. PMID:23569212

  12. Checkpoint immunotherapy for cancer: superior survival, unaccustomed toxicities.

    PubMed

    Gedye, C; van der Westhuizen, A; John, T

    2015-07-01

    Novel cancer immunotherapy antibodies are moving from clinical trials into routine practice, delivering sustained benefits and prolonged survival to patients with melanoma, lung, kidney and other cancers. These immunostimulatory antibodies non-specifically activate the patient's own immune system by inhibiting immune system checkpoint proteins. This mechanism of action is entirely different to traditional cancer treatments, such as chemotherapy. While there are virtually no immediate toxicities, serious life-threatening autoimmune side-effects such as colitis, dermatitis, hypophysitis, pneumonitis and hepatitis can occur, sometimes starting long after the treatment has been given. Recognition, referral and prompt treatment with immunosuppressive drugs like corticosteroids can control these immune-related side-effects without compromising efficacy. This exciting new class of drugs is defining a new paradigm in cancer therapy.

  13. Genetic instability in cancer cells by impaired cell cycle checkpoints.

    PubMed

    Nakanishi, Makoto; Shimada, Midori; Niida, Hiroyuki

    2006-10-01

    Cells continuously encounter DNA damage caused either by damaging agents, including oxygen radicals and DNA replication errors caused by stalled replication forks, or by extracellular environments such as ultraviolet or ionizing irradiation. Such DNA damage poses a great threat to genome stability, potentially leading to loss or amplification of chromosome activity, which may result in cellular senescence, cancer or apoptosis. The DNA damage checkpoints coordinate an arrest in cell cycle progression with the DNA repair process, suppressing either mitotic catastrophe or proliferation of cells with damaged DNA. Numerous key players have been identified in terms of damage sensor proteins, transducer kinases and effectors, but their coordination and interconnectedness in damage control have only recently become evident. In this review, we discuss changes in chromatin structure, recruitment of mediator proteins and activation of transducer kinases in response to DNA damage. These cellular responses are important for determining the potential effects of current cancer therapies in terms of toxicity and efficacy.

  14. VISTA is an immune checkpoint molecule for human T cells

    PubMed Central

    Lines, J. Louise; Sempere, Lorenzo F.; Wang, Li; Pantazi, Eirini; Mak, Justin; O’Connell, Samuel; Ceeraz, Sabrina; Suriawinata, Arief A.; Yan, Shaofeng; Ernstoff, Marc S.; Noelle, Randolph

    2014-01-01

    VISTA is a potent negative regulator of T cell function that is expressed on hematopoietic cells and leukocytes. VISTA levels are heightened within the tumor microenvironment where its blockade can enhance anti-tumor immune responses in mice. In humans, blockade of the related PD-1 pathway has shown great potential in clinical immunotherapy trials. Here we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells, blunted production of T cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T cell activation, induces Foxp3 expression and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer. PMID:24691993

  15. 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

  16. Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes.

    PubMed

    Kaisari, Sharon; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Teichner, Adar; Hershko, Avram

    2017-01-31

    The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a mitotic checkpoint complex (MCC), composed of the checkpoint proteins Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), whose action is necessary for anaphase initiation. When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31(comet) Now we have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as chaperonin containing TCP1 or TCP1-Ring complex (CCT/TRiC chaperonin), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homooligomers [Sergeeva, et al. (2013) J Biol Chem 288(24):17734-17744], also promote the disassembly of MCC. CCT chaperonin further binds and disassembles subcomplexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.

  17. 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

  18. Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction.

    PubMed

    Santaguida, Stefano; Vernieri, Claudio; Villa, Fabrizio; Ciliberto, Andrea; Musacchio, Andrea

    2011-04-20

    Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

  19. 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.

  20. KSRP: A Checkpoint for Inflammatory Cytokine Production in Astrocytes

    PubMed Central

    LI, XUELIN; LIN, WEI-JYE; CHEN, CHING-YI; SI, YING; ZHANG, XIAOWEN; LU, LIANG; SUSWAM, ESTHER; ZHENG, LEI; KING, PETER H.

    2013-01-01

    Chronic inflammation in the central nervous system (CNS) is a central feature of many neurodegenerative and autoimmune diseases. As an immunologically competent cell, the astrocyte plays an important role in CNS inflammation. It is capable of expressing a number of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) that promote inflammation directly and through the recruitment of immune cells. Checkpoints are therefore in place to keep tight control over cytokine production. Adenylate/uridylate-rich elements (ARE) in the 3′ untranslated region of cytokine mRNAs serve as a major checkpoint by regulating mRNA stability and translational efficiency. Here, we examined the impact of KH-type splicing regulatory protein (KSRP), an RNA binding protein which destabilizes mRNAs via the ARE, on cytokine expression and paracrine phenotypes of primary astrocytes. We identified a network of inflammatory mediators, including TNF-α and IL-1β, whose expression increased 2 to 4-fold at the RNA level in astrocytes isolated from KSRP−/− mice compared to littermate controls. Upon activation, KSRP−/− astrocytes produced TNF-α and IL-1β at levels that exceeded control cells by 15-fold or more. Conditioned media from KSRP−/− astrocytes induced chemotaxis and neuronal cell death in vitro. Surprisingly, we observed a prolongation of half-life in only a subset of mRNA targets and only after selective astrocyte activation. Luciferase reporter studies indicated that KSRP regulates cytokine gene expression at both transcriptional and post-transcriptional levels. Our results outline a critical role for KSRP in regulating pro-inflammatory mediators and have implications for a wide range of CNS inflammatory and autoimmune diseases. PMID:22847996

  1. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    PubMed Central

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.

    2011-01-01

    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  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. Impact of histone H4K16 acetylation on the meiotic recombination checkpoint in Saccharomyces cerevisiae

    PubMed Central

    Cavero, Santiago; Herruzo, Esther; Ontoso, David; San-Segundo, Pedro A.

    2016-01-01

    In meiotic cells, the pachytene checkpoint or meiotic recombination checkpoint is a surveillance mechanism that monitors critical processes, such as recombination and chromosome synapsis, which are essential for proper distribution of chromosomes to the meiotic progeny. Failures in these processes lead to the formation of aneuploid gametes. Meiotic recombination occurs in the context of chromatin; in fact, the histone methyltransferase Dot1 and the histone deacetylase Sir2 are known regulators of the pachytene checkpoint in Saccharomyces cerevisiae. We report here that Sas2-mediated acetylation of histone H4 at lysine 16 (H4K16ac), one of the Sir2 targets, modulates meiotic checkpoint activity in response to synaptonemal complex defects. We show that, like sir2, the H4-K16Q mutation, mimicking constitutive acetylation of H4K16, eliminates the delay in meiotic cell cycle progression imposed by the checkpoint in the synapsis-defective zip1 mutant. We also demonstrate that, like in dot1, zip1-induced phosphorylation of the Hop1 checkpoint adaptor at threonine 318 and the ensuing Mek1 activation are impaired in H4-K16 mutants. However, in contrast to sir2 and dot1, the H4-K16R and H4-K16Q mutations have only a minor effect in checkpoint activation and localization of the nucleolar Pch2 checkpoint factor in ndt80-prophase-arrested cells. We also provide evidence for a cross-talk between Dot1-dependent H3K79 methylation and H4K16ac and show that Sir2 excludes H4K16ac from the rDNA region on meiotic chromosomes. Our results reveal that proper levels of H4K16ac orchestrate this meiotic quality control mechanism and that Sir2 impinges on additional targets to fully activate the checkpoint. PMID:28357333

  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. Planar silicon patch-clamp electrodes integrated with polydimethylsiloxane microfluidics

    NASA Astrophysics Data System (ADS)

    Nagarah, John Michael

    The patch-clamp technique allows one to probe single ion channels and macroscopic ion channel activity in their native environment and resolve their activity as their physical and chemical surroundings are varied. The traditional method of patch-clamping cells involves bringing a clean, flame-polished glass pipette tip with a 1-2 mum diameter pore into contact with a cell membrane to form a high electrical resistance seal. This technique is the gold standard for cellular electrophysiology investigations because it allows the observation of single ion channel protein dynamics as well as activity from an ensemble ion channels from a single cell. Furthermore, any drug approved by federal drug agencies must be screened against particular ion channels with the patch-clamp technique. However, this technique by its nature is serial, time consuming, difficult when exchanging pipette solutions, and difficult to integrate with other technologies. These reasons have prompted several investigators to explore alternative approaches to traditional pipette patch-clamping to increase the throughput of measurements. Herein, I describe the development of a silicon-wafer based device platform that enables the measurement of ion channel activities. The electrical nature of the cell/wafer seal is characterized for several pore design variations. The majority of gigaohm seals obtained falls in the range of 10-20GO. The cell-attached and whole cell configurations are demonstrated. Whole cell ion channel activity originating from various cell fines is consistent with the more traditional micropipette patch-clamp recordings. The silicon fabrication methods developed, although novel, utilize established semiconductor technologies, making them amenable to batch fabrication techniques. I integrate these silicon devices with PDMS microfluidics with monolithic valves, allowing ultra-fast solution exchange as low as tens of milliseconds for the extracellular solution. Furthermore, I developed a

  6. Extracellular potassium accumulation in voltage-clamped frog ventricular muscle.

    PubMed Central

    Cleemann, L; Morad, M

    1979-01-01

    1. Application of voltage clamp pulses (1--10 sec) to frog ventricular strips causes temporary changes in the extracellular K concentration. 2. The changes in the extracellular K concentration can be estimated from (a) slowly decaying post-clamp after-potentials, (b) changes in the action potential duration, and (c) measurements with a K-selective micro-electrode. 3. The depolarization of the resting potential and the shortening of the action potential are present in approximately the same proportions during voltage-clamp induced extracellular K accumulation and during perfusion with a K-ricn Ringer solution but small consistent differences are noticed. 4. The measurements of the after-potential, the action potential shortening, and the K-electrode response were analysed as indicators of extracellular K+ activity and it was concluded that the after-potential provides the most convenient and reliable estimate of the absolute magnitude of the voltage-clamp induced extracellular K accumulation. 5. The depolarizing after-potentials decay more slowly than the hyperpolarizing after-potentials but it is found that this reflects the selectivity of the membrane to K+ concentrations as predicted by the Nernst or the Goldman equations. 6. Analysis of the redistribution of accumulated K+ from the decay of the after-potential suggests that the major part of the redistribution process can be described by a single time constant (2--4 sec). A much longer time constant is required for a smaller component of the 'tail' in order to bring [K]o to the normal resting state. 7. N-shaped relations similar to the 'steady state' current-voltage relation are obtained when the post-clamp after-potential, the action potential shortening, and the K-electrode response are plotted versus the clamped membrane potential. The maxima of these curves are located around -40 mV and the minima around -20 mV. 8. In spite of a significant outward membrane current (1--1.5 microamperemeter) in the minimum

  7. Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices.

    PubMed

    Perkins, Katherine L

    2006-06-30

    Cell-attached recording provides a way to record the activity of - and to stimulate - neurons in brain slices without rupturing the cell membrane. This review uses theory and experimental data to address the proper application of this technique and the correct interpretation of the data. Voltage-clamp mode is best-suited for recording cell firing activity, and current-clamp mode is best-suited for recording resting membrane potential and synaptic potentials. The magnitude of the seal resistance determines what types of experiments can be accomplished with a cell-attached recording: a loose seal is adequate for recording action potential currents, and a tight seal is required for evoking action potentials in the attached cell and for recording resting and synaptic potentials. When recording action potential currents, if the researcher does not want to change the firing activity of the cell, then it is important that no current passes from the amplifier through the patch resistance. In order to accomplish this condition, the recording pipette should be held at the potential that gives a holding current of 0. An advantage of cell-attached current-clamp over whole-cell recording is that it accurately depicts whether a synaptic potential is hyperpolarizing or depolarizing without the risk of changing its polarity.

  8. Clamping stiffness and its influence on load distribution between paired internal spinal fixation devices.

    PubMed

    Rohlmann, A; Calisse, J; Bergmann, G; Radvan, J; Mayer, H M

    1996-06-01

    The load distribution between two internal spinal fixation devices depends, besides other factors, on their stiffness. The stiffness ranges were determined experimentally for the clamps of the AO internal fixator with lateral nut and with posterior nut as well as for the clamps of the SOCON fixator. The stiffness of eight devices each differed by a factor of 3.1 for the clamp with lateral nut, by a factor of 1.5 for the clamp with posterior nut, and by a factor of 1.4 for the clamp of the SOCON fixator. For the AO clamp with lateral nut, the influence of the nut-tightening torque on the stiffness was determined. Using instrumented internal spinal fixation devices mounted to plastic vertebrae and simulating a corpectomy, the load distribution between the implants was measured for different tightening torques. It could be shown that, for the AO internal fixator whose clamps have a lateral nut, a nut-tightening torque > 5 Nm has only a negligible influence on load-sharing between the implants. Tooth damage occurs when the teeth of the clamp body and clamping jaw of the clamp with lateral nut do not gear together exactly, which leads to changes in the clamping stiffness and load-sharing between the two implants.

  9. Expression of immune checkpoints in T cells of esophageal cancer patients

    PubMed Central

    Xie, Jinhua; Wang, Ji; Cheng, Shouliang; Zheng, Liangfeng; Ji, Feiyue; Yang, Lin; Zhang, Yan; Ji, Haoming

    2016-01-01

    Inhibition of immune checkpoint proteins (checkpoints) has become a promising anti-esophageal cancer strategy. We here tested expressions of immune checkpoints in human esophageal cancers. Our results showed the expressions of many immune checkpoints, including CD28, CD27, CD137L, programmed death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), T cell Ig and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte antigen 4 (CTLA-4), CD200, CD137 and CD158, were dysregulated in peripheral T cells of esophageal cancer patients. Further, the expressions of PD-1, TIM-3 and TIGIT were upregulated in tumor infiltrating lymphocytes (TILs), which might be associated with TILs exhaustion. Meanwhile, the expressions of PD-1 and TIM-3 on CD4+ T cells were closely associated with clinic pathological features of esophageal cancer patients. These results indicate that co-inhibitory receptors PD-1, TIM-3 and TIGIT may be potential therapeutic oncotargets for esophageal cancer. PMID:27577071

  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. Immune-Checkpoint Inhibitors in the Era of Precision Medicine: What Radiologists Should Know

    PubMed Central

    Tirumani, Sree Harsha; Hodi, Frank Stephen; Nishino, Mizuki

    2017-01-01

    Over the past five years immune-checkpoint inhibitors have dramatically changed the therapeutic landscape of advanced solid and hematologic malignancies. The currently approved immune-checkpoint inhibitors include antibodies to cytotoxic T-lymphocyte antigen-4, programmed cell death (PD-1), and programmed cell death ligand (PD-L1 and PD-L2). Response to immune-checkpoint inhibitors is evaluated on imaging using the immune-related response criteria. Activation of immune system results in a unique toxicity profile termed immune-related adverse events. This article will review the molecular mechanism, clinical applications, imaging of immune-related response patterns and adverse events associated with immune-checkpoint inhibitors. PMID:28096717

  12. Lymphocyte-activation gene-3, an important immune checkpoint in cancer.

    PubMed

    He, Yayi; Rivard, Christopher J; Rozeboom, Leslie; Yu, Hui; Ellison, Kim; Kowalewski, Ashley; Zhou, Caicun; Hirsch, Fred R

    2016-09-01

    Immunotherapy has recently become widely used in lung cancer. Many oncologists are focused on cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death ligand-1 (PD-L1) and programmed cell death-1 (PD-1). Immunotherapy targeting the PD-1/PD-L1 checkpoints has shown promising efficacy in non-small cell lung cancer (NSCLC), but questions remain to be answered. Among them is whether the simultaneous inhibition of other checkpoints could improve outcomes. Lymphocyte-activation gene-3 (LAG-3) is another vital checkpoint that may have a synergistic interaction with PD-1/PD-L1. Here we review the LAG-3 function in cancer, clinical trials with agents targeting LAG-3 and the correlation of LAG-3 with other checkpoints.

  13. Immune-Checkpoint Inhibitors in the Era of Precision Medicine: What Radiologists Should Know.

    PubMed

    Braschi-Amirfarzan, Marta; Tirumani, Sree Harsha; Hodi, Frank Stephen; Nishino, Mizuki

    2017-01-01

    Over the past five years immune-checkpoint inhibitors have dramatically changed the therapeutic landscape of advanced solid and hematologic malignancies. The currently approved immune-checkpoint inhibitors include antibodies to cytotoxic T-lymphocyte antigen-4, programmed cell death (PD-1), and programmed cell death ligand (PD-L1 and PD-L2). Response to immune-checkpoint inhibitors is evaluated on imaging using the immune-related response criteria. Activation of immune system results in a unique toxicity profile termed immune-related adverse events. This article will review the molecular mechanism, clinical applications, imaging of immune-related response patterns and adverse events associated with immune-checkpoint inhibitors.

  14. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy

    PubMed Central

    Topalian, Suzanne L.; Taube, Janis M.; Anders, Robert A.; Pardoll, Drew M.

    2017-01-01

    With recent approvals for multiple therapeutic antibodies that block cytotoxic T lymphocyte associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) in melanoma, non-small-cell lung cancer and kidney cancer, and additional immune checkpoints being targeted clinically, many questions still remain regarding the optimal use of drugs that block these checkpoint pathways. Defining biomarkers that predict therapeutic effects and adverse events is a crucial mandate, highlighted by recent approvals for two PDL1 diagnostic tests. Here, we discuss biomarkers for anti-PD1 therapy based on immunological, genetic and virological criteria. The unique biology of the CTLA4 immune checkpoint, compared with PD1, requires a different approach to biomarker development. Mechanism-based insights from such studies may guide the design of synergistic treatment combinations based on immune checkpoint blockade. PMID:27079802

  15. M. tuberculosis Sliding β-Clamp Does Not Interact Directly with the NAD+ -Dependent DNA Ligase

    PubMed Central

    Kukshal, Vandna; Khanam, Taran; Chopra, Deepti; Singh, Nidhi; Sanyal, Sabyasachi; Ramachandran, Ravishankar

    2012-01-01

    The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C2221 with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent Kd of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD+ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria. PMID:22545130

  16. Analysis of motion during the breast clamping phase of mammography

    PubMed Central

    McEntee, Mark F; Mercer, Claire; Kelly, Judith; Millington, Sara; Hogg, Peter

    2016-01-01

    Objective: To measure paddle motion during the clamping phase of a breast phantom for a range of machine/paddle combinations. Methods: A deformable breast phantom was used to simulate a female breast. 12 mammography machines from three manufacturers with 22 flexible and 20 fixed paddles were evaluated. Vertical motion at the paddle was measured using two calibrated linear potentiometers. For each paddle, the motion in millimetres was recorded every 0.5 s for 40 s, while the phantom was compressed with 80 N. Independent t-tests were used to determine differences in paddle motion between flexible and fixed, small and large, GE Senographe Essential (General Electric Medical Systems, Milwaukee, WI) and Hologic Selenia Dimensions paddles (Hologic, Bedford, MA). Paddle tilt in the medial–lateral plane for each machine/paddle combination was calculated. Results: All machine/paddle combinations demonstrate highest levels of motion during the first 10 s of the clamping phase. The least motion is 0.17 ± 0.05 mm/10 s (n = 20) and the most motion is 0.51 ± 0.15 mm/10 s (n = 80). There is a statistical difference in paddle motion between fixed and flexible (p < 0.001), GE Senographe Essential and Hologic Selenia Dimensions paddles (p < 0.001). Paddle tilt in the medial–lateral plane is independent of time and varied from 0.04 ° to 0.69 °. Conclusion: All machine/paddle combinations exhibited motion and tilting, and the extent varied with machine and paddle sizes and types. Advances in knowledge: This research suggests that image blurring will likely be clinically insignificant 4 s or more after the clamping phase commences. PMID:26739577

  17. 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.

  18. Potassium Chloride Versus Voltage Clamp Contractures in Ventricular Muscle

    NASA Astrophysics Data System (ADS)

    Morad, M.; Reeck, S.; Rao, M.

    1981-01-01

    In frog ventricle, developed tension was markedly larger in response to depolarization caused by a voltage clamp step than to depolarization induced by high concentrations of potassium chloride. Measurement of extracellular potassium activity at the surface and at the depth of muscle during the development of contractures showed that the diffusion of potassium is much slower than the spread of depolarization through the cross section of muscle. These two observations suggest that competition between the depolarizing and the negative inotropic effects of an increase in the extracellular potassium ion concentration may determine the time course and magnitude of contractile tension in heart muscle.

  19. From unwinding to clamping - the DEAD box RNA helicase family.

    PubMed

    Linder, Patrick; Jankowsky, Eckhard

    2011-07-22

    RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.

  20. 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 from 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 permanent set as a function of the dimensions of the plate and the tensile properties of the material.

  1. 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.

  2. Nanomechanics of PCNA: A protein-made DNA sliding clamp

    NASA Astrophysics Data System (ADS)

    Rydzewski, Jakub; Strzalka, Wojciech; Nowak, Wieslaw

    2015-08-01

    Proliferating Cell Nuclear Antigen (PCNA) is a homotrimeric DNA sliding clamp involved in DNA replication, repair and cell cycle control. Understanding determinants of mechanical stability of a PCNA monomer composed of two N- and C-terminal domains requires all-atom and coarse-grained steered molecular dynamics simulations. Three schemes of unfolding at a single molecule level indicate that the conserved curvature of the PCNA monomer is due to aggregation of side β-sheets. The force spectra suggest that rupturing an interface between the domains is less probable than opening an interface between the two monomers during PCNA loading onto DNA by Replication Factor C.

  3. Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast.

    PubMed Central

    Forbes, K C; Humphrey, T; Enoch, T

    1998-01-01

    Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA. PMID:9832516

  4. The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation.

    PubMed

    Pérez-Hidalgo, Livia; Moreno, Sergio; San-Segundo, Pedro A

    2008-12-01

    In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1(+) as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.

  5. Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA.

    PubMed

    Lewis, Cody W; Golsteyn, Roy M

    2016-11-16

    We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 μM). Nearly 100% of M059K cells were positive for histone γH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone γH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.

  6. 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

  7. Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy

    PubMed Central

    Lee, Ju Yeon; Lee, Hyun Tae; Shin, Woori; Chae, Jongseok; Choi, Jaemo; Kim, Sung Hyun; Lim, Heejin; Won Heo, Tae; Park, Kyeong Young; Lee, Yeon Ji; Ryu, Seong Eon; Son, Ji Young; Lee, Jee Un; Heo, Yong-Seok

    2016-01-01

    Cancer cells express tumour-specific antigens derived via genetic and epigenetic alterations, which may be targeted by T-cell-mediated immune responses. However, cancer cells can avoid immune surveillance by suppressing immunity through activation of specific inhibitory signalling pathways, referred to as immune checkpoints. In recent years, the blockade of checkpoint molecules such as PD-1, PD-L1 and CTLA-4, with monoclonal antibodies has enabled the development of breakthrough therapies in oncology, and four therapeutic antibodies targeting these checkpoint molecules have been approved by the FDA for the treatment of several types of cancer. Here, we report the crystal structures of checkpoint molecules in complex with the Fab fragments of therapeutic antibodies, including PD-1/pembrolizumab, PD-1/nivolumab, PD-L1/BMS-936559 and CTLA-4/tremelimumab. These complex structures elucidate the precise epitopes of the antibodies and the molecular mechanisms underlying checkpoint blockade, providing useful information for the improvement of monoclonal antibodies capable of attenuating checkpoint signalling for the treatment of cancer. PMID:27796306

  8. Binding inhibitors of the bacterial sliding clamp by design.

    PubMed

    Wijffels, Gene; Johnson, Wynona M; Oakley, Aaron J; Turner, Kathleen; Epa, V Chandana; Briscoe, Susan J; Polley, Mitchell; Liepa, Andris J; Hofmann, Albert; Buchardt, Jens; Christensen, Caspar; Prosselkov, Pavel; Dalrymple, Brian P; Alewood, Paul F; Jennings, Philip A; Dixon, Nicholas E; Winkler, David A

    2011-07-14

    The bacterial replisome is a target for the development of new antibiotics to combat drug resistant strains. The β(2) sliding clamp is an essential component of the replicative machinery, providing a platform for recruitment and function of other replisomal components and ensuring polymerase processivity during DNA replication and repair. A single binding region of the clamp is utilized by its binding partners, which all contain conserved binding motifs. The C-terminal Leu and Phe residues of these motifs are integral to the binding interaction. We acquired three-dimensional structural information on the binding site in β(2) by a study of the binding of modified peptides. Development of a three-dimensional pharmacophore based on the C-terminal dipeptide of the motif enabled identification of compounds that on further development inhibited α-β(2) interaction at low micromolar concentrations. We report the crystal structure of the complex containing one of these inhibitors, a biphenyl oxime, bound to β(2), as a starting point for further inhibitor design.

  9. Image processing techniques in computer-assisted patch clamping

    NASA Astrophysics Data System (ADS)

    Azizian, Mahdi; Patel, Rajni; Gavrilovici, Cezar; Poulter, Michael O.

    2010-02-01

    Patch clamping is used in electrophysiology to study single or multiple ion channels in cells. Multiple micropipettes are used as electrodes to collect data from several cells. Placement of these electrodes is a time consuming and complicated task due to the lack of depth perception, limited view through the microscope lens and the possibility of collisions between micro-pipettes. To aid in this process, a computer-assisted approach is developed using image processing techniques applied to images obtained through the microscope. Image processing algorithms are applied to perform autofocusing, relative depth estimation, distance estimation and tracking of the micro-pipettes in the images without making any major changes in the existing patch clamp equipment. An autofocusing algorithm with a micrometer precision is developed and the relative depth estimation is performed based on autofocusing. A micro-pipette tip detection algorithm is developed which can be used to initialize or reset the tracking algorithm and to calibrate the system by registering the relative image and micro-manipulator coordinates. An image-based tracking algorithm is also developed to track a micro-pipette tip in real time. The real-time tracking data is then used for visual servoing the micro-pipette tips and updating the calibration information.

  10. Clamping of fine Kirschner wires in external fixators.

    PubMed

    Zamani, Ahmad R; Oyadiji, S Olutunde

    2016-11-01

    In Ilizarov circular (ring) external fixators, fine Kirschner wires are used to fix the bone to the fixator. Clamping of the wires to the rings with different bolt torques has been studied. However, the relation between the bolt torque and the fixation load applied to the wire was not investigated. In this work, finite element method is used to address this problem. Here, a fully three-dimensional model of the wire fixation assembly was built, with geometric details like threads on the bolt to produce a realistic simulation of the clamping of the wire. Both cannulated and slotted bolt types were studied and values of 0.2, 0.25, 0.3 and 0.45 were used for coefficient of friction. A torque was applied to the nut while the ring section and bolt kept in place. The results for bolt load, nut rotation as well as axial and radial wire deformations were obtained. The results demonstrated a linear relation between the bolt load and the bolt torque. The coefficient of this relationship was shown to be inversely proportional to the coefficient of friction. For all results, the bolt load (N) was approximately 124 times the bolt torque (N m) divided by the friction coefficient. The results highlighted the difference between the cannulated and the slotted bolts in terms of their grip on the wire.

  11. Umbilical cord clamping and preterm infants: a randomised trial.

    PubMed Central

    Kinmond, S; Aitchison, T C; Holland, B M; Jones, J G; Turner, T L; Wardrop, C A

    1993-01-01

    OBJECTIVE--To investigate the clinical effects of regulating umbilical cord clamping in preterm infants. DESIGN--A prospective randomised study. SETTING--The Queen Mother's Hospital, Glasgow. SUBJECTS--36 vaginally delivered infants over 27 and under 33 weeks' gestation. INTERVENTION--Holding the infant 20 cm below the introitus for 30 seconds before clamping the umbilical cord ("regulated" group, 17 patients), or conventional management ("random" group, 19 patients). MAIN OUTCOME MEASURES--Initial packed cell volume, peak serum bilirubin concentrations, red cell transfusion requirements, and respiratory impairment (assessed by ventilatory requirements, arterial-alveolar oxygen tension ratio over the first day in ventilated infants, and duration of dependence on supplemental oxygen). RESULTS--There were statistically significant differences between the two groups in mean initial packed cell volume (regulated group 0.564, random group 0.509) and median red cell transfusion requirements (regulated group zero, random group 23 ml/kg). 13 infants from each group underwent mechanical ventilation and showed significant differences in mean minimum arterial-alveolar oxygen tension ratio on the first day (regulated group 0.42, random group 0.22) and in median duration of dependence on supplemental oxygen (regulated group three days, random group 10 days). Differences in final outcome measures such as duration of supplemental oxygen dependence and red cell transfusion requirements were mediated primarily through arterial-alveolar oxygen tension ratio and also packed cell volume. CONCLUSIONS--This intervention at preterm deliveries produces clinical and economic benefits. PMID:8443480

  12. Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

    PubMed Central

    Kitagawa, Mayumi; Caldez, Matias J.; Gunaratne, Jayantha; Lee, Sang Hyun

    2016-01-01

    The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing. PMID:27631493

  13. Retinoids regulate a developmental checkpoint for tissue regeneration in Drosophila

    PubMed Central

    Halme, Adrian; Cheng, Michelle; Hariharan, Iswar K.

    2010-01-01

    Summary Drosophila melanogaster larvae have a remarkable capacity for regenerative growth: Damage to their imaginal discs, the larval precursors of adult structures, elicits a robust proliferative response from the surviving tissue [1–4]. However, as in other organisms, developmental progression and differentiation can restrict regenerative capacity of Drosophila tissues. Experiments in Drosophila and other holometabolous insects have demonstrated that either damage to imaginal tissues [5, 6] or transplantation of a damaged imaginal disc [7, 8] delays the onset of metamorphosis, a time when the imaginal discs undergo morphogenesis and differentiation into their adult structures. Therefore, in Drosophila there appears to be a mechanism that senses tissue damage and extends the larval phase to coordinate tissue regeneration with the overall developmental program of the organism. However, how such a pathway functions remains unknown. Here we demonstrate that a developmental checkpoint extends larval growth after imaginal disc damage by inhibiting the transcription of the gene encoding PTTH, a neuropeptide that promotes the release of the steroid hormone ecdysone. Using a genetic screen, we identify a previously unsuspected role for retinoid biosynthesis in regulating PTTH expression and delaying development in response to tissue damage. Retinoid signaling plays an important, but poorly defined role in several vertebrate regeneration models [9–11]. Our findings demonstrate that retinoid biosynthesis in Drosophila is important for the maintenance of a permissive condition for regenerative growth. PMID:20189388

  14. 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.

  15. SOCS: potential immune checkpoint molecules for cancer immunotherapy.

    PubMed

    Chikuma, Shunsuke; Kanamori, Mitsuhiro; Mise-Omata, Setsuko; Yoshimura, Akihiko

    2017-02-11

    Inhibition of immune checkpoint molecules, PD-1 and CTLA4, has been shown to be a promising cancer treatment. PD-1 and CTLA4 inhibit TCR and co-stimulatory signals, respectively. The third T cell activation signal represents the signals from the cytokine receptors. The cytokine Interferon-γ (IFNγ) plays an important role in anti-tumor immunity by activating cytotoxic T cells (CTLs). Most cytokines use the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and the suppressors of cytokine signaling (SOCS) family of proteins are major negative regulators of the JAK/STAT pathway. Among SOCS proteins, CIS, SOCS1, and SOCS3 proteins can be considered the third immunocheckpoint molecules since they regulate cytokine signals that control the polarization of CD4(+) T cells and the maturation of CD8(+) T cells. This review summarizes recent progress on CIS, SOCS1, and SOCS3 in terms of their anti-tumor immunity and potential applications. This article is protected by copyright. All rights reserved.

  16. A dynamical model of the spindle position checkpoint.

    PubMed

    Caydasi, Ayse Koca; Lohel, Maiko; Grünert, Gerd; Dittrich, Peter; Pereira, Gislene; Ibrahim, Bashar

    2012-05-08

    The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother-to-daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase-activating protein (GAP) complex Bfa1-Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1-Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1-Bub2 and Tem1 localization at the SPBs. Based on the measured SPB-bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition.

  17. Resistance to PD1/PDL1 checkpoint inhibition.

    PubMed

    O'Donnell, Jake S; Long, Georgina V; Scolyer, Richard A; Teng, Michele W L; Smyth, Mark J

    2017-01-01

    For the first time in decades, patients with difficult-to-treat cancers such as advanced stage metastatic melanoma are being offered a glimpse of hope in the form of immunotherapies. By targeting factors that foster the development and maintenance of an immunosuppressive microenvironment within tumors, these therapies release the brakes on the host's own immune system; allowing cure of disease. Indeed, phase III clinical trials have revealed that therapies such as ipilimumab and pembrolizumab which target the CTLA4 and PD-1 immune checkpoints, respectively, have raised the three-year survival of patients with melanoma to ∼70%, and overall survival (>5years) to ∼30%. Despite this unprecedented efficacy, many patients fail to respond, and more concerning, some patients who demonstrate encouraging initial responses to immunotherapy, can acquire resistance over time. There is now an urgent need to identify mechanisms of resistance, to predict outcome and to identify targets for combination therapy. Here, with the aim of guiding future combination trials that target specific resistance mechanisms to immunotherapies, we have summarised and discussed the current understanding of mechanisms promoting resistance to anti-PD1/PDL1 therapies, and how combination strategies which target these pathways might yield better outcomes for patients.

  18. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints

    PubMed Central

    Zhao, Hui; Watkins, Janis L.; Piwnica-Worms, Helen

    2002-01-01

    Checkpoint kinase (Chk)1 is an evolutionarily conserved protein kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint. In mice, Chk1 provides an essential function in the absence of environmentally imposed genotoxic stress. Here we show that human cells lacking Chk1 exhibit defects in both the ionizing radiation (IR)-induced S and G2 checkpoints. In addition, loss of Chk1 resulted in the accumulation of a hypophosphorylated form of the Cdc25A protein phosphatase, and Chk1-deficient cells failed to degrade Cdc25A after IR. The IR-induced S and G2 checkpoints were partially restored in Chk1-deficient cells when Cdc25A accumulation was interfered with. Finally, Cdc25A was phosphorylated by Chk1 in vitro on similar sites phosphorylated in vivo, including serine-123. These findings indicate that Chk1 directly phosphorylates Cdc25A during an unperturbed cell cycle, and that phosphorylation of Cdc25A by Chk1 is required for cells to delay cell cycle progression in response to double-strand DNA breaks. PMID:12399544

  19. Bir1 deletion causes malfunction of the spindle assembly checkpoint and apoptosis in yeast.

    PubMed

    Ren, Qun; Liou, Liang-Chun; Gao, Qiuqiang; Bao, Xiaoming; Zhang, Zhaojie

    2012-01-01

    Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H(2)O(2) treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

  20. Concordance of immune checkpoints within tumor immune contexture and their prognostic significance in gastric cancer.

    PubMed

    Dai, Congqi; Geng, Ruixuan; Wang, Chenchen; Wong, Angela; Qing, Min; Hu, Jianjun; Sun, Yu; Lo, A W I; Li, Jin

    2016-12-01

    Checkpoint blockade therapy has emerged as a novel approach for cancer immunotherapy in several malignancies. However, patient prognosis and disease progression relevant to immune checkpoints in gastric tumor microenvironment are not defined. This study aims to investigate the expression and prognostic significance of immune checkpoints within gastric cancer. In the study, a cohort of 398 cancer tissues from stage I to IV gastric cancer patients were assessed for programmed cell death 1 ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) infiltration using immunohistochemistry to ascertain their survival correlation. The data revealed that higher TIL density correlated with less risk of disease progression, and exhibited survival benefits in gastric cancer patients, and PD-L1 positivity showed a significant association with the presence of high TIL infiltration. Furthermore, real-time quantitative polymerase chain reaction was performed to detect expression of multiple immune checkpoints with the relation to clinical outcome in 139 samples randomly selected from the same cohort, and higher messenger RNA levels of most immune checkpoints were associated with favorable outcome, while consistently showing a positive correlation with interferon gamma levels. In situ hybridization was used to determine the localization of Epstein-Barr virus (EBV) in 97 specimens, and showed EBV-positive gastric cancer samples correlated with PD-L1 expression and increased TIL density. These results suggest that induction of immune checkpoint within gastric cancer patients reflects a high immune infiltration density, especially in those with EBV-associated gastric cancer, which may direct patient selection for checkpoint blockade therapy.

  1. Hyperinsulinemic-euglycemic clamp in the conscious rat.

    PubMed

    Hughey, Curtis C; Hittel, Dustin S; Johnsen, Virginia L; Shearer, Jane

    2011-02-07

    Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet

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

    PubMed Central

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

    2014-01-01

    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

  3. Modeling considerations for the analysis of LMFBR steam generator tube clamps

    SciTech Connect

    Lay, D.M.; Piper, R.M.

    1982-01-01

    In the design of the Babcock and Wilcox Helical Coil Liquid Metal Fast Breeder Reactor (LMFBR) Steam Generator, the tube bundle is connected to the feedwater and steam plenums via ''inlet/outlet tubes''. Of prime importance in the design of these tubes is the tube-to-tube and tube-to-shell clamps which are provided to prevent detrimental vibration. This paper presents a method of modeling the tube-to-tube clamps to accurately predict tube-to-clamp interaction in the finite element analysis. It also demonstrates the validity of specific modeling assumptions in determining stresses in the clamp assembly.

  4. Electrically integrated SU-8 clamped graphene drum resonators for strain engineering

    NASA Astrophysics Data System (ADS)

    Lee, Sunwoo; Chen, Changyao; Deshpande, Vikram V.; Lee, Gwan-Hyoung; Lee, Ilkyu; Lekas, Michael; Gondarenko, Alexander; Yu, Young-Jun; Shepard, Kenneth; Kim, Philip; Hone, James

    2013-04-01

    Graphene mechanical resonators are the ultimate two-dimensional nanoelectromechanical systems (NEMS) with applications in sensing and signal processing. While initial devices have shown promising results, an ideal graphene NEMS resonator should be strain engineered, clamped at the edge without trapping gas underneath, and electrically integratable. In this Letter, we demonstrate fabrication and direct electrical measurement of circular SU-8 polymer-clamped chemical vapor deposition graphene drum resonators. The clamping increases device yield and responsivity, while providing a cleaner resonance spectrum from eliminated edge modes. Furthermore, the clamping induces a large strain in the resonator, increasing its resonant frequency.

  5. Mechanism of ATP-driven PCNA clamp loading by S. cerevisiae RFC.

    PubMed

    Chen, Siying; Levin, Mikhail K; Sakato, Miho; Zhou, Yayan; Hingorani, Manju M

    2009-05-08

    Circular clamps tether polymerases to DNA, serving as essential processivity factors in genome replication, and function in other critical cellular processes as well. Clamp loaders catalyze clamp assembly onto DNA, and the question of how these proteins construct a topological link between a clamp and DNA, especially the mechanism by which ATP is utilized for the task, remains open. Here we describe pre-steady-state analysis of ATP hydrolysis, proliferating cell nuclear antigen (PCNA) clamp opening, and DNA binding by Saccharomyces cerevisiae replication factor C (RFC), and present the first kinetic model of a eukaryotic clamp-loading reaction validated by global data analysis. ATP binding to multiple RFC subunits initiates a slow conformational change in the clamp loader, enabling it to bind and open PCNA and to bind DNA as well. PCNA opening locks RFC into an active state, and the resulting RFC.ATP.PCNA((open)) intermediate is ready for the entry of DNA into the clamp. DNA binding commits RFC to ATP hydrolysis, which is followed by PCNA closure and PCNA.DNA release. This model enables quantitative understanding of the multistep mechanism of a eukaryotic clamp loader and furthermore facilitates comparative analysis of loaders from diverse organisms.

  6. 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.

  7. 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,

  8. Self-clamping arc light reflector for welding torch

    NASA Astrophysics Data System (ADS)

    Gordon, Stephen S.

    1987-07-01

    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,

  9. Priming and testing silicon patch-clamp neurochips.

    PubMed

    Py, Christophe; Denhoff, Michael W; Sabourin, Nicaulas; Weber, John; Shiu, Matthew; Zhao, Ping

    2014-09-25

    We report on the systematic and automated priming and testing of silicon planar patch-clamp chips after their assembly in Plexiglas packages and sterilization in an air plasma reactor. We find that almost 90% of the chips are successfully primed by our automated setup, and have a shunt capacitance of between 10 pF and 30 pF. Blocked chips are mostly due to glue invasion in the well, and variability in the manual assembly process is responsible for the distribution in shunt capacitance value. Priming and testing time with our automated setup is less than 5 min per chip, which is compatible with the production of large series for use in electrophysiology experiments.

  10. 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

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

    DOE PAGES

    Cuellar-Hengartner, Leticia; Watkins, Daniel; Kubicek, Deborah A.; ...

    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

  12. Caffeine stabilizes Cdc25 independently of Rad3 in Schizosaccharomyces pombe contributing to checkpoint override.

    PubMed

    Alao, John P; Sjölander, Johanna J; Baar, Juliane; Özbaki-Yagan, Nejla; Kakoschky, Bianca; Sunnerhagen, Per

    2014-05-01

    Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both Schizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S. pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S. pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25.

  13. The nucleoporin Nup153 affects spindle checkpoint activity due to an association with Mad1

    PubMed Central

    Shimi, Takeshi

    2010-01-01

    The nucleoporin Nup153 is known to play pivotal roles in nuclear import and export in interphase cells and as the cell transitions into mitosis, Nup153 is involved in nuclear envelope breakdown. In this study, we demonstrate that the interaction of Nup153 with the spindle assembly checkpoint protein Mad1 is important in the regulation of the spindle checkpoint. Overexpression of human Nup153 in HeLa cells leads to the appearance of multinucleated cells and induces the formation of multipolar spindles. Importantly, it causes inactivation of the spindle checkpoint due to hypophosphorylation of Mad1. Depletion of Nup153 using RNA interference results in the decline of Mad1 at nuclear pores during interphase and more significantly causes a delayed dissociation of Mad1 from kinetochores in metaphase and an increase in the number of unresolved midbodies. In the absence of Nup153 the spindle checkpoint remains active. In vitro studies indicate direct binding of Mad1 to the N-terminal domain of Nup153. Importantly, Nup153 binding to Mad1 affects Mad1's phosphorylation status, but not its ability to interact with Mad2. Our data suggest that Nup153 levels regulate the localization of Mad1 during the metaphase/anaphase transition thereby affecting its phoshorylation status and in turn spindle checkpoint activity and mitotic exit. PMID:21327106

  14. Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?D⃞

    PubMed Central

    Vigneron, Suzanne; Prieto, Susana; Bernis, Cyril; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2004-01-01

    The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other. PMID:15269280

  15. 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

  16. TIGIT and CD96: new checkpoint receptor targets for cancer immunotherapy.

    PubMed

    Dougall, William C; Kurtulus, Sema; Smyth, Mark J; Anderson, Ana C

    2017-03-01

    While therapies targeting the co-inhibitory or immune checkpoint receptors PD-1 and CTLA-4 have shown remarkable success in many cancers, not all patients benefit from these therapies. This has catalyzed enormous interest in the targeting of other immune checkpoint receptors. In this regard, TIGIT and CD96 have recently entered the limelight as novel immune checkpoint receptor targets. TIGIT and CD96 together with the co-stimulatory receptor CD226 form a pathway that is analogous to the CD28/CTLA-4 pathway, in which shared ligands and differential receptor:ligand affinities fine-tune the immune response. Although the roles of TIGIT and CD96 as immune checkpoint receptors in T cell and natural killer cell biology are just beginning to be uncovered, accumulating data support the targeting of these receptors for improving anti-tumor immune responses. A clear understanding of the immune cell populations regulated by TIGIT and CD96 is key to the design of immunotherapies that target these receptors in combination with other existing immune checkpoint blockade therapies.

  17. Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control.

    PubMed

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K; O'Hanlon, Karen; Quaas, Marianne; Larsen, Brian D; Rolland, Baptiste; Rösner, Heike I; Walter, David; Kousholt, Arne Nedergaard; Menzel, Tobias; Lees, Michael; Johansen, Jens Vilstrup; Rappsilber, Juri; Engeland, Kurt; Sørensen, Claus Storgaard

    2015-01-05

    Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.

  18. Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

    PubMed

    Cordeiro-Stone, Marila; McNulty, John J; Sproul, Christopher D; Chastain, Paul D; Gibbs-Flournoy, Eugene; Zhou, Yingchun; Carson, Craig; Rao, Shangbang; Mitchell, David L; Simpson, Dennis A; Thomas, Nancy E; Ibrahim, Joseph G; Kaufmann, William K

    2016-01-01

    The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

  19. 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.

  20. FAP positive fibroblasts induce immune checkpoint blockade resistance in colorectal cancer via promoting immunosuppression.

    PubMed

    Chen, Lingling; Qiu, Xiangting; Wang, Xinhua; He, Jian

    2017-03-13

    Immune checkpoint blockades that significantly prolonged survival of melanoma patients have been less effective on colorectal cancer (CRC) patients. Growing evidence suggested that fibroblast activation protein-alpha (FAP) on cancer associate fibroblasts (CAFs) has critical roles in regulating antitumor immune response by inducing tumor-promoting inflammation. In this study, we explored the roles of FAP in regulating the tumor immunity and immune checkpoint blockades resistance in CRC experimental systems. We found that CAFs with high FAP expression could induce immune checkpoint blockade resistance in CRC mouse model. Mechanistically, CAFs with high FAP expression promoted immunosuppression in the CRC tumor immune microenvironment by up-regulating CCL2 secretion, recruiting myeloid cells, and decreasing T-cell activity. In human CRC samples, FAP expression was proportional to myeloid cells number, but inversely related to T-cell number. High FAP expression also predicted poor survival of CRC patients. Taken together, our study suggested that high FAP expression in CAFs is one reason leading to immune checkpoint blockades resistance in CRC patients and FAP is an optional target for reversing immune checkpoint blockades resistance.

  1. Information Sharing from 9-1-1 Centers

    DTIC Science & Technology

    2014-09-01

    P. Simpson 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Naval Postgraduate School Monterey, CA 93943-5000 8. PERFORMING ORGANIZATION REPORT...describe it is, if it is not broken, don’t fix it. In the early 1970s, psychologists Amos Tversky and Daniel Kahneman demonstrated three heuristics

  2. 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.

  3. Dynactin is involved in a checkpoint to monitor cell wall synthesis in Saccharomyces cerevisiae.

    PubMed

    Suzuki, Masaya; Igarashi, Ryoji; Sekiya, Mizuho; Utsugi, Takahiko; Morishita, Shinichi; Yukawa, Masashi; Ohya, Yoshikazu

    2004-09-01

    Checkpoint controls ensure the completion of cell cycle events with high fidelity in the correct order. Here we show the existence of a novel checkpoint that ensures coupling of cell wall synthesis and mitosis. In response to a defect in cell wall synthesis, S. cerevisiae cells arrest the cell-cycle before spindle pole body separation. This arrest results from the regulation of the M-phase cyclin Clb2p at the transcriptional level through the transcription factor Fkh2p. Components of the dynactin complex are required to achieve the G2 arrest whilst keeping cells highly viable. Thus, the dynactin complex has a function in a checkpoint that monitors cell wall synthesis.

  4. Challenges and opportunities for checkpoint blockade in T-cell lymphoproliferative disorders.

    PubMed

    Phillips, Tycel; Devata, Sumana; Wilcox, Ryan A

    2016-01-01

    The T-cell lymphoproliferative disorders are a heterogeneous group of non-Hodgkin's lymphomas (NHL) for which current therapeutic strategies are inadequate, as most patients afflicted with these NHL will succumb to disease progression within 2 years of diagnosis. Appreciation of the genetic and immunologic landscape of these aggressive NHL, including PD-L1 (B7-H1, CD274) expression by malignant T cells and within the tumor microenvironment, provides a strong rationale for therapeutic targeting this immune checkpoint. While further studies are needed, the available data suggests that responses with PD-1 checkpoint blockade alone will unlikely approach those achieved in other lymphoproliferative disorders. Herein, we review the unique challenges posed by the T-cell lymphoproliferative disorders and discuss potential strategies to optimize checkpoint blockade in these T-cell derived malignancies.

  5. DGK-α: A Checkpoint in Cancer-Mediated Immuno-Inhibition and Target for Immunotherapy

    PubMed Central

    Noessner, Elfriede

    2017-01-01

    Immunotherapy is moving to the forefront of cancer treatments owing to impressive durable responses achieved with checkpoint blockade antibodies and adoptive T-cell therapy. Still, improvements are necessary since, overall, only a small percentage of patients benefit from current therapies. Here, I summarize evidence that DGK-α may represent an immunological checkpoint suppressing the activity of cytotoxic immunocytes in the tumor microenvironment. DGK-inhibitors can restore the antitumor function of tumor-suppressed adaptive and innate cytotoxic immunocytes. The activity of DGK-inhibitors lays downstream of current checkpoint blockade antibodies. Thus, synergistic effects are expected from combination strategies. Moreover, DGK-inhibitors may permit a double-strike attack on tumor cells as DGK-inhibition may not only re-instate immunological tumor attack but also may harm tumor cells directly by interfering with oncogenic survival pathways. Together, DGK-inhibitors have very promising characteristics and may be beneficially included into the armamentarium of cancer immunotherapeutics. PMID:28316970

  6. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

    PubMed Central

    Chauvier, Adrien; Picard-Jean, Frédéric; Berger-Dancause, Jean-Christophe; Bastet, Laurène; Naghdi, Mohammad Reza; Dubé, Audrey; Turcotte, Pierre; Perreault, Jonathan; Lafontaine, Daniel A.

    2017-01-01

    On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation. PMID:28071751

  7. Is there a role for therapeutic cancer vaccines in the age of checkpoint inhibitors?

    PubMed Central

    Dillman, Robert O.

    2017-01-01

    ABSTRACT Because of the recent success of monoclonal antibody checkpoint inhibitors, and the disappointing results of most therapeutic cancer vaccine trials, it has been questioned whether there is any potential role for such products going forward. In my opinion the answer is “yes” based on the following: [1] there is a persistent unmet clinical need because the majority of patients do not benefit from anti-checkpoint therapy, [2] there is evidence that not all patients make immune responses to their tumors, [3] there is evidence that immune responses to autologous tumor antigens can be induced by patient-specific vaccines, [4] there is clinical evidence from the pre-checkpoint era that suggests survival can be positively impacted by such patient-specific vaccines, and [5] the 2 available therapeutic vaccines that have received regulatory approval are quite limited in terms of their therapeutic benefit. PMID:27808593

  8. Modulation of DNA damage checkpoint; patenting and possible application for cancer medicine.

    PubMed

    Ishikawa, Kazuhiro; Ishii, Hideshi; Ichimura, Keiichi

    2008-01-01

    Eukaryote cells survey genomic integrity for DNA damage or incomplete replication. Aberrant structures being detected, checkpoint mechanisms are activated to slow down or arrest cell cycle progression, which allow the DNA damage to be repaired and the replication to be completed. In cancer development, precancerous cells overcome selective pressure to escape from blocked cell cycle progression, induced by checkpoint responses to DNA damage. Medical applications targeting the process of DNA damage would lead to efficient repairs of DNA damage or induction of cell death, which contributes to cancer detection, diagnosis and therapeutic approaches. In this article, the recent progress of our knowledge and patenting in modulation of DNA damage checkpoint especially by Rad9-Chk1 pathway is noted and possible application for cancer medicine is discussed.

  9. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    PubMed

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  10. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

    PubMed Central

    1996-01-01

    M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity. PMID:8567717

  11. Contribution of Growth and Cell Cycle Checkpoints to Radiation Survival in Drosophila

    PubMed Central

    Jaklevic, Burnley; Uyetake, Lyle; Lemstra, Willy; Chang, Julia; Leary, William; Edwards, Anthony; Vidwans, Smruti; Sibon, Ody; Tin Su, Tin

    2006-01-01

    Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy. PMID:17028317

  12. Regulation of AURORA B function by mitotic checkpoint protein MAD2.

    PubMed

    Shandilya, Jayasha; Medler, Kathryn F; Roberts, Stefan G E

    2016-08-17

    Cell cycle checkpoint signaling stringently regulates chromosome segregation during cell division. MAD2 is one of the key components of the spindle and mitotic checkpoint complex that regulates the fidelity of cell division along with MAD1, CDC20, BUBR1, BUB3 and MAD3. MAD2 ablation leads to erroneous attachment of kinetochore-spindle fibers and defective chromosome separation. A potential role for MAD2 in the regulation of events beyond the spindle and mitotic checkpoints is not clear. Together with active spindle assembly checkpoint signaling, AURORA B kinase activity is essential for chromosome condensation as cells enter mitosis. AURORA B phosphorylates histone H3 at serine 10 and serine 28 to facilitate the formation of condensed metaphase chromosomes. In the absence of functional AURORA B cells escape mitosis despite the presence of misaligned chromosomes. In this study we report that silencing of MAD2 results in a drastic reduction of metaphase-specific histone H3 phosphorylation at serine 10 and serine 28. We demonstrate that this is due to mislocalization of AURORA B in the absence of MAD2. Conversely, overexpression of MAD2 concentrated the localization of AURORA B at the metaphase plate and caused hyper-phosphorylation of histone H3. We find that MAD1 plays a minor role in influencing the MAD2-dependent regulation of AURORA B suggesting that the effects of MAD2 on AURORA B are independent of the spindle checkpoint complex. Our findings reveal that, in addition to its role in checkpoint signaling, MAD2 ensures chromosome stability through the regulation of AURORA B.

  13. 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

    2016-08-08

    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.

  14. A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques

    PubMed Central

    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

  15. Assessing clamp-related vascular injuries by measurement of associated vascular dysfunction.

    PubMed

    Barone, G W; Conerly, J M; Farley, P C; Flanagan, T L; Kron, I L

    1989-04-01

    The development of vascular clamps requires a reliable method to quantitate clamp-related vascular injuries. The degree of vessel damage usually is estimated subjectively from photomicrographs made with scanning electron microscopy. In order to test whether the use of vascular rings to assess residual vascular function may be a better method, rabbit thoracic aortas were occluded by five types of clamps: a Fogarty softjaw bulldog, a Fogarty Hydragrip, a 6-inch Satinsky clamp, an Edslab bulldog, and a silicone vessel band. Each area of clamp injury was sectioned into a vascular ring and suspended in a tissue bath. Residual vascular function was determined by contraction in response to phenylephrine and by relaxation in response to methacholine chloride and sodium nitroprusside. Morphologic studies with use of Evans blue dye and scanning electron microscopy complemented the vascular-function studies. The Fogarty bulldog clamp was the best at preserving vascular contraction and relaxation; the more crushing Satinsky clamp was the worst. The testing of vascular rings for residual function appears to be a useful technique for objectively quantitating vascular clamp-related vascular damage.

  16. Application of active electrode compensation to perform continuous voltage-clamp recordings with sharp microelectrodes

    NASA Astrophysics Data System (ADS)

    Gómez-González, J. F.; Destexhe, A.; Bal, T.

    2014-10-01

    Objective. Electrophysiological recordings of single neurons in brain tissues are very common in neuroscience. Glass microelectrodes filled with an electrolyte are used to impale the cell membrane in order to record the membrane potential or to inject current. Their high resistance induces a high voltage drop when passing current and it is essential to correct the voltage measurements. In particular, for voltage clamping, the traditional alternatives are two-electrode voltage-clamp technique or discontinuous single electrode voltage-clamp (dSEVC). Nevertheless, it is generally difficult to impale two electrodes in a same neuron and the switching frequency is limited to low frequencies in the case of dSEVC. We present a novel fully computer-implemented alternative to perform continuous voltage-clamp recordings with a single sharp-electrode. Approach. To reach such voltage-clamp recordings, we combine an active electrode compensation algorithm (AEC) with a digital controller (AECVC). Main results. We applied two types of control-systems: a linear controller (proportional plus integrative controller) and a model-based controller (optimal control). We compared the performance of the two methods to dSEVC using a dynamic model cell and experiments in brain slices. Significance. The AECVC method provides an entirely digital method to perform continuous recording and smooth switching between voltage-clamp, current clamp or dynamic-clamp configurations without introducing artifacts.

  17. 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

  18. 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

  19. CAR T-cell intrinsic PD-1 checkpoint blockade: A two-in-one approach for solid tumor immunotherapy.

    PubMed

    Chen, Nan; Morello, Aurore; Tano, Zachary; Adusumilli, Prasad S

    2017-01-01

    PD-L1/2 expression in solid tumors inhibits chimeric antigen receptor (CAR) T-cell efficacy. A PD-1 dominant negative receptor expressed in CAR T cells provides cell-intrinsic checkpoint blockade and augments antitumor efficacy. A combinatorial immunotherapeutic strategy of combining CAR T cells with checkpoint blockade is a promising treatment approach for solid tumors.

  20. Vaccines combined with immune checkpoint antibodies promote cytotoxic T cell activity and tumor eradication

    PubMed Central

    Ali, Omar A.; Lewin, Sarah A.; Dranoff, Glenn; Mooney, David J.

    2015-01-01

    We demonstrate that a poly(lactide-co-glycolide) (PLG) cancer vaccine can be used in combination with immune checkpoint antibodies, anti–CTLA-4 or anti–PD-1, to enhance cytotoxic T cell (CTL) activity and induce the regression of solid B16 tumors in mice. Combination therapy obviated the need for vaccine boosting and significantly skewed intratumoral reactions toward CTL activity, resulting in the regression of B16 tumors up to 50mm2 in size and 75% survival rates. These data suggests that combining material-based cancer vaccines with checkpoint antibodies has the potential to mediate tumor regression in humans. PMID:26669718

  1. FAK-inhibition opens the door to checkpoint immunotherapy in Pancreatic Cancer.

    PubMed

    Symeonides, Stefan N; Anderton, Stephen M; Serrels, Alan

    2017-01-01

    Immunotherapy has had remarkable success in the treatment of some cancer types. However, pancreatic cancer has remained largely refractory to immunotherapy, including immune checkpoint inhibitors. Recently, Jiang and colleagues identified a key role for FAK in regulating the composition of the fibrotic and immuno-suppressive pancreatic tumour niche, and showed that FAK inhibitors can be used in combination with immune checkpoint blockade and gemcitabine chemotherapy to significantly delay pancreatic tumour progression. This study further supports the use of FAK inhibitors in combination with immunotherapy.

  2. PD-1 and PD-L1 Immune Checkpoint Blockade to Treat Breast Cancer.

    PubMed

    Hartkopf, Andreas D; Taran, Florin-Andrei; Wallwiener, Markus; Walter, Christina B; Krämer, Bernhard; Grischke, Eva-Maria; Brucker, Sara Y

    2016-12-01

    Immune checkpoint inhibition represents a major recent breakthrough in the treatment of malignant diseases including breast cancer. Blocking the programmed death receptor-1 (PD-1) and its ligand, PD-L1, has shown impressive antitumor activity and may lead to durable long-term disease control, especially in the triple-negative subtypes of breast cancer (TNBC). Although immune checkpoint blockade is generally well tolerated, specific immune-related adverse events (irAEs) may occur. This review summarizes the clinical efficacy, perspectives, and future challenges of using PD-1/PD-L1-directed antibodies in the treatment of breast cancer.

  3. Use of common time base for checkpointing and rollback recovery in a distributed system

    NASA Technical Reports Server (NTRS)

    Ramanathan, Parameswaran; Shin, Kang G.

    1993-01-01

    An approach to checkpointing and rollback recovery in a distributed computing system using a common time base is proposed. A common time base is established in the system using a hardware clock synchronization algorithm. This common time base is coupled with the idea of pseudo-recovery points to develop a checkpointing algorithm that has the following advantages: reduced wait for commitment for establishing recovery lines, fewer messages to be exchanged, and less memory requirement. These advantages are assessed quantitatively by developing a probabilistic model.

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

    PubMed

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

    2014-02-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

    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.

  7. On the mechanism of loading the PCNA sliding clamp by RFC.

    PubMed

    Dionne, Isabelle; Brown, Nicola J; Woodgate, Roger; Bell, Stephen D

    2008-04-01

    Sliding clamps play central roles in a broad range of DNA replication and repair processes. The clamps form circular molecules that must be opened and resealed around DNA by the clamp loader complex to fulfil their function. While most eukaryotes and many archea possess a homo-trimeric PCNA, the PCNA of Sulfolobus solfataricus is a heterotrimer. Here, we exploit the asymmetry of S. solfataricus PCNA to create a series of circularly permuted PCNA subunit fusions, thereby covalently closing defined interfaces within the heterotrimer. Using these concatamers, we investigate the requirements for loading the clamp onto DNA and reveal that a single defined interface within the heterotrimer is opened during the loading process. Subunit-specific interactions between S. solfataricus RFC clamp loader and PCNA permit us to superimpose our data upon the structure of yeast RFC-PCNA complex, thereby presenting a general model for PCNA loading by RFC in archaea and eukaryotes.

  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. Calcium-activated conductance in skate electroreceptors: current clamp experiments.

    PubMed

    Clusin, W T; Bennett, M V

    1977-02-01

    When current clamped, skate electroreceptor epithelium produces large action potentials in response to stimuli that depolarize the lumenal faces of the receptor cells. With increasing stimulus strength these action potentials become prolonged. When the peak voltage exceeds about 140 mV the repolarizing phase is blocked until the end of the stimulus. Perfusion experiments show that the rising phase of the action potential results from an increase in calcium permeability in the lumenal membranes. Perfusion of the lumen with cobalt or with a zero calcium solution containing EGTA blocks the action potential. Perfusion of the lumen with a solution containing 10 mM Ca and 20 mM EGTA initially slows the repolarizing process at all voltages and lowers the potential at which it is blocked. With prolonged perfusion, repolarization is blocked at all voltages. When excitability is abolished by perfusion with cobalt, or with a zero calcium solution containing EGTA, no delayed rectification occurs. We suggest that repolarization during the action potential depends on an influx of calcium into the cytoplasm, and that the rate of repolarization depends on the magnitude of the inward calcium current. Increasingly large stimuli reduce the rate of repolarization by reducing the driving force for calcium, and then block repolarization by causing the lumenal membrane potential to exceed ECa. Changes in extracellular calcium affect repolarization in a manner consistent with the resulting change in ECa.

  10. Non-Hookean statistical mechanics of clamped graphene ribbons

    NASA Astrophysics Data System (ADS)

    Bowick, Mark J.; Košmrlj, Andrej; Nelson, David R.; Sknepnek, Rastko

    2017-03-01

    Thermally fluctuating sheets and ribbons provide an intriguing forum in which to investigate strong violations of Hooke's Law: Large distance elastic parameters are in fact not constant but instead depend on the macroscopic dimensions. Inspired by recent experiments on free-standing graphene cantilevers, we combine the statistical mechanics of thin elastic plates and large-scale numerical simulations to investigate the thermal renormalization of the bending rigidity of graphene ribbons clamped at one end. For ribbons of dimensions W ×L (with L ≥W ), the macroscopic bending rigidity κR determined from cantilever deformations is independent of the width when W <ℓth , where ℓth is a thermal length scale, as expected. When W >ℓth , however, this thermally renormalized bending rigidity begins to systematically increase, in agreement with the scaling theory, although in our simulations we were not quite able to reach the system sizes necessary to determine the fully developed power law dependence on W . When the ribbon length L >ℓp , where ℓp is the W -dependent thermally renormalized ribbon persistence length, we observe a scaling collapse and the beginnings of large scale random walk behavior.

  11. 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

  12. Indentation metrology of clamped, ultra-thin elastic sheets.

    PubMed

    Vella, Dominic; Davidovitch, Benny

    2017-03-06

    We study the indentation of ultrathin elastic sheets clamped to the edge of a circular hole. This classical setup has received considerable attention lately, being used by various experimental groups as a probe to measure the surface properties and stretching modulus of thin solid films. Despite the apparent simplicity of this method, the geometric nonlinearity inherent in the mechanical response of thin solid objects renders the analysis of the resulting data a nontrivial task. Importantly, the essence of this difficulty is in the geometric coupling between in-plane stress and out-of-plane deformations, and hence is present in the behaviour of Hookean solids even when the slope of the deformed membrane remains small. Here we take a systematic approach to address this problem, using the membrane limit of the Föppl-von-Kármán equations. This approach highlights some of the dangers in the use of approximate formulae in the metrology of solid films, which can introduce large errors; we suggest how such errors may be avoided in performing experiments and analyzing the resulting data.

  13. Simultaneous patch-clamping and calcium imaging in developing dendrites.

    PubMed

    Kleindienst, Thomas; Lohmann, Christian

    2014-03-01

    Calcium imaging has been used extensively to explore the role of action potential (AP) firing in the development of neuronal structure and synaptic function because increases in intracellular calcium ([Ca(2+)]i) reliably and, within a certain range, linearly reflect neuronal spiking activity. Patterns of APs in individual cells can be deduced from calcium recordings, which have typically been performed at the level of cell bodies. However, neurons are particularly susceptible to phototoxicity when they are illuminated at the soma. Furthermore, for some imaging experiments (e.g., those that address the interactions between dendrites and axons during synapse formation), the cell body of a given neuron may simply not be in the field of view. In these situations, it would be helpful to determine the spiking patterns of a neuron from the calcium activity in its subcellular compartments such as stretches of dendrites or axons. Here, we describe an approach for determining the relationship between AP firing and dendritic calcium transients by simultaneously imaging calcium transients in small dendritic stretches of hippocampal pyramidal neurons in slice cultures from neonatal rats and recording spiking activity with whole-cell patch-clamp recordings in these neurons. These experiments allow us to correlate the electrophysiological spiking pattern with the accompanying changes in the calcium concentration in individual dendritic segments.

  14. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    PubMed

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  15. A leukemia fusion protein attenuates the spindle checkpoint and promotes aneuploidy

    PubMed Central

    Boyapati, Anita; Yan, Ming; Peterson, Luke F.; Biggs, Joseph R.; Le Beau, Michelle M.

    2007-01-01

    The 8;21 chromosomal translocation occurs in 15% to 40% of patients with the FAB M2 subtype of acute myeloid leukemia (AML). This chromosomal abnormality fuses part of the AML1/RUNX1 gene to the ETO/MTG8 gene and generates the AML1-ETO protein. We previously identified a C-terminal truncated AML1-ETO protein (AEtr) in a mouse leukemia model. AEtr is almost identical to the AML1-ETO exon 9a isoform expressed in leukemia patients. Here, we describe a novel function of AEtr in the development of aneuploidy through spindle checkpoint attenuation. AEtr cells had a reduced mitotic index following nocodazole treatment, suggesting a failure in a subset of cells to arrest in mitosis with a functional spindle checkpoint. Additionally, primary leukemia cells and cell lines expressing AEtr were aneuploid. Moreover, AEtr cells had reduced levels of several spindle checkpoint proteins including BubR1 and securin following treatment with the spindle poison nocodazole. These results suggest that inactivation of the spindle checkpoint may contribute to the development of aneuploidy described in t(8;21) leukemia patients. PMID:17197431

  16. The pre-B-cell receptor checkpoint in acute lymphoblastic leukaemia.

    PubMed

    Eswaran, J; Sinclair, P; Heidenreich, O; Irving, J; Russell, L J; Hall, A; Calado, D P; Harrison, C J; Vormoor, J

    2015-08-01

    The B-cell receptor (BCR) and its immature form, the precursor-BCR (pre-BCR), have a central role in the control of B-cell development, which is dependent on a sequence of cell-fate decisions at specific antigen-independent checkpoints. Pre-BCR expression provides the first checkpoint, which controls differentiation of pre-B to immature B-cells in normal haemopoiesis. Pre-BCR signalling regulates and co-ordinates diverse processes within the pre-B cell, including clonal selection, proliferation and subsequent maturation. In B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), B-cell development is arrested at this checkpoint. Moreover, malignant blasts avoid clonal extinction by hijacking pre-BCR signalling in favour of the development of BCP-ALL. Here, we discuss three mechanisms that occur in different subtypes of BCP-ALL: (i) blocking pre-BCR expression; (ii) activating pre-BCR-mediated pro-survival and pro-proliferative signalling, while inhibiting cell cycle arrest and maturation; and (iii) bypassing the pre-BCR checkpoint and activating pro-survival signalling through pre-BCR independent alternative mechanisms. A complete understanding of the BCP-ALL-specific signalling networks will highlight their application in BCP-ALL therapy.

  17. An assessment of the checkpoint bioassay concept for full scale wastewater UV reactor validation.

    PubMed

    Maka, P P; Lawryshyn, Y A

    2011-01-01

    In an effort to help policy makers and manufacturers understand the impact of parameter uncertainties on UV reactor performance, a numerical bioassay model was developed by integrating a UV reactor model based on computational fluid dynamics with a Monte Carlo model developed to account for parameter uncertainty. For the model implemented, it was determined that reactor performance uncertainty was less than 6%. The integrated model was used to evaluate several checkpoint bioassay criteria including one currently used by the California Department of Public Health. The model showed that these criteria failed to take into account the fact that in an ideal case, a full scale reactor will pass a single checkpoint test 50% of the time. In reality, differences in equipment measurement errors between the system validation and checkpoint bioassay, and limitations of the power law form of the dose monitoring equation in accurately representing system validation data will result in poorer than expected performance. It was suggested that such checkpoint criteria be modified by crediting the inherent over-sizing of full scale reactors.

  18. Noncoding RNAs and immune checkpoints-clinical implications as cancer therapeutics.

    PubMed

    Smolle, Maria A; Calin, Horatiu N; Pichler, Martin; Calin, George A

    2017-01-28

    A major mechanism of tumor development and progression is silencing of the patient's immune response to cancer-specific antigens. Defects in the so-called cancer immunity cycle may occur at any stage of tumor development. Within the tumor microenvironment, aberrant expression of immune checkpoint molecules with activating or inhibitory effects on T lymphocytes induces immune tolerance and cellular immune escape. Targeting immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and its ligand PD-L1 with specific antibodies has proven to be a major advance in the treatment of several types of cancer. Another way to therapeutically influence the tumor microenvironment is by modulating the levels of microRNAs (miRNAs), small noncoding RNAs that shuttle bidirectionally between malignant and tumor microenvironmental cells. These small RNA transcripts have two features: (a) their expression is quite specific to distinct tumors, and (b) they are involved in early regulation of immune responses. Consequently, miRNAs may be ideal molecules for use in cancer therapy. Many miRNAs are aberrantly expressed in human cancer cells, opening new opportunities for cancer therapy, but the exact functions of these miRNAs and their interactions with immune checkpoint molecules have yet to be investigated. This review summarizes recently reported findings about miRNAs as modulators of immune checkpoint molecules and their potential application as cancer therapeutics in clinical practice.

  19. 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

  20. 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

  1. Sources of errors in different single-electrode voltage-clamp techniques: a computer simulation study.

    PubMed

    Sala, F; Sala, S

    1994-08-01

    The use of voltage clamp with a single electrode has been useful in estimating kinetic parameters for a number of ionic whole-cell currents. There are two main types of such a technique: discontinuous voltage clamp (dSEVC) (Brennecke and Lindemann, 1974), and continuous voltage clamp (cSEVC) (Hamill et al., 1981). We have studied, by means of computer simulations, the performance of both types of clamp on estimating activation kinetics parameters of a typical neuronal Ca2+ current. Deviations from the theoretical values are shown to be sensitive on both set-up and cell properties. Both types of clamp are shown to lose voltage control when either access resistance or absolute membrane conductance are increased. In contrast, changes in membrane capacitance affect differently to the estimates obtained by the two types of clamp. Cell size is also shown to affect cSEVC performance but not that of dSEVC. The nature and magnitude of errors obtained by using both types of clamp in different situations are discussed.

  2. Endovascular balloon versus transthoracic aortic clamping for minimally invasive mitral valve surgery: impact on cerebral microemboli.

    PubMed

    Maselli, Daniele; Pizio, Raffaella; Borelli, Gabriele; Musumeci, Francesco

    2006-04-01

    To evaluate micro embolic events occurrence during minimally invasive mitral valve procedures, comparing balloon endovascular aortic occlusion (Group I) and transthoracic aortic clamping technique (Group II), 36 patients (20 in Group I and 16 in Group II) undergoing minimally invasive mitral valve surgery were selected by CT scan and Doppler studies for absence of atherosclerotic disease at aortic, coronary or peripheral level. Assignment to one of the two groups was made on the basis of surgeon's preference. Continuous automated intra-operative transcranial Doppler was used to monitor micro embolic events during five operative steps: cardiopulmonary bypass (CPB) setup, time interval from CPB start until aortic clamp positioning, first minute after clamp-on, first minute after clamp-off, first ten minutes after CPB weaning start. More embolic events were observed in Group II than in Group I (total 143.4+/-30.6 per patient vs. 78.9+/-28.6 per patient). A large amount of embolic events occurring mainly when the aortic clamp was positioned and released accounted for the observed differences. In a low risk population for embolic events occurrence, endovascular balloon aortic clamping determined less embolic signals than transthoracic aortic clamping.

  3. [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.

  4. Programmed death-1 immune checkpoint blockade in the treatment of hematological malignancies.

    PubMed

    Tsirigotis, Panagiotis; Savani, Bipin N; Nagler, Arnon

    2016-09-01

    The use of tumor-specific monoclonal antibodies (MAbs) has revolutionize the field of cancer immunotherapy. Although treatment of malignant diseases with MAbs is promising, many patients fail to respond or relapse after an initial response. Both solid tumors and hematological malignancies develop mechanisms that enable them to evade the host immune system by usurping immune checkpoint pathways such as PD-1, PD-2, PDL-1, or PDL-2 (programmed cell death protein-1 or 2 and PD-Ligand 1 or 2), which are expressed on activated T cells and on T-regulatory, B cells, natural killers, monocytes, and dendritic cells. One of the most exciting anticancer development in recent years has been the immune checkpoint blockade therapy by using MAbs against immune checkpoint receptor and/or ligands. Anti-PD1 antibodies have been tested in clinical studies that included patients with hematological malignancies and showed remarkable efficacy in Hodgkin lymphoma (HL). In our review, we will focus on the effect of PD-1 activation on hematological malignancies and its role as a therapeutic target. Key messages The programmed death 1 (PD1) immune checkpoint is an important homeostatic mechanism of the immune system that helps in preventing autoimmunity and uncontrolled inflammation in cases of chronic infections. However, PD1 pathway is also operated by a wide variety of malignancies and represents one of the most important mechanisms by which tumor cells escape from the surveillance of the immune system. Blocking of immune checkpoints by the use of monoclonal antibodies opened a new era in the field of cancer immunotherapy. Results from clinical trials are promising, and currently, this approach has been proven effective and safe in patients with solid tumors and hematological malignancies.

  5. A Fhit-ing role in the DNA damage checkpoint response.

    PubMed

    Ishii, Hideshi; Wang, Ya; Huebner, Kay

    2007-05-02

    The FHIT gene encompasses the most active common fragile site of the human genome and is thus exquisitely sensitive to intragenic alterations by DNA damaging agents, alterations that can lead to FHIT allele loss very early in the preneoplastic phase of cancer development, before or coincident with activation of the DNA damage checkpoint. Fhit protein expression is lost or reduced in many preneoplastic lesions and in >50% of cancers, Fhit knockout mice are highly susceptible to carcinogen induction of tumors and Fhit replacement in these mice by gene therapy induces apoptosis and significantly reduces tumor burden. But learning how Fhit induces apoptosis and suppresses tumors has been a challenge because interacting proteins, effectors of Fhit signals, have not been discovered. Nevertheless, the study of Fhit deficient mouse and human tissue-derived and cancer-derived cells in vitro has led to several important conclusions: repair protein-deficient cancers are more likely to be Fhit-deficient; Fhit-deficient cells show enhanced resistance to UVC, mitomycin C, camptothecin and ionizing radiation-induced cell killing, possibly due to strong activation of the ATR pathway following DNA damage; Fhit-deficient cells show higher efficiency of homologous recombination repair, a double-strand break repair pathway in mammalian cells; Fhit protein indirectly affects S-phase checkpoint and DNA repair. Finally, results of a recent study have suggested that the DNA damage-susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein, Fhit, that is necessary for protecting cells from accumulation of DNA damage, through modulation of checkpoint proteins Hus1 and phosphoChk1. Thus, inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. It will be very important to determine mechanisms employed by Fhit in modulating checkpoint pathways, and to define consequences of Fhit loss in specific preneoplastic and

  6. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

    PubMed Central

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers. PMID:24955955

  7. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints.

    PubMed

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

  8. Evolution of Neoantigen Landscape during Immune Checkpoint Blockade in Non-Small Cell Lung Cancer.

    PubMed

    Anagnostou, Valsamo; Smith, Kellie N; Forde, Patrick M; Niknafs, Noushin; Bhattacharya, Rohit; White, James; Zhang, Theresa; Adleff, Vilmos; Phallen, Jillian; Wali, Neha; Hruban, Carolyn; Guthrie, Violeta B; Rodgers, Kristen; Naidoo, Jarushka; Kang, Hyunseok; Sharfman, William; Georgiades, Christos; Verde, Franco; Illei, Peter; Li, Qing Kay; Gabrielson, Edward; Brock, Malcolm V; Zahnow, Cynthia A; Baylin, Stephen B; Scharpf, Robert B; Brahmer, Julie R; Karchin, Rachel; Pardoll, Drew M; Velculescu, Victor E

    2017-03-01

    Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non-small cell lung cancer after initial response to immune checkpoint blockade with anti-PD-1 or anti-PD-1/anti-CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens.Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264-76. ©2017 AACR.See related commentary by Yang, p. 250This article is highlighted in the In This Issue feature, p. 235.

  9. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    PubMed Central

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired. PMID:21060809

  10. Caenorhabditis elegans histone methyltransferase MET-2 shields the male X chromosome from checkpoint machinery and mediates meiotic sex chromosome inactivation.

    PubMed

    Checchi, Paula M; Engebrecht, JoAnne

    2011-09-01

    Meiosis is a specialized form of cellular division that results in the precise halving of the genome to produce gametes for sexual reproduction. Checkpoints function during meiosis to detect errors and subsequently to activate a signaling cascade that prevents the formation of aneuploid gametes. Indeed, asynapsis of a homologous chromosome pair elicits a checkpoint response that can in turn trigger germline apoptosis. In a heterogametic germ line, however, sex chromosomes proceed through meiosis with unsynapsed regions and are not recognized by checkpoint machinery. We conducted a directed RNAi screen in Caenorhabditis elegans to identify regulatory factors that prevent recognition of heteromorphic sex chromosomes as unpaired and uncovered a role for the SET domain histone H3 lysine 9 histone methyltransferase (HMTase) MET-2 and two additional HMTases in shielding the male X from checkpoint machinery. We found that MET-2 also mediates the transcriptional silencing program of meiotic sex chromosome inactivation (MSCI) but not meiotic silencing of unsynapsed chromatin (MSUC), suggesting that these processes are distinct. Further, MSCI and checkpoint shielding can be uncoupled, as double-strand breaks targeted to an unpaired, transcriptionally silenced extra-chromosomal array induce checkpoint activation in germ lines depleted for met-2. In summary, our data uncover a mechanism by which repressive chromatin architecture enables checkpoint proteins to distinguish between the partnerless male X chromosome and asynapsed chromosomes thereby shielding the lone X from inappropriate activation of an apoptotic program.

  11. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    PubMed Central

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  12. Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

    PubMed Central

    López de Saro, Francisco J.; O'Donnell, Mike

    2001-01-01

    The β and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli β clamp is known to function with DNA polymerases II and V, indicating that β also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein–protein interactions with the β clamp. Thus, β interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of β interactive proteins suggests that the prokaryotic β ring participates in a wide variety of reactions beyond its role in chromosomal replication. PMID:11459978

  13. Shock Response of the Clamped Disk in Small Form Factor Hard Disk Drive

    NASA Astrophysics Data System (ADS)

    Gu, Bin; Shu, Dongwei; Shi, Baojun; Lu, Guoxing

    As small form factor (one-inch and smaller) hard disk drives are widely used in portable consumer appliances and gadgets, their mechanical robustness is of greater concern. In the previous work, it is found that when the disk is more tightly clamped, it helps to decrease the shock response of the disk and then avoid the head slap. In this paper, the real boundary condition of the disk for a small form factor hard disk drive from Seagate is investigated numerically. The disk is clamped between the clamp and the hub. The shock response of the disk under a half-sine acceleration pulse is simulated by using the finite element method. In the finite element model, both contact between disk and clamp and contact between disk and hub are considered. According to the simulation results, how to decrease the shock response of the disk is suggested.

  14. Modeling considerations for the analysis of LMFBR steam generator tube clamps

    SciTech Connect

    Lay, D.M.; Piper, R.M.

    1983-10-01

    In the design of the Babcock and Wilcox Helical Coil Liquid Metal Fast Breeder Reactor (LMFBR) Steam Generator, the tube bundle is connected to the feedwater and steam plenums via ''inlet/outlet tubes.'' Of prime importance in the design of these tubes is the tube-to-tube and tube-to-shell clamps which are provided to prevent detrimental vibration. This paper presents a method of modeling the tubeto-tube clamps to accurately predict tube-to-clamp interaction in the finite element analysis. It is also the objective of this paper to demonstrate the validity of specific modeling assumptions in determining stresses in the clamp assembly. As this paper deals only with the analytical approach taken, no detailed results are presented.

  15. 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.

  16. 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

  17. Tenderization of hot-boned broiler breast meat by clamping during chilling.

    PubMed

    Cason, J A; Lyon, C E; Dickens, J A

    2002-01-01

    Hot-boned broiler breast fillets were tightly clamped between rigid aluminum plates during chilling to determine whether tenderness is increased if breast fillets are not allowed to shorten during rigor. In two experiments, 6-wk-old broilers were processed in a pilot plant. Approximately 5 min after evisceration, the breast fillets (pectoralis major) were deboned, and each fillet was subjected to one of two treatments while chilling for 2 h in ice slush. Fillets were placed in perforated plastic bags (hot-boned control) or clamped between rigid aluminum plates that compressed the meat to a uniform thickness of 7.2 mm during chilling. In Experiment 2, chilling time in ice slush was 1 h, and a third treatment was added to make an incomplete block design in which one breast half was left intact on the carcass and was deboned immediately after chilling. All breast fillets were sealed in plastic bags after the chilling period, held overnight at 4 C, and then cooked at 85 C for 30 min in a steam kettle. In Experiment 1, clamping for 2 h reduced Warner-Bratzler shear values of hot-boned fillets from 11.4 to 2.7 kg. In Experiment 2, shear values for the treatments were 13.0, 9.2, and 5.1 kg for the hot-boned, cold-boned, and hot-boned clamped treatments, respectively, with significantly lower shear values for the clamped fillets. Clamped fillets were significantly thinner than the control fillets in both experiments. Cooked yield as a percentage of postchill weight was significantly higher for the clamped compared to the hot-boned control pieces, 81.1 versus 77.3%, with cold-boned pieces being intermediate and not different from the other treatments. Shear values were reduced, and cooked yield was increased by clamping hot-boned fillets during chilling.

  18. Patch Clamp: A Powerful Technique for Studying the Mechanism of Acupuncture

    PubMed Central

    Zhang, D.

    2012-01-01

    Cellular and molecular events can be investigated using electrophysiological techniques. In particular, the patch-clamp method provides detailed information. In addition, the patch-clamp technique has become a powerful method for investigating the mechanisms underlying the effects of acupuncture. In this paper, recent researches on how acupuncture might modulate electrophysiological responses in the central nervous system (CNS) and affect peripheral structures are reviewed. PMID:23133497

  19. 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

  20. Clamping of the Linewidth Enhancement Factor in Narrow Quantum-Well GRINSCH Semiconductor Lasers

    SciTech Connect

    Bossert, D.; Chow, W.W.; Hader, J.; Koch, S.W.; Moloney, J.V.; Stohls, J.

    1999-01-20

    The linewidth enhancement factor in single quantum-well GRINSCH semiconductor lasers is investigated theoretically and experimentally. For thin wells a small linewidth enhancement factor is obtained which clamps with increasing carrier density, in contrast to the monotonous increase observed for thicker wells. Microscopic many-body calculations reproduce the experimental observations attributing the clamping to a subtle interplay between excitation dependent gain shifts and carrier population distributions.

  1. Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from Methanosarcina acetivorans▿ †

    PubMed Central

    Chen, Yi-Hsing; Lin, Yuyen; Yoshinaga, Aya; Chhotani, Benazir; Lorenzini, Jenna L.; Crofts, Alexander A.; Mei, Shou; Mackie, Roderick I.; Ishino, Yoshizumi; Cann, Isaac K. O.

    2009-01-01

    Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFCMa), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFCMa complex. PMID:19717601

  2. Effect of non-ideal clamping shape on the resonance frequencies of silicon nanocantilevers.

    PubMed

    Guillon, Samuel; Saya, Daisuke; Mazenq, Laurent; Perisanu, Sorin; Vincent, Pascal; Lazarus, Arnaud; Thomas, Olivier; Nicu, Liviu

    2011-06-17

    In this paper, we investigate the effects of non-ideal clamping shapes on the dynamic behavior of silicon nanocantilevers. We fabricated silicon nanocantilevers using silicon on insulator (SOI) wafers by employing stepper ultraviolet (UV) lithography, which permits a resolution of under 100 nm. The nanocantilevers were driven by electrostatic force inside a scanning electron microscope (SEM). Both lateral and out-of-plane resonance frequencies were visually detected with the SEM. Next, we discuss overhanging of the cantilever support and curvature at the clamping point in the silicon nanocantilevers, which generally arises in the fabrication process. We found that the fundamental out-of-plane frequency of a realistically clamped cantilever is always lower than that for a perfectly clamped cantilever, and depends on the cantilever width and the geometry of the clamping point structure. Using simulation with the finite-elements method, we demonstrate that this discrepancy is attributed to the particular geometry of the clamping point (non-zero joining curvatures and a flexible overhanging) that is obtained in the fabrication process. The influence of the material orthotropy is also investigated and is shown to be negligible.

  3. 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.

  4. 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.

  5. 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.

  6. Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells.

    PubMed

    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

    2014-08-12

    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.

  7. 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.

  8. 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.

  9. Dynamic versus static biomarkers in cancer immune checkpoint blockade: unravelling complexity.

    PubMed

    Lesterhuis, W Joost; Bosco, Anthony; Millward, Michael J; Small, Michael; Nowak, Anna K; Lake, Richard A

    2017-04-01

    Recently, there has been a coordinated effort from academic institutions and the pharmaceutical industry to identify biomarkers that can predict responses to immune checkpoint blockade in cancer. Several biomarkers have been identified; however, none has reliably predicted response in a sufficiently rigorous manner for routine use. Here, we argue that the therapeutic response to immune checkpoint blockade is a critical state transition of a complex system. Such systems are highly sensitive to initial conditions, and critical transitions are notoriously difficult to predict far in advance. Nevertheless, warning signals can be detected closer to the tipping point. Advances in mathematics and network biology are starting to make it possible to identify such warning signals. We propose that these dynamic biomarkers could prove to be useful in distinguishing responding from non-responding patients, as well as facilitate the identification of new therapeutic targets for combination therapy.

  10. 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

  11. The spindle checkpoint in plants-a green variation over a conserved theme?

    PubMed

    Komaki, Shinichiro; Schnittger, Arp

    2016-12-01

    The spindle checkpoint, also called spindle assembly checkpoint (SAC), is a crucial control instance in animals and yeast that surveys the correct attachment of chromosomes to the spindle assuring their equal distribution in mitosis and meiosis. The presence of homologs of all core SAC components in plants indicates that these regulators have an ancient function. However, the fact that mutants of SAC components in plants are usually fully viable together with the observation that plants can be readily made polyploid raises the question whether plants have an efficient SAC. Recently, the role and regulation of a putative SAC in plants has been addressed. Interestingly, these studies also revealed that SAC genes are involved in several other cellular and developmental processes outside of chromosome distribution control.

  12. 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

  13. 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.

  14. 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

  15. Essential functions for ID proteins at multiple checkpoints in invariant NKT cell development.

    PubMed

    Verykokakis, Mihalis; Krishnamoorthy, Veena; Iavarone, Antonio; Lasorella, Anna; Sigvardsson, Mikael; Kee, Barbara L

    2013-12-15

    Invariant NKT (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. In this study, 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 postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.

  16. 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

  17. 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

  18. Subverting the adaptive immune resistance mechanism to improve clinical responses to immune checkpoint blockade therapy

    PubMed Central

    Kim, Young J

    2015-01-01

    The correlation between tumor-infiltrating lymphocyte (TIL)-expression of programmed cell death ligand 1 (PD-L1) and clinical responsiveness to the PD-1 blocking antibody nivolumab implicates adaptive immune evasion mechanisms in cancer. We review our findings that tumor cell PD-L1 expression is induced by interferon γ (IFNγ) producing TILs. We provide a mechanistic rationale for combining IFNγ+ T helper type 1 (Th1)-inducing cancer vaccines with PD-1 immune checkpoint blockade. PMID:25964860

  19. The Mre11 complex: at the crossroads of dna repair and checkpoint signalling.

    PubMed

    D'Amours, Damien; Jackson, Stephen P

    2002-05-01

    The Mre11 complex is a multisubunit nuclease that is composed of Mre11, Rad50 and Nbs1/Xrs2. Mutations in the genes that encode components of this complex result in DNA- damage sensitivity, genomic instability, telomere shortening and aberrant meiosis. The molecular defect that underlies these phenotypes has long been thought to be related to a DNA repair deficiency. However, recent studies have uncovered functions for the Mre11 complex in checkpoint signalling and DNA replication.

  20. T-cell-based Immunotherapy: Adoptive Cell Transfer and Checkpoint Inhibition.

    PubMed

    Houot, Roch; Schultz, Liora Michal; Marabelle, Aurélien; Kohrt, Holbrook

    2015-10-01

    Tumor immunotherapy has had demonstrable efficacy in patients with cancer. The most promising results have been with T-cell-based therapies. These include adoptive cell transfer of tumor-infiltrating lymphocytes, genetically engineered T cells, and immune checkpoint inhibitor antibodies. In this review, we describe the different T-cell-based strategies currently in clinical trials and put their applications, present and future, into perspective.

  1. 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

  2. Angiopoietin-2 as a Biomarker and Target for Immune Checkpoint Therapy.

    PubMed

    Wu, Xinqi; Giobbie-Hurder, Anita; Liao, Xiaoyun; Connelly, Courtney; Connolly, Erin M; Li, Jingjing; Manos, Michael P; Lawrence, Donald; McDermott, David; Severgnini, Mariano; Zhou, Jun; Gjini, Evisa; Lako, Ana; Lipschitz, Mikel; Pak, Christine J; Abdelrahman, Sara; Rodig, Scott; Hodi, F Stephen

    2017-01-01

    Immune checkpoint therapies targeting CTLA-4 and PD-1 have proven effective in cancer treatment. However, the identification of biomarkers for predicting clinical outcomes and mechanisms to overcome resistance remain as critical needs. Angiogenesis is increasingly appreciated as an immune modulator with potential for combinatorial use with checkpoint blockade. Angiopoietin-2 (ANGPT2) is an immune target in patients and is involved in resistance to anti-VEGF treatment with the monoclonal antibody bevacizumab. We investigated the predictive and prognostic value of circulating ANGPT2 in metastatic melanoma patients receiving immune checkpoint therapy. High pretreatment serum ANGPT2 was associated with reduced overall survival in CTLA-4 and PD-1 blockade-treated patients. These treatments also increased serum ANGPT2 in many patients early after treatment initiation, whereas ipilimumab plus bevacizumab treatment decreased serum concentrations. ANGPT2 increases were associated with reduced response and/or overall survival. Ipilimumab increased, and ipilimumab plus bevacizumab decreased, tumor vascular ANGPT2 expression in a subset of patients, which was associated with increased and decreased tumor infiltration by CD68(+) and CD163(+) macrophages, respectively. In vitro, bevacizumab blocked VEGF-induced ANGPT2 expression in tumor-associated endothelial cells, whereas ANGPT2 increased PD-L1 expression on M2-polarized macrophages. Treatments elicited long-lasting and functional antibody responses to ANGPT2 in a subset of patients receiving clinical benefit. Our findings suggest that serum ANGPT2 may be considered as a predictive and prognostic biomarker for immune checkpoint therapy and may contribute to treatment resistance via increasing proangiogenic and immunosuppressive activities in the tumor microenvironment. Targeting ANGPT2 provides a rational combinatorial approach to improve the efficacy of immune therapy. Cancer Immunol Res; 5(1); 17-28. ©2016 AACR.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Improvement in cardioplegic perfusion technique in single aortic clamping - initial results

    PubMed Central

    Sobral, Marcelo Luiz Peixoto; dos Santos Júnior, Sérgio Francisco; de Sá, Juliano Cavalcante; Terrazas, Anderson da Silva; Trompieri, Daniel Francisco de Mendonça; de Sousa, Thierry Araújo Nunes; dos Santos, Gilmar Geraldo; Stolf, Noedir Antonio Groppo

    2014-01-01

    Introduction The most common method used for myocardial protection is administering cardioplegic solution in the coronary circulation. Nevertheless, protection may be achieved by intermittent perfusion of the coronary system with patient's own blood. The intermittent perfusion may be performed by multiple sequences of clamping and opening of the aortic clamp or due single clamping and accessory cannulation of the aortic root as in the improved technique proposed in this study, reperfusion without the need for multiple clamping of the aorta. Objective To evaluate the clinical outcome and the occurrence of neurological events in in-hospital patients submitted to myocardial revascularization surgery with the "improved technique" of intermittent perfusion of the aortic root with single clamping. Methods This is a prospective, cross-sectional, observational study that describes a myocardial management technique that consists of intermittent perfusion of the aortic root with single clamping in which 50 patients (mean age 58.5±7.19 years old) have been submitted to the myocardial revasculrization surgery under the proposed technique. Clinical and laboratory variables, pre- and post-surgery, have been assessed. Results The mean peak level of post-surgery CKMB was 51.64±27.10 U/L in the second post-surgery and of troponin I was 3.35±4.39 ng/ml in the fourth post-surgery, within normal limits. No deaths have occurred and one patient presented mild neurological disorder. Hemodynamic monitoring has not indicated any changes. Conclusion The myocardial revascularization surgery by perfusion with the improved technique with intermittent aortic root with single clamping proved to be safe, enabling satisfactory clinical results. PMID:25140473

  8. A central swivel point in the RFC clamp loader controls PCNA opening and loading on DNA.

    PubMed

    Sakato, Miho; O'Donnell, Mike; Hingorani, Manju M

    2012-02-17

    Replication factor C (RFC) is a five-subunit complex that loads proliferating cell nuclear antigen (PCNA) clamps onto primer-template DNA (ptDNA) during replication. RFC subunits belong to the AAA(+) superfamily, and their ATPase activity drives interactions between the clamp loader, the clamp, and the ptDNA, leading to topologically linked PCNA·ptDNA. We report the kinetics of transient events in Saccharomyces cerevisiae RFC-catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing, and PCNA·ptDNA release. This detailed perspective enables assessment of individual RFC-A, RFC-B, RFC-C, RFC-D, and RFC-E subunit functions in the reaction mechanism. Functions have been ascribed to RFC subunits previously based on a steady-state analysis of 'arginine-finger' ATPase mutants; however, pre-steady-state analysis provides a different view. The central subunit RFC-C serves as a critical swivel point in the clamp loader. ATP binding to this subunit initiates RFC activation, and the clamp loader adopts a spiral conformation that stabilizes PCNA in a corresponding open spiral. The importance of RFC subunit response to ATP binding decreases as RFC-C>RFC-D>RFC-B, with RFC-A being unnecessary. RFC-C-dependent activation of RFC also enables ptDNA binding, leading to the formation of the RFC·ATP·PCNA(open)·ptDNA complex. Subsequent ATP hydrolysis leads to complex dissociation, with RFC-D activity contributing the most to rapid ptDNA release. The pivotal role of the RFC-B/C/D subunit ATPase core in clamp loading is consistent with the similar central location of all three ATPase active subunits of the Escherichia coli clamp loader.

  9. WEE1 inhibition targets cell cycle checkpoints for triple negative breast cancers to overcome cisplatin resistance

    PubMed Central

    Zheng, Hongping; Shao, Fangyuan; Martin, Scots; Xu, Xiaoling; Deng, Chu-Xia

    2017-01-01

    Cisplatin is one of the most commonly used therapeutic drugs for cancer therapy, yet prolonged cisplatin treatment frequently results in drug resistance. To enhance therapeutic effect of cisplatin, we conducted a high throughput screening using a kinase library containing 704 kinases against triple negative breast cancer (TNBC) cells. We demonstrated that cisplatin activates ATR, CHK1 and WEE1, which shut down DNA replication and attenuate cisplatin induced-lethality. WEE1 inhibition sensitizes TNBCs and cisplatin resistant cancer cells to cisplatin-induced lethality, because it not only impairs DNA replication checkpoint more profoundly than inhibition of ATR or CHK1, but also defects G2-M cell cycle checkpoint. Finally, we demonstrated that combined cisplatin treatment and WEE1 inhibition synergistically inhibits xenograft cancer growth accompanied by markedly reduced expression of TNBC signature genes. Thus targeting DNA replication and G2-M cell cycle checkpoint simultaneously by cisplatin and WEE1 inhibition is promising for TNBCs treatment, and for overcoming their cisplatin resistance. PMID:28262781

  10. Molecular checkpoints controlling natural killer cell activation and their modulation for cancer immunotherapy

    PubMed Central

    Kwon, Hyung-Joon; Kim, Nayoung; Kim, Hun Sik

    2017-01-01

    Natural killer (NK) cells have gained considerable attention as promising therapeutic tools for cancer therapy due to their innate selectivity against cancer cells over normal healthy cells. With an array of receptors evolved to sense cellular alterations, NK cells provide early protection against cancer cells by producing cytokines and chemokines and exerting direct cytolytic activity. These effector functions are governed by signals transmitted through multiple receptor–ligand interactions but are not achieved by engaging a single activating receptor on resting NK cells. Rather, they require the co-engagement of different activating receptors that use distinct signaling modules, due to a cell-intrinsic inhibition mechanism. The redundancy of synergizing receptors and the inhibition of NK cell function by a single class of inhibitory receptor suggest the presence of common checkpoints to control NK cell activation through different receptors. These molecular checkpoints would be therapeutically targeted to harness the power of NK cells against diverse cancer cells that express heterogeneous ligands for NK cell receptors. Recent advances in understanding the activation of NK cells have revealed promising candidates in this category. Targeting such molecular checkpoints will facilitate NK cell activation by lowering activation thresholds, thereby providing therapeutic strategies that optimize NK cell reactivity against cancer. PMID:28360428

  11. 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)

  12. DNA damage checkpoint adaptation genes are required for division of cells harbouring eroded telomeres

    PubMed Central

    Mersaoui, Sofiane Y.; Gravel, Serge; Karpov, Victor; Wellinger, Raymund J.

    2015-01-01

    In budding yeast, telomerase and the Cdc13p protein are two key players acting to ensure telomere stability. In the absence of telomerase, cells eventually enter a growth arrest which only few can overcome via a conserved process; such cells are called survivors. Survivors rely on homologous recombination-dependent mechanisms for telomeric repeat addition. Previously, we showed that such survivor cells also manage to bypass the loss of the essential Cdc13p protein to give rise to Cdc13-independent (or cap-independent) strains. Here we show that Cdc13-independent cells grow with persistently recognized DNA damage, which does not however result in a checkpoint activation; thus no defect in cell cycle progression is detectable. The absence of checkpoint signalling rather is due to the accumulation of mutations in checkpoint genes such as RAD24 or MEC1. Importantly, our results also show that cells that have lost the ability to adapt to persistent DNA damage, also are very much impaired in generating cap-independent cells. Altogether, these results show that while the capping process can be flexible, it takes a very specific genetic setup to allow a change from canonical capping to alternative capping. We hypothesize that in the alternative capping mode, genome integrity mechanisms are abrogated, which could cause increased mutation frequencies. These results from yeast have clear parallels in transformed human cancer cells and offer deeper insights into processes operating in pre-cancerous human cells that harbour eroded telomeres.

  13. Combining Immune Checkpoint Inhibitors and Kinase-Inhibiting Supramolecular Therapeutics for Enhanced Anticancer Efficacy.

    PubMed

    Kulkarni, Ashish; Natarajan, Siva Kumar; Chandrasekar, Vineethkrishna; Pandey, Prithvi Raj; Sengupta, Shiladitya

    2016-09-29

    A major limitation of immune checkpoint inhibitors is that only a small subset of patients achieve durable clinical responses. This necessitates the development of combinatorial regimens with immunotherapy. However, some combinations, such as MEK- or PI3K-inhibitors with a PD1-PDL1 checkpoint inhibitor, are pharmacologically challenging to implement. We rationalized that such combinations can be enabled using nanoscale supramolecular targeted therapeutics, which spatially home into tumors and exert temporally sustained inhibition of the target. Here we describe two case studies where nanoscale MEK- and PI3K-targeting supramolecular therapeutics were engineered using a quantum mechanical all-atomistic simulation-based approach. The combinations of nanoscale MEK- and PI3K-targeting supramolecular therapeutics with checkpoint PDL1 and PD1 inhibitors exert enhanced antitumor outcome in melanoma and breast cancers in vivo, respectively. Additionally, the temporal sequence of administration impacts the outcome. The combination of supramolecular therapeutics and immunotherapy could emerge as a paradigm shift in the treatment of cancer.

  14. 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

  15. WEE1 inhibition targets cell cycle checkpoints for triple negative breast cancers to overcome cisplatin resistance.

    PubMed

    Zheng, Hongping; Shao, Fangyuan; Martin, Scots; Xu, Xiaoling; Deng, Chu-Xia

    2017-03-06

    Cisplatin is one of the most commonly used therapeutic drugs for cancer therapy, yet prolonged cisplatin treatment frequently results in drug resistance. To enhance therapeutic effect of cisplatin, we conducted a high throughput screening using a kinase library containing 704 kinases against triple negative breast cancer (TNBC) cells. We demonstrated that cisplatin activates ATR, CHK1 and WEE1, which shut down DNA replication and attenuate cisplatin induced-lethality. WEE1 inhibition sensitizes TNBCs and cisplatin resistant cancer cells to cisplatin-induced lethality, because it not only impairs DNA replication checkpoint more profoundly than inhibition of ATR or CHK1, but also defects G2-M cell cycle checkpoint. Finally, we demonstrated that combined cisplatin treatment and WEE1 inhibition synergistically inhibits xenograft cancer growth accompanied by markedly reduced expression of TNBC signature genes. Thus targeting DNA replication and G2-M cell cycle checkpoint simultaneously by cisplatin and WEE1 inhibition is promising for TNBCs treatment, and for overcoming their cisplatin resistance.

  16. Both Cyclin B levels and DNA-replication checkpoint control the early embryonic mitoses in Drosophila

    PubMed Central

    Ji, Jun-Yuan; Squirrell, Jayne M.; Schubiger, Gerold

    2013-01-01

    Summary The earliest embryonic mitoses in Drosophila, as in other animals except mammals, are viewed as synchronous and of equal duration. However, we observed that total cell-cycle length steadily increases after cycle 7, solely owing to the extension of interphase. Between cycle 7 and cycle 10, this extension is DNA-replication checkpoint independent, but correlates with the onset of Cyclin B oscillation. In addition, nuclei in the middle of embryos have longer metaphase and shorter anaphase than nuclei at the two polar regions. Interestingly, sister chromatids move faster in anaphase in the middle than the posterior region. These regional differences correlate with local differences in Cyclin B concentration. After cycle 10, interphase and total cycle duration of nuclei in the middle of the embryo are longer than at the poles. Because interphase also extends in checkpoint mutant (grapes) embryo after cycle 10, although less dramatic than wild-type embryos, interphase extension after cycle 10 is probably controlled by both Cyclin B limitation and the DNA-replication checkpoint. PMID:14681192

  17. Prognostic and predictive aspects of the tumor immune microenvironment and immune checkpoints in malignant pleural mesothelioma.

    PubMed

    Marcq, Elly; Siozopoulou, Vasiliki; De Waele, Jorrit; van Audenaerde, Jonas; Zwaenepoel, Karen; Santermans, Eva; Hens, Niel; Pauwels, Patrick; van Meerbeeck, Jan P; Smits, Evelien L J

    2017-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and an increasing incidence, for which novel therapeutic strategies are urgently required. Since the immune system has been described to play a presumed role in the protection against MPM, characterization of its tumor immune microenvironment (TME) and immune checkpoints can identify new immunotherapeutic targets and their predictive and/or prognostic value. To characterize the TME and the immune checkpoint expression profile, we performed immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue sections from 54 MPM patients (40 at time of diagnosis; 14 treated with chemotherapy). We stained for PD-1, PD-L1, TIM-3, LAG-3, CD4, CD8, CD45RO, granzyme B, FoxP3 and CD68. Furthermore, we analyzed the relationship between the immunological parameters and survival, as well as response to chemotherapy. We found that TIM-3, PD-1 and PD-L1 were expressed on both immune and tumor cells. Strikingly, PD-1 and PD-L1 expression on tumor cells was only seen in unpretreated samples. No LAG-3 expression was observed. CD45RO expression in the stroma was an independent negative predictive factor for response on chemotherapy, while CD4 and TIM-3 expression in lymphoid aggregates were independent prognostic factors for better outcome. Our data propose TIM-3 as a promising new target in mesothelioma. Chemotherapy influences the expression of immune checkpoints and therefore further research on the best combination treatment schedule is required.

  18. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.

    PubMed

    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

    2016-02-17

    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.

  19. Functional dissection of synaptic circuits: in vivo patch-clamp recording in neuroscience

    PubMed Central

    Tao, Can; Zhang, Guangwei; Xiong, Ying; Zhou, Yi

    2015-01-01

    Neuronal activity is dominated by synaptic inputs from excitatory or inhibitory neural circuits. With the development of in vivo patch-clamp recording, especially in vivo voltage-clamp recording, researchers can not only directly measure neuronal activity, such as spiking responses or membrane potential dynamics, but also quantify synaptic inputs from excitatory and inhibitory circuits in living animals. This approach enables researchers to directly unravel different synaptic components and to understand their underlying roles in particular brain functions. Combining in vivo patch-clamp recording with other techniques, such as two-photon imaging or optogenetics, can provide even clearer functional dissection of the synaptic contributions of different neurons or nuclei. Here, we summarized current applications and recent research progress using the in vivo patch-clamp recording method and focused on its role in the functional dissection of different synaptic inputs. The key factors of a successful in vivo patch-clamp experiment and possible solutions based on references and our experiences were also discussed. PMID:26052270

  20. Clamping instability and van der Waals forces in carbon nanotube mechanical resonators.

    PubMed

    Aykol, Mehmet; Hou, Bingya; Dhall, Rohan; Chang, Shun-Wen; Branham, William; Qiu, Jing; Cronin, Stephen B

    2014-05-14

    We investigate the role of weak clamping forces, typically assumed to be infinite, in carbon nanotube mechanical resonators. Due to these forces, we observe a hysteretic clamping and unclamping of the nanotube device that results in a discrete drop in the mechanical resonance frequency on the order of 5-20 MHz, when the temperature is cycled between 340 and 375 K. This instability in the resonant frequency results from the nanotube unpinning from the electrode/trench sidewall where it is bound weakly by van der Waals forces. Interestingly, this unpinning does not affect the Q-factor of the resonance, since the clamping is still governed by van der Waals forces above and below the unpinning. For a 1 μm device, the drop observed in resonance frequency corresponds to a change in nanotube length of approximately 50-65 nm. On the basis of these findings, we introduce a new model, which includes a finite tension around zero gate voltage due to van der Waals forces and shows better agreement with the experimental data than the perfect clamping model. From the gate dependence of the mechanical resonance frequency, we extract the van der Waals clamping force to be 1.8 pN. The mechanical resonance frequency exhibits a striking temperature dependence below 200 K attributed to a temperature-dependent slack arising from the competition between the van der Waals force and the thermal fluctuations in the suspended nanotube.

  1. Implementation of a fast 16-Bit dynamic clamp using LabVIEW-RT.

    PubMed

    Kullmann, Paul H M; Wheeler, Diek W; Beacom, Joshua; Horn, John P

    2004-01-01

    The dynamic-clamp method provides a powerful electrophysiological tool for creating virtual ionic conductances in living cells and studying their influence on membrane potential. Here we describe G-clamp, a new way to implement a dynamic clamp using the real-time version of the Lab-VIEW programming environment together with a Windows host, an embedded microprocessor that runs a real-time operating system and a multifunction data-acquisition board. The software includes descriptions of a fast voltage-dependent sodium conductance, delayed rectifier, M-type and A-type potassium conductances, and a leak conductance. The system can also read synaptic conductance waveforms from preassembled data files. These virtual conductances can be reliably implemented at speeds < or =43 kHz while simultaneously saving two channels of data with 16-bit precision. G-clamp also includes utilities for measuring current-voltage relations, synaptic strength, and synaptic gain. Taking an approach built on a commercially available software/hardware platform has resulted in a system that is easy to assemble and upgrade. In addition, the graphical programming structure of LabVIEW should make it relatively easy for others to adapt G-clamp for new experimental applications.

  2. Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

    PubMed

    Brueggemann, A; George, M; Klau, M; Beckler, M; Steindl, J; Behrends, J C; Fertig, N

    2004-01-01

    Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

  3. A Pleiotropic RNA-Binding Protein Controls Distinct Cell Cycle Checkpoints to Drive Resistance of p53-Defective Tumors to Chemotherapy.

    PubMed

    Cannell, Ian G; Merrick, Karl A; Morandell, Sandra; Zhu, Chang-Qi; Braun, Christian J; Grant, Robert A; Cameron, Eleanor R; Tsao, Ming-Sound; Hemann, Michael T; Yaffe, Michael B

    2015-11-09

    In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.

  4. Large-deflection theory for end compression of long rectangular plates rigidly clamped along two edges

    NASA Technical Reports Server (NTRS)

    Levy, Samuel; Krupen, Philip

    1943-01-01

    The von Karman equations for flat plates are solved beyond the buckling load up to edge strains equal to eight time the buckling strain, for the extreme case of rigid clamping along the edges parallel to the load. Deflections, bending stresses, and membrane stresses are given as a function of end compressive load. The theoretical values of effective width are compared with the values derived for simple support along the edges parallel to the load. The increases in effective width due to rigid clamping drops from about 20 percent near the buckling strain to about 8 percent at an edge strain equal to eight times the buckling strain. Experimental values of effective width in the elastic range reported in NACA Technical Note No. 684 are between the theoretical curves for the extremes of simple support and rigid clamping.

  5. 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

  6. 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.

  7. Sequential dissection of multiple ionic currents in single cardiac myocytes under action potential-clamp

    PubMed Central

    Banyasz, Tamas; Horvath, Balazs; Jian, Zhong; Izu, Leighton T.; Chen-Izu, Ye

    2011-01-01

    The cardiac action potential (AP) is shaped by myriad ionic currents. In this study we develop an innovative AP-clamp Sequential Dissection technique to enable recording of multiple ionic currents in the single cell under AP-clamp. This new technique presents a significant step beyond the traditional way of recording only one current in any one cell. The ability to measure many currents in a single cell has revealed two hitherto unknown characteristics of the ionic currents in cardiac cells: coordination of currents within a cell and large variation of currents between cells. Hence, the AP-clamp Sequential Dissection method provides a unique and powerful tool for studying individual cell electrophysiology. PMID:21215755

  8. Sequential dissection of multiple ionic currents in single cardiac myocytes under action potential-clamp.

    PubMed

    Banyasz, Tamas; Horvath, Balazs; Jian, Zhong; Izu, Leighton T; Chen-Izu, Ye

    2011-03-01

    The cardiac action potential (AP) is shaped by myriad ionic currents. In this study, we develop an innovative AP-clamp Sequential Dissection technique to enable the recording of multiple ionic currents in the single cell under AP-clamp. This new technique presents a significant step beyond the traditional way of recording only one current in any one cell. The ability to measure many currents in a single cell has revealed two hitherto unknown characteristics of the ionic currents in cardiac cells: coordination of currents within a cell and large variation of currents between cells. Hence, the AP-clamp Sequential Dissection method provides a unique and powerful tool for studying individual cell electrophysiology.

  9. 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

  10. 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.

  11. 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

  12. A Randomized Trial of Mogen Clamp versus Plastibell for Neonatal Male Circumcision in Botswana

    PubMed Central

    Plank, Rebeca M; Ndubuka, Nnamdi O; Wirth, Kathleen E; Mwambona, Janet T; Kebaabetswe, Poloko; Bassil, Barbara; Lesetedi, Chiapo; Hussein, Fatma M; Magetse, Jane; Nkgau, Maggie; Makhema, Joseph; Mmalane, Mompati; Creek, Tracy; Powis, Kathleen M; Shapiro, Roger; Lockman, Shahin

    2013-01-01

    Background Male circumcision can reduce the risk of heterosexually-acquired HIV-1 infection in men. Neonatal male circumcision (NMC) has many potential advantages over circumcision at older ages but little is known about its feasibility and safety in resource-limited settings. Methods We performed a randomized trial in southeastern Botswana of Mogen clamp and Plastibell, two commonly used devices for NMC. Follow-up visits occurred at six weeks and four months postpartum. Adverse events, parental satisfaction and staff impressions were recorded. Results Of 302 male neonates randomized, 300 (99%) underwent circumcision, 153 (51%) with Mogen clamp and 147 (49%) with Plastibell. There were no major adverse events in the Mogen clamp arm but there were two major adverse events in the Plastibell arm (both were a proximally migrated ring that had to be removed by study staff). Minor adverse events were more common with the Mogen clamp compared with the Plastibell, specifically removal of too little skin and formation of skin bridges or adhesions (12 vs. 1 and 11 vs. 3, respectively, all P<0.05). Five (3%) infants in the Mogen clamp arm and none in the Plastibell arm had minor bleeding (P=0.03). More than 94% of mothers reported being highly or completely satisfied with the procedure. Conclusions NMC can be performed in Botswana with a low rate of adverse events and high parental satisfaction. Although the risk of migration and retention of the Plastibell is small, the Mogen clamp may be safer for NMC in regions where immediate emergent medical attention is not available. PMID:23314413

  13. Analysis and testing of the DIII-D ohmic heating coil lead repair clamp

    SciTech Connect

    Reis, E.E.; Anderson, P.M.; Chin, E.; Robinson, J.I.

    1997-11-01

    DIII-D has been operating for the last year with limited volt-second capabilities due to structural failure of a conductor lead to one of the ohmic heating (OH) solenoids. The conductor failure was due to poor epoxy impregnation of the overwrap of the lead pack, resulting in copper fatigue and a water leak. A number of structural analyses were performed to assist in determining the failure scenario and to evaluate various repair options. A fatigue stress analysis of the leads with a failed epoxy overwrap indicated crack initiation after 1,000 cycles at the maximum operating conditions. The failure occurred in a very inaccessible area which restricted design repair options to concepts which could be implemented remotely. Several design options were considered for repairing the lead so that it can sustain the loads for 7.5 Vs conditions at full toroidal field. A clamp, along with preloaded banding straps and shim bags, provides a system that guarantees that the stress at the crack location is always compressive and prevents further crack growth in the conductor. Due to the limited space available for the repair, it was necessary to design the clamp system to operate at the material yield stress. The primary components of the clamp system were verified by load tests prior to installation. The main body of the clamp contains a load cell and potentiometer for monitoring the load-deflection characteristics of the clamp and conductors during plasma operation. Strain gages provides redundant instrumentation. If required, the preload on the conductors can be increased remotely by a special wrench attached to the clamp assembly.

  14. Growth hormone response to GHRH + GHRP-6 in type 2 diabetes during euglycemic and hyperglycemic clamp.

    PubMed

    Micic, Dragan; Kendereski, Aleksandra; Sumarac-Dumanovic, Mirjana; Cvijovic, Goran; Popovic, Vera; Dieguez, Carlos; Casanueva, Felipe

    2004-01-01

    The aim of this study was to investigate the effect of two different glucose levels on GH response to the combined administration of GHRH+GHRP-6 in patients with type 2 diabetes. GH response to i.v. bolus of GHRH+GHRP-6 (100 mcg, each) was measured in 12 male patients with type 2 diabetes (mean age: 53.9+/-1.59 years; BMI: 25.58+/-0.39 kg/m(2); mean HbA(1c): 8.7+/-0.42%), during a euglycemic (mean glucose: 4.92+/-0.08 mmol) hyperinsulinemic clamp (insulin infusion rate of 100 mU/kg/h) and a hyperglycemic clamp (mean glucose: 12.19+/-0.11 mmol/l). There was no difference in basal GH levels between the hyperglycemic and euglycemic clamps (2.9+/-0.99 mU/l versus 1.48+/-0.44 mU/l; P>0.05). Peak GH response to GHRH+GHRP-6 during the hyperglycemic clamp was lower than in the englycemic clamp (112.45+/-14.45 mU/l versus 151.06+/-16.87 mU/l; P<0.05). Area under the GH curve was lower in the hyperglycemic than in the euglycemic clamp (6974.49+/-1001.95 mU/l/min versus 9560.75+/-1140.65 mU/l/min; P<0.05). It is concluded that hyperglycemia significantly reduces GH response to combined administration of GHRH+GHRP-6 in normal weight patients with type 2 diabetes. It is suggested that ambient glucose levels should be taken into account during interpretation of GH response to combined administration of GHRH+GHRP-6 in patients with type 2 diabetes.

  15. Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation.

    PubMed

    Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

    2010-06-01

    Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.

  16. A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

    PubMed Central

    DeMoe, Joanna H.; Santaguida, Stefano; Daum, John R.; Musacchio, Andrea; Gorbsky, Gary J.

    2008-01-01

    In mitosis the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic spindle checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate (1). The spindle checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened checkpoint signaling system that may contribute to chromosome instability in tumors. Since complete absence of the spindle checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the checkpoint might provide a therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel. PMID:19190331

  17. Design and fabrication of a planar patch-clamp substrate using a silicon-on-insulator wafer

    NASA Astrophysics Data System (ADS)

    Zhenlong, Zhang; Xiangyang, Liu; Yanli, Mao

    2009-09-01

    The planar patch-clamp technique has been applied to high throughput screening in drug discovery. The key feature of this technique is the fabrication of a planar patch-clamp substrate using appropriate materials. In this study, a planar patch-clamp substrate was designed and fabricated using a silicon-on-insulator (SOI) wafer. The access resistance and capacitance of SOI-based planar patch-clamp substrates are smaller than those of bulk silicon-based planar substrates, which will reduce the distributed RC noise.

  18. 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

  19. Soft-Switched Neutral-Point-Clamped Single-Phase Boost Rectifier

    NASA Astrophysics Data System (ADS)

    Itoh, Ryozo; Ishizaka, Kouichi

    A soft-switched neutral-point-clamped single-phase boost rectifier capable of compensating the imbalance load voltage is studied. This is based on a single-phase rectifier, in which an inductor is placed in series with the AC supply to resonate with a capacitor connected across the DC output of a full-bridge rectifier and the switching transition is mainly governed by a series resonance. The experimental prototype using insulated-gate bipolar transistors is implemented to investigate the operation under the charge control. The experimental results confirm that the rectifier has a neutral-point-clamp feature providing a good quality AC current.

  20. Patch-clamp array with on-chip electronics, optics, flow control and mechanical actuation.

    SciTech Connect

    James, Conrad D.; Okandan, Murat; Draper, Bruce Leroy; Mani, Seethambal S.

    2003-07-01

    Fast and quantitative analysis of cellular activity, signaling and responses to external stimuli is a crucial capability and it has been the goal of several projects focusing on patch clamp measurements. To provide the maximum functionality and measurement options, we have developed a patch clamp array device that incorporates on-chip electronics, mechanical, optical and microfluidic coupling as well as cell localization through fluid flow. The preliminary design, which integrated microfluidics, electrodes and optical access, was fabricated and tested. In addition, new designs which further combine mechanical actuation, on-chip electronics and various electrode materials with the previous designs are currently being fabricated.

  1. 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.

  2. Clamping drainage is unnecessary after minimally invasive total knee arthroplasty in patients with tranexamic acid

    PubMed Central

    Wu, Yuangang; Yang, Timin; Zeng, Yi; Li, Canfeng; Shen, Bin; Pei, Fuxing

    2017-01-01

    Abstract Background: Drainage and tranexamic acid (TXA) have been widely used in total knee arthroplasty (TKA). However, it remains unclear whether it is necessary to clamp the drain after minimally invasive TKA (MIS-TKA) when TXA is used. We therefore conducted a randomized controlled trial to compare the effects of clamping versus not clamping drainage following MIS-TKA in patients in whom TXA was used. Methods: From January 2015 to December 2015, 121 patients undergoing unilateral primary MIS-TKA were enrolled and randomly divided into 2 groups. In the clamping group (N = 60), drainage was clamped for the 1st 4 postoperative hours. In the nonclamping group (N = 61), drainage was not clamped. All patients underwent a minimidvastus approach and received 10 mg/kg TXA intravenously before tourniquet deflation. We recorded the total blood loss, drainage volume, and transfusion requirements in the postoperative period. We also measured the hemoglobin (Hb) and hematocrit (Hct) levels on postoperative days 1, 3, and 5. Other factors, including range of motion (ROM), visual analog scale (VAS), and occurrence of wound-related complications, deep vein thrombosis (DVT), and pulmonary embolism (PE) were recorded at the time of discharge and 1 and 6 months postoperatively. No statistically significant differences were found between the 2 groups with regard to age, gender, weight, BMI, preoperative Hb and Hct levels, preoperative ROM, VAS, duration of surgery, anesthesia method, and the American Society of Anesthesiologists classification. Results: The clamping group experienced better drainage volume results than the nonclamping group (P < 0.001). There were no statistically significant differences in TBL and transfusion requirements (P = 0.105 and 0.276, respectively); Hb and Hct levels on postoperative days 1, 3, and 5 were similar between the 2 groups. No significant differences were found for ROM, VAS, DVT, PE, wound-related complications, and hospital

  3. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    PubMed Central

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  4. Contrasting roles of checkpoint proteins as recombination modulators at Fob1-Ter complexes with or without fork arrest.

    PubMed

    Mohanty, Bidyut K; Bairwa, Narendra K; Bastia, Deepak

    2009-04-01

    The replication terminator protein Fob1 of Saccharomyces cerevisiae specifically interacts with two tandem Ter sites (replication fork barriers) located in the nontranscribed spacer of ribosomal DNA (rDNA) to cause polar fork arrest. The Fob1-Ter complex is multifunctional and controls other DNA transactions such as recombination by multiple mechanisms. Here, we report on the regulatory roles of the checkpoint proteins in the initiation and progression of recombination at Fob1-Ter complexes. The checkpoint adapter proteins Tof1 and Csm3 either positively or negatively controlled recombination depending on whether it was provoked by polar fork arrest or by transcription, respectively. The absolute requirements for these proteins for inducing recombination at an active replication terminus most likely masked their negative modulatory role at a later step of the process. Other checkpoint proteins of the checkpoint adapter/mediator class such as Mrc1 and Rad9, which channel signals from the sensor to the effector kinase, tended to suppress recombination at Fob1-Ter complexes regardless of how it was initiated. We have also discovered that the checkpoint sensor kinase Mec1 and the effector Rad53 were positive modulators of recombination initiated by transcription but had little effect on recombination at Ter. The work also showed that the two pathways were Rad52 dependent but Rad51 independent. Since Ter sites occur in the intergenic spacer of rDNA from yeast to humans, the mechanism is likely to be of widespread occurrence.

  5. 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

  6. TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus of Tetrahymena

    PubMed Central

    Yakisich, J. Sebastian; Sandoval, Pamela Y.; Morrison, Tara L.

    2006-01-01

    The ribosomal DNA origin binding protein Tif1p regulates the timing of rDNA replication and is required globally for proper S-phase progression and division of the Tetrahymena thermophila macronucleus. Here, we show that Tif1p safeguards chromosomes from DNA damage in the mitotic micronucleus and amitotic macronucleus. TIF1p localization is dynamically regulated as it moves into the micro- and macronucleus during the respective S phases. TIF1 disruption mutants are hypersensitive to hydroxyurea and methylmethanesulfonate, inducers of DNA damage and intra-S-phase checkpoint arrest in all examined eukaryotes. TIF1 mutants incur double-strand breaks in the absence of exogenous genotoxic stress, destabilizing all five micronuclear chromosomes. Wild-type Tetrahymena elicits an intra-S-phase checkpoint response that is induced by hydroxyurea and suppressed by caffeine, an inhibitor of the apical checkpoint kinase ATR/MEC1. In contrast, hydroxyurea-challenged TIF1 mutants fail to arrest in S phase or exhibit caffeine-sensitive Rad51 overexpression, indicating the involvement of TIF1 in checkpoint activation. Although aberrant micro- and macronuclear division occurs in TIF1 mutants and caffeine-treated wild-type cells, TIF1p bears no similarity to ATR or its substrates. We propose that TIF1 and ATR function in the same epistatic pathway to regulate checkpoint responses in the diploid mitotic micronucleus and polyploid amitotic macronucleus. PMID:17005912

  7. Systematic analysis in Caenorhabditis elegans reveals that the spindle checkpoint is composed of two largely independent branches.

    PubMed

    Essex, Anthony; Dammermann, Alexander; Lewellyn, Lindsay; Oegema, Karen; Desai, Arshad

    2009-02-01

    Kinetochores use the spindle checkpoint to delay anaphase onset until all chromosomes have formed bipolar attachments to spindle microtubules. Here, we use controlled monopolar spindle formation to systematically define the requirements for spindle checkpoint signaling in the Caenorhabditis elegans embryo. The results, when interpreted in light of kinetochore assembly epistasis analysis, indicate that checkpoint activation is coordinately directed by the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB-1-three components independently targeted to the outer kinetochore by the scaffold protein KNL-1. These components orchestrate the integration of a core Mad1(MDF-1)/Mad2(MDF-2)-based signal, with a largely independent Mad3(SAN-1)/BUB-3 pathway. Evidence for independence comes from the fact that subtly elevating Mad2(MDF-2) levels bypasses the requirement for BUB-3 and Mad3(SAN-1) in kinetochore-dependent checkpoint activation. Mad3(SAN-1) does not accumulate at unattached kinetochores and BUB-3 kinetochore localization is independent of Mad2(MDF-2). We discuss the rationale for a bipartite checkpoint mechanism in which a core Mad1(MDF-1)/Mad2(MDF-2) signal generated at kinetochores is integrated with a separate cytoplasmic Mad3(SAN-1)/BUB-3-based pathway.

  8. NONO regulates the intra-S-phase checkpoint in response to UV radiation.

    PubMed

    Alfano, L; Costa, C; Caporaso, A; Altieri, A; Indovina, P; Macaluso, M; Giordano, A; Pentimalli, F

    2016-02-04

    The main risk factor for skin cancer is ultraviolet (UV) exposure, which causes DNA damage. Cells respond to UV-induced DNA damage by activating the intra-S-phase checkpoint, which prevents replication fork collapse, late origin firing and stabilizes fragile sites. Recently, the 54-kDa multifunctional protein NONO was found to be involved in the non-homologous end-joining DNA repair process and in poly ADP-ribose polymerase 1 activation. Interestingly, NONO is mutated in several tumour types and emerged as a crucial factor underlying both melanoma development and progression. Therefore, we set out to evaluate whether NONO could be involved in the DNA-damage response to UV radiations. We generated NONO-silenced HeLa cell clones and found that lack of NONO decreased cell growth rate. Then, we challenged NONO-silenced cells with exposure to UV radiations and found that NONO-silenced cells, compared with control cells, continued to synthesize DNA, failed to block new origin firing and impaired CHK1S345 phosphorylation showing a defective checkpoint activation. Consistently, NONO is present at the sites of UV-induced DNA damage where it localizes to RAD9 foci. To position NONO in the DNA-damage response cascade, we analysed the loading onto chromatin of various intra-S-phase checkpoint mediators and found that NONO favours the loading of topoisomerase II-binding protein 1 acting upstream of the ATM and Rad3-related kinase activity. Strikingly, re-expression of NONO, through an sh-resistant mRNA, rescued CHK1S345 phosphorylation in NONO-silenced cells. Interestingly, NONO silencing affected cell response to UV radiations also in a melanoma cell line. Overall, our data uncover a new role for NONO in mediating the cellular response to UV-induced DNA damage.

  9. Beyond pattern recognition: five immune checkpoints for scaling the microbial threat.

    PubMed

    Blander, J Magarian; Sander, Leif E

    2012-02-24

    Pattern recognition by the innate immune system enables the detection of microorganisms, but how the level of microbial threat is evaluated - a process that is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage - is less well understood. New evidence has shown that features of microbial viability can be detected by the immune system and thereby induce robust responses that are not warranted for dead microorganisms. Here, we propose five immune checkpoints that, as defined here, collectively determine the gravity of microbial encounters.

  10. 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

  11. Tumor Microenvironment Metabolism: A New Checkpoint for Anti-Tumor Immunity

    PubMed Central

    Scharping, Nicole E.; Delgoffe, Greg M.

    2016-01-01

    When a T cell infiltrates a tumor, it is subjected to a variety of immunosuppressive and regulatory signals in the microenvironment. However, it is becoming increasingly clear that due to the proliferative and energetically-deregulated nature of tumor cells, T cells also operate at a metabolic disadvantage. The nutrient dearth of the tumor microenvironment (TME) creates “metabolic checkpoints” upon infiltrating T cells, impacting their ability to survive, proliferate and function effectively. In this review, we summarize the basics of tumor cell and T cell metabolism and discuss recent advances elucidating the individual metabolic checkpoints exerted on T cells that drive their dysfunction in the TME. PMID:27929420

  12. BMI1 attenuates etoposide-induced G2/M checkpoints via reducing ATM activation.

    PubMed

    Wei, F; Ojo, D; Lin, X; Wong, N; He, L; Yan, J; Xu, S; Major, P; Tang, D

    2015-06-04

    The BMI1 protein contributes to stem cell pluripotency and oncogenesis via multiple functions, including its newly identified role in DNA damage response (DDR). Although evidence clearly demonstrates that BMI1 facilitates the repair of double-stranded breaks via homologous recombination (HR), it remains unclear how BMI1 regulates checkpoint activation during DDR. We report here that BMI1 has a role in G2/M checkpoint activation in response to etoposide (ETOP) treatment. Ectopic expression of BMI1 in MCF7 breast cancer and DU145 prostate cancer cells significantly reduced ETOP-induced G2/M arrest. Conversely, knockdown of BMI1 in both lines enhanced the arrest. Consistent with ETOP-induced activation of the G2/M checkpoints via the ATM pathway, overexpression and knockdown of BMI1, respectively, reduced and enhanced ETOP-induced phosphorylation of ATM at serine 1981 (ATM pS1981). Furthermore, the phosphorylation of ATM targets, including γH2AX, threonine 68 (T68) on CHK2 (CHK2 pT68) and serine 15 (S15) on p53 were decreased in overexpression and increased in knockdown BMI1 cells in response to ETOP. In line with the requirement of NBS1 in ATM activation, we were able to show that BMI1 associates with NBS1 and that this interaction altered the binding of NBS1 with ATM. BMI1 consists of a ring finger (RF), helix-turn-helix-turn-helix-turn (HT), proline/serine (PS) domain and two nuclear localization signals (NLS). Although deletion of either RF or HT did not affect the association of BMI1 with NBS1, the individual deletions of PS and one NLS (KRMK) robustly reduced the interaction. Stable expression of these BMI1 mutants decreased ETOP-induced ATM pS1981 and CHK2 pT68, but not ETOP-elicited γH2AX in MCF7 cells. Furthermore, ectopic expression of BMI1 in non-transformed breast epithelial MCF10A cells also compromised ETOP-initiated ATM pS1981 and γH2AX. Taken together, we provide compelling evidence that BMI1 decreases ETOP-induced G2/M checkpoint activation via

  13. 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…

  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. Experiments on vertical slender flexible cylinders clamped at both ends and subjected to axial flow.

    PubMed

    Modarres-Sadeghi, Y; Païdoussis, M P; Semler, C; Grinevich, E

    2008-04-13

    Three series of experiments were conducted on vertical clamped-clamped cylinders in order to observe experimentally the dynamical behaviour of the system, and the results are compared with theoretical predictions. In the first series of experiments, the downstream end of the clamped-clamped cylinder was free to slide axially, while in the second, the downstream end was fixed; the influence of externally applied axial compression was also studied in this series of experiments. The third series of experiments was similar to the second, except that a considerably more slender, hollow cylinder was used. In these experiments, the cylinder lost stability by divergence at a sufficiently high flow velocity and the amplitude of buckling increased thereafter. At higher flow velocities, the cylinder lost stability by flutter (attainable only in the third series of experiments), confirming experimentally the existence of a post-divergence oscillatory instability, which was previously predicted by both linear and nonlinear theory. Good quantitative agreement is obtained between theory and experiment for the amplitude of buckling, and for the critical flow velocities.

  16. Enhancement of heat transfer by clamped flags in a Poiseuille channel flow

    NASA Astrophysics Data System (ADS)

    Lee, Jae Bok; Park, Sung Goon; Kim, Boyoung; Sung, Hyung Jin

    2016-11-01

    A pair of flexible flags clamped vertically in a heated channel was numerically modeled to study an enhancement of heat transfer by the clamped flags in a Poiseuille channel flow. The penalty immersed boundary method was adopted to analyze the fluid-structure-thermal interaction between the surrounding fluid and the clamped flags. The dynamics of the clamped flags was categorized into three distinctive modes: a flapping mode, a fully deflected mode, and an irregular mode. The distinctive modes that depended on the relationship between the hydrodynamic force and the restoring force displayed different movement patterns. The flapping mode provided superior thermal performance to the other modes. Vortices generated from the flapping flags swept out the thermal boundary layer and entrained the fluid near channel walls into the channel core flow while passing through the wake periodically. Compared to rigid flags, the flapping flags significantly improved the thermal efficiency. In addition, the effects of channel height and Reynolds number on the thermal efficiency were explored to obtain an optimal parameter set, which presented the highest thermal performance in present study. The flexible flags regarding the optimal parameter set showed an increase of up to 230% in net heat flux, compared to the baseline flow. Dynamic modes decomposition (DMD) method was adopted to examine the correlation between the vorticity and temperature fields.

  17. Influence of clamp-up force on the strength of bolted composite joints

    NASA Astrophysics Data System (ADS)

    Horn, Walter J.; Schmitt, Ron R.

    1994-03-01

    Composite materials offer the potential for a reduction in the number of individual parts and joints in a structure because large one-piece components can replace multipart assemblies. Nevertheless, there are many situations where composite parts must be joined and often mechanical fasteners provide the only practical method of joining those parts. The long-term strength of mechanically fastened joints of composite members can be directly affected by the clamp-up force of the fastener and thus perhaps by the relaxation of this force due to the viscoelastic character of the composite materials of the joint. Methods for predicting the effect of bolt clamp-up force relaxation on the strength of mechanically fastened joints of thermoplastic composite materials were investigated during the present study. A test program, using two thermoplastic composite materials, was conducted to determine the influence of clamp-up force on joint strength, to measure the relaxation of the joint clamp-up force with time, and to measure the change of joint strength as a function of time.

  18. Chromosomal Replication Dynamics and Interaction with the β Sliding Clamp Determine Orientation of Bacterial Transposable Elements

    PubMed Central

    Gómez, Manuel J.; Díaz-Maldonado, Héctor; González-Tortuero, Enrique; López de Saro, Francisco J.

    2014-01-01

    Insertion sequences (ISs) are small transposable elements widespread in bacterial genomes, where they play an essential role in chromosome evolution by stimulating recombination and genetic flow. Despite their ubiquity, it is unclear how ISs interact with the host. Here, we report a survey of the orientation patterns of ISs in bacterial chromosomes with the objective of gaining insight into the interplay between ISs and host chromosomal functions. We find that a significant fraction of IS families present a consistent and family-specific orientation bias with respect to chromosomal DNA replication, especially in Firmicutes. Additionally, we find that the transposases of up to nine different IS families with different transposition pathways interact with the β sliding clamp, an essential replication factor, suggesting that this is a widespread mechanism of interaction with the host. Although we find evidence that the interaction with the β sliding clamp is common to all bacterial phyla, it also could explain the observed strong orientation bias found in Firmicutes, because in this group β is asymmetrically distributed during synthesis of the leading or lagging strands. Besides the interaction with the β sliding clamp, other asymmetries also play a role in the biased orientation of some IS families. The utilization of the highly conserved replication sliding clamps suggests a mechanism for host regulation of IS proliferation and also a universal platform for IS dispersal and transmission within bacterial populations and among phylogenetically distant species. PMID:24614824

  19. Efficacy of Combined Histone Deacetylase and Checkpoint Kinase Inhibition in a Preclinical Model of Human Burkitt Lymphoma

    PubMed Central

    Kong, YanGuo; Barisone, Gustavo A; Sidhu, Ranjit S; O’Donnell, Robert T; Tuscano, Joseph M

    2015-01-01

    Checkpoint kinase inhibition has been studied as a way of enhancing the effectiveness of DNA-damaging agents. More recently, histone deacetylase inhibitors have shown efficacy in several cancers, including non-Hodgkin lymphoma. To evaluate the effectiveness of this combination for the treatment of lymphoma, we examined the combination of AR42, a histone deacetylase inhibitor, and checkpoint kinase 2 (CHEK2) inhibitor II in vitro and in vivo. The combination resulted in up to 10-fold increase in potency in five Burkitt lymphoma cell lines when compared with either drug alone. Both drugs inhibited tumor progression in xenograft models, but the combination was more effective than either agent alone, resulting in regression of established tumors. No toxicity was observed. These results suggest that the combination of histone deacetylase inhibition and checkpoint kinase inhibition represent an effective and nontoxic treatment option that should be further explored in preclinical and clinical studies. PMID:26322845

  20. PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint.

    PubMed

    Baumann, Christoph; Körner, Roman; Hofmann, Kay; Nigg, Erich A

    2007-01-12

    We identify PICH (Plk1-interacting checkpoint "helicase"), a member of the SNF2 ATPase family, as an interaction partner and substrate of Plk1. Following phosphorylation of PICH on the Cdk1 site T1063, Plk1 is recruited to PICH and controls its localization. Starting in prometaphase, PICH accumulates at kinetochores and inner centromeres. Moreover, it decorates threads that form during metaphase before increasing in length and progressively diminishing during anaphase. PICH-positive threads connect sister kinetochores and are dependent on tension, sensitive to DNase, and exacerbated in response to premature loss of cohesins or inhibition of topoisomerase II, suggesting that they represent stretched centromeric chromatin. Depletion of PICH causes the selective loss of Mad2 from kinetochores and completely abrogates the spindle checkpoint, resulting in massive chromosome missegregation. These data identify PICH as a novel essential component of checkpoint signaling. We propose that PICH binds to catenated centromere-related DNA to monitor tension developing between sister kinetochores.

  1. A lysine deacetylase Hos3 is targeted to the bud neck and involved in the spindle position checkpoint.

    PubMed

    Wang, Mengqiao; Collins, Ruth N

    2014-09-15

    An increasing number of cellular activities can be regulated by reversible lysine acetylation. Targeting the enzymes responsible for such posttranslational modifications is instrumental in defining their substrates and functions in vivo. Here we show that a Saccharomyces cerevisiae lysine deacetylase, Hos3, is asymmetrically targeted to the daughter side of the bud neck and to the daughter spindle pole body (SPB). The morphogenesis checkpoint member Hsl7 recruits Hos3 to the neck region. Cells with a defect in spindle orientation trigger Hos3 to load onto both SPBs. When associated symmetrically with both SPBs, Hos3 functions as a spindle position checkpoint (SPOC) component to inhibit mitotic exit. Neck localization of Hos3 is essential for its symmetric association with SPBs in cells with misaligned spindles. Our data suggest that Hos3 facilitates cross-talk between the morphogenesis checkpoint and the SPOC as a component of the intricate monitoring of spindle orientation after mitotic entry and before commitment to mitotic exit.

  2. Exaggerated natriuresis during clamping of systemic NO supply in healthy young men.

    PubMed

    Simonsen, Jane A; Rasmussen, Mona S; Vach, Werner; Høilund-Carlsen, Poul F; Bie, Peter

    2012-01-01

    NO (nitric oxide) may be involved in fluid homoeostasis. We hypothesized that increases in NO synthesis contribute to acute, saline-induced natriuresis, which, therefore, should be blunted when NO availability is stabilized. Young men were studied during simultaneous infusions of L-NAME [NG-nitro-L-arginine methyl ester; bolus of 750 μg·kg⁻¹ of body weight and 8.3 μg·min⁻¹·kg⁻¹ of body weight] and SNP (sodium nitroprusside), the latter at a rate preventing L-NAME from increasing total peripheral resistance ('NO-clamping'). Slow volume expansion (saline, 20 μmol of NaCl·min⁻¹·kg⁻¹ of body weight for 3 h) was performed with and without concomitant NO-clamping. NO-clamping itself decreased RPF (renal plasma flow; P~0.02) and tended to decrease arterial blood pressure [MABP (mean arterial blood pressure)]. Volume expansion markedly decreased the plasma levels of renin, AngII (angiotensin II) and aldosterone (all P<0.001), while MABP (oscillometry), heart rate, cardiac output (impedance cardiography), RPF (by p-aminohippurate), GFR [glomerular filtration rate; by using 51Cr-labelled EDTA] and plasma [Na+] and [K+] remained constant. Volume expansion increased sodium excretion (P<0.02) at constant filtered load, but more so during NO-clamping than during control (+184% compared with 52%; P<0.0001). Urinary nitrate/nitrite excretion increased during volume expansion; plasma cGMP and plasma vasopressin were unchanged. The results demonstrate that NO-clamping augments sodium excretion in response to volume expansion at constant MABP and GFR, reduced RPF and decreased renin system activity, a response termed hypernatriuresis. The results indicate that mediator(s) other than MABP, RPF, GFR and renin system activity contribute significantly to the homoeostatic response to saline loading, but the specific mechanisms of hypernatriuresis remain obscure.

  3. 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.

  4. Ventilation before Umbilical Cord Clamping Improves the Physiological Transition at Birth

    PubMed Central

    Bhatt, Sasmira; Polglase, Graeme R.; Wallace, Euan M.; te Pas, Arjan B.; Hooper, Stuart B.

    2014-01-01

    The transition from a fetus to a neonate at birth represents a critical phase in our life. Most infants make this transition without complications, but preterm infants usually require some form of assistance due to immature cardiopulmonary systems that predispose them to lifelong sequelae. As the incidence of preterm birth is increasing, there is now an urgent need for the development of management strategies that facilitate this transition, which will likely include improved strategies for the management of the maternal third stage of labor. For instance, recent studies on the physiological transition at birth have led to the discovery that establishing ventilation in the infant before the umbilical cord is clamped greatly stabilizes the cardiovascular transition at birth. While most benefits of delayed clamping previously have been attributed to an increase in placenta to infant blood transfusion, clearly there are other significant benefits for the infant, which are not well understood. Nevertheless, if ventilation can be established before cord clamping in a preterm infant, the large adverse changes in cardiac function that normally accompanies umbilical cord clamping can be avoided. As preterm infants have an immature cerebral vascular bed, large swings in cardiovascular function places them at high risk of cerebral vascular rupture and the associated increased risk of mortality and morbidity. In view of the impact that cord clamping has on the cardiovascular transition at birth, it is also time to re-examine some of the strategies used in the management of the third stage of labor. These include the appropriate timing of uterotonic administration in relation to delivery of the infant and placenta. As there is a lack of evidence on the effects these individual practices have on the infant, there is a necessity to improve our understanding of their impact in order to develop strategies that facilitate the transition to newborn life. PMID:25368858

  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. 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

  7. C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

    PubMed

    Singh, Gaurav Kumar; Karade, Sharanbasappa Shrimant; Ranjan, Rajeev; Ahamad, Nafees; Ahmed, Shakil

    2017-02-01

    The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

  8. 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…

  9. 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

  10. 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.

  11. The interplay among chromatin dynamics, cell cycle checkpoints and repair mechanisms modulates the cellular response to DNA damage.

    PubMed

    Lazzaro, Federico; Giannattasio, Michele; Muzi-Falconi, Marco; Plevani, Paolo

    2007-06-01

    Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.

  12. Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy

    NASA Astrophysics Data System (ADS)

    Chen, Qian; Xu, Ligeng; Liang, Chao; Wang, Chao; Peng, Rui; Liu, Zhuang

    2016-10-01

    A therapeutic strategy that can eliminate primary tumours, inhibit metastases, and prevent tumour relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clinically approved components can be used for near-infrared laser-triggered photothermal ablation of primary tumours, generating tumour-associated antigens, which in the presence of R837-containing nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunological responses will be able to attack remaining tumour cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumour models. Furthermore, such strategy offers a strong immunological memory effect, which can provide protection against tumour rechallenging post elimination of their initial tumours.

  13. Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy.

    PubMed

    Zamarin, Dmitriy; Holmgaard, Rikke B; Subudhi, Sumit K; Park, Joon Seok; Mansour, Mena; Palese, Peter; Merghoub, Taha; Wolchok, Jedd D; Allison, James P

    2014-03-05

    Preexisting lymphocytic infiltration of tumors is associated with superior prognostic outcomes in a variety of cancers. Recent studies also suggest that lymphocytic responses may identify patients more likely to benefit from therapies targeting immune checkpoints, suggesting that therapeutic efficacy of immune checkpoint blockade can be enhanced through strategies that induce tumor inflammation. To achieve this effect, we explored the immunotherapeutic potential of oncolytic Newcastle disease virus (NDV). We find that localized intratumoral therapy of B16 melanoma with NDV induces inflammatory responses, leading to lymphocytic infiltrates and antitumor effect in distant (nonvirally injected) tumors without distant virus spread. The inflammatory effect coincided with distant tumor infiltration with tumor-specific CD4(+) and CD8(+) T cells, which was dependent on the identity of the virus-injected tumor. Combination therapy with localized NDV and systemic CTLA-4 blockade led to rejection of preestablished distant tumors and protection from tumor rechallenge in poorly immunogenic tumor models, irrespective of tumor cell line sensitivity to NDV-mediated lysis. Therapeutic effect was associated with marked distant tumor infiltration with activated CD8(+) and CD4(+) effector but not regulatory T cells, and was dependent on CD8(+) cells, natural killer cells, and type I interferon. Our findings demonstrate that localized therapy with oncolytic NDV induces inflammatory immune infiltrates in distant tumors, making them susceptible to systemic therapy with immunomodulatory antibodies, which provides a strong rationale for investigation of such combination therapies in the clinic.

  14. Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma.

    PubMed

    Beug, Shawn T; Beauregard, Caroline E; Healy, Cristin; Sanda, Tarun; St-Jean, Martine; Chabot, Janelle; Walker, Danielle E; Mohan, Aditya; Earl, Nathalie; Lun, Xueqing; Senger, Donna L; Robbins, Stephen M; Staeheli, Peter; Forsyth, Peter A; Alain, Tommy; LaCasse, Eric C; Korneluk, Robert G

    2017-02-15

    Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.

  15. Anti-TNF-refractory colitis after checkpoint inhibitor therapy: Possible role of CMV-mediated immunopathogenesis

    PubMed Central

    Lankes, Katharina; Hundorfean, Gheorghe; Harrer, Thomas; Pommer, Ansgar J.; Agaimy, Abbas; Angelovska, Irena; Tajmir-Riahi, Azadeh; Göhl, Jonas; Schuler, Gerold; Neurath, Markus F.; Hohenberger, Werner; Heinzerling, Lucie

    2016-01-01

    ABSTRACT Immune-related adverse events (irAEs) induced by checkpoint inhibitors are well known. Since fatal outcomes have been reported early detection and adequate management are crucial. In particular, colitis is frequently observed and can result in intestinal perforation. This is the first report of an autoimmune colitis that was treated according to algorithms but became resistant due to a CMV reactivation. The 32-y-old male patient with metastatic melanoma treated within an anti-PD-1/ipilimumab combination study developed severe immune-mediated colitis (CTCAE grade 3) with up to 18 watery stools per day starting 2 weeks after treatment initiation. After improving upon therapy with immunosuppressive treatment (high dose steroids and infliximab) combined with parenteral nutrition diarrhea again exacerbated. Additionally, the patient had asymptomatic grade 3 CTCAE amylase and lipase elevation. Colitis was monitored by weekly endoscopies and colon biopsies were analyzed histologically with CMV staining, multi-epitope ligand cartography (MELC) and qRT-PCR for inflammatory genes. In the course, CMV reactivation was detected in the colon and treated with antiviral medication in parallel to a reduction of corticosteroids. Subsequently, symptoms improved. The patient showed a complete response for 2 y now including regression of bone metastases. CMV reactivation under checkpoint inhibitor therapy in combination with immunosuppressive treatment for autoimmune side effects has to be considered in these patients and if present treated. Potentially, CMV reactivation is underdiagnosed. Treatment algorithms should include CMV diagnostics. PMID:27471608

  16. Crystal Structure of Checkpoint Kinase 2 in Complex with Nsc 109555, a Potent and Selective Inhibitor

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Zhang, Di; Jobson, Andrew G.; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2009-03-05

    Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC{sub 50} = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

  17. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins.

    PubMed

    Lisby, Michael; Barlow, Jacqueline H; Burgess, Rebecca C; Rothstein, Rodney

    2004-09-17

    DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

  18. Host-Pathogen Checkpoints and Population Bottlenecks in Persistent and Intracellular Uropathogenic E. coli Bladder Infection

    PubMed Central

    Hannan, Thomas J.; Totsika, Makrina; Mansfield, Kylie J.; Moore, Kate H.; Schembri, Mark A.; Hultgren, Scott J.

    2013-01-01

    Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multi-drug resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic E. coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the Quiescent Intracellular Reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection: QIR, ASB, or chronic cystitis, is determined within the first 24 hours of infection and constitutes a putative host-pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies. PMID:22404313

  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. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1

    PubMed Central

    Breit, Claudia; Bange, Tanja; Petrovic, Arsen; Weir, John R.; Müller, Franziska; Vogt, Doro; Musacchio, Andrea

    2015-01-01

    The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint. PMID:26658523

  1. Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe.

    PubMed

    Cornago, M; Garcia-Alberich, C; Blasco-Angulo, N; Vall-Llaura, N; Nager, M; Herreros, J; Comella, J X; Sanchis, D; Llovera, M

    2014-10-02

    Glioblastoma multiforme is resistant to conventional anti-tumoral treatments due to its infiltrative nature and capability of relapse; therefore, research efforts focus on characterizing gliomagenesis and identifying molecular targets useful on therapy. New therapeutic strategies are being tested in patients, such as Histone deacetylase inhibitors (HDACi) either alone or in combination with other therapies. Here two HDACi included in clinical trials have been tested, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), to characterize their effects on glioma cell growth in vitro and to determine the molecular changes that promote cancer cell death. We found that both HDACi reduce glioma cell viability, proliferation and clonogenicity. They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh. Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1). In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures. Therefore, HDACi treatment causes glioma cell entry into mitosis before DNA damage could be repaired and to the formation of an aberrant mitotic spindle that results in glioma cell death through mitotic catastrophe-induced apoptosis.

  2. Immune Checkpoint Inhibition in Hepatocellular Carcinoma: Basics and Ongoing Clinical Trials.

    PubMed

    Kudo, Masatoshi

    2017-01-01

    Clinical trials of antibodies targeting the immune checkpoint inhibitors programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) for the treatment of advanced hepatocellular carcinoma (HCC) are ongoing. Expansion cohorts of a phase I/II trial of the anti-PD-1 antibody nivolumab in advanced HCC showed favorable results. Two phase III studies are currently ongoing: a comparison of nivolumab and sorafenib in the first-line setting for advanced HCC, and a comparison of the anti-PD-1 antibody pembrolizumab and a placebo in the second-line setting for patients with advanced HCC who progressed on sorafenib therapy. The combination of anti-PD-1/PD-L1 and anti-CTLA-4 antibodies is being evaluated in other phase I/II trials, and the results suggest that an anti-PD-1 antibody combined with locoregional therapy or other molecular targeted agents is an effective treatment strategy for HCC. Immune checkpoint inhibitors may therefore open new doors to the treatment of HCC.

  3. Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication.

    PubMed

    Piunti, Andrea; Rossi, Alessandra; Cerutti, Aurora; Albert, Mareike; Jammula, Sriganesh; Scelfo, Andrea; Cedrone, Laura; Fragola, Giulia; Olsson, Linda; Koseki, Haruhiko; Testa, Giuseppe; Casola, Stefano; Helin, Kristian; d'Adda di Fagagna, Fabrizio; Pasini, Diego

    2014-04-14

    The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human tumours, and PcG inhibition has been proposed as a strategy for cancer treatment. However, the recurrent inactivation of pRb/p53 responses in human cancers raises a question regarding the ability of PcG proteins to affect cellular proliferation independently from this checkpoint. Here we demonstrate that PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel activity by which PcGs can regulate cell proliferation independently of major cell cycle restriction checkpoints.

  4. Molecular basis of APC/C regulation by the spindle assembly checkpoint

    PubMed Central

    Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-01-01

    In the dividing eukaryotic cell the spindle assembly checkpoint (SAC) ensures each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), a multimeric assembly that inhibits the APC/C, delaying chromosome segregation. Here, using cryo-electron microscopy we determined the near-atomic resolution structure of an APC/C-MCC complex (APC/CMCC). We reveal how degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit (Cdc20APC/C) responsible for substrate interactions. BubR1 also obstructs binding of UbcH10 (APC/C’s initiating E2) to repress APC/C ubiquitination activity. Conformational variability of the complex allows for UbcH10 association, and we show from a structure of APC/CMCC in complex with UbcH10 how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. PMID:27509861

  5. Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade

    PubMed Central

    Zhang, Fei; Wei, Hudie; Wang, Xiaoxiao; Bai, Yu; Wang, Pilin; Wu, Jiawei; Jiang, Xiaoyong; Wang, Yugang; Cai, Haiyan; Xu, Ting; Zhou, Aiwu

    2017-01-01

    The use of antibodies to target immune checkpoints, particularly PD-1/PD-L1, has made a profound impact in the field of cancer immunotherapy. Here, we identified KN035, an anti-PD-L1 nanobody that can strongly induce T-cell responses and inhibit tumor growth. The crystal structures of KN035 complexed with PD-L1 and free PD-L1, solved here at 1.7 and 2.7 Å resolution, respectively, show that KN035 competes with PD-1 (programmed death protein 1) for the same flat surface on PD-L1, mainly through a single surface loop of 21 amino acids. This loop forms two short helices and develops key hydrophobic and ionic interactions with PD-L1 residues, such as Ile54, Tyr56 and Arg113, which are also involved in PD-1 binding. The detailed mutagenesis study identified the hotspot residues of the PD-L1 surface and provides an explanation for the stronger (~1 000-fold) binding of KN035 to PD-L1 than PD-1 and its lack of binding to PD-L2. Overall, this study reveals how a single immunoglobulin-variable scaffold of KN035 or PD-1 can bind to a flat protein surface through either a single surface loop or beta-sheet strands; and provides a basis for designing new immune checkpoint blockers and generating bi-specific antibodies for combination therapy. PMID:28280600

  6. SNM1A Acts Downstream of ATM to Promote the G1 Cell Cycle Checkpoint

    PubMed Central

    Akhter, Shamima; Legerski, Randy J.

    2008-01-01

    We have shown previously that SNM1A co-localizes with 53BP1 at sites of double-strand breaks (DSBs) induced by IR, and that these proteins interact with or without DNA damage. However, the role of SNM1A in the DNA damage response has not been elucidated. Here, we show that SNM1A is required for an efficient G1 checkpoint arrest after IR exposure. Interestingly, the localization of SNM1A to sites of DSBs does not require either 53BP1 or H2AX, nor does the localization of 53BP1 require SNM1A. However, the localization of SNM1A does require ATM. Furthermore, SNM1A is shown to be a phosphorylation substrate of ATM in vitro, and to interact with ATM in vivo particularly after exposure of cells to IR. In addition, in the absence of SNM1A the activation of the downstream ATM target p53 is reduced. These findings suggest that SNM1A acts with ATM to promote the G1 cell cycle checkpoint. PMID:18848520

  7. A2aR antagonists: Next generation checkpoint blockade for cancer immunotherapy.

    PubMed

    Leone, Robert D; Lo, Ying-Chun; Powell, Jonathan D

    2015-01-01

    The last several years have witnessed exciting progress in the development of immunotherapy for the treatment of cancer. This has been due in great part to the development of so-called checkpoint blockade. That is, antibodies that block inhibitory receptors such as CTLA-4 and PD-1 and thus unleash antigen-specific immune responses against tumors. It is clear that tumors evade the immune response by usurping pathways that play a role in negatively regulating normal immune responses. In this regard, adenosine in the immune microenvironment leading to the activation of the A2a receptor has been shown to represent one such negative feedback loop. Indeed, the tumor microenvironment has relatively high concentrations of adenosine. To this end, blocking A2a receptor activation has the potential to markedly enhance anti-tumor immunity in mouse models. This review will present data demonstrating the ability of A2a receptor blockade to enhance tumor vaccines, checkpoint blockade and adoptive T cell therapy. Also, as several recent studies have demonstrated that under certain conditions A2a receptor blockade can enhance tumor progression, we will also explore the complexities of adenosine signaling in the immune response. Despite important nuances to the A2a receptor pathway that require further elucidation, studies to date strongly support the development of A2a receptor antagonists (some of which have already been tested in phase III clinical trials for Parkinson Disease) as novel modalities in the immunotherapy armamentarium.

  8. Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma

    PubMed Central

    Beug, Shawn T.; Beauregard, Caroline E.; Healy, Cristin; Sanda, Tarun; St-Jean, Martine; Chabot, Janelle; Walker, Danielle E.; Mohan, Aditya; Earl, Nathalie; Lun, Xueqing; Senger, Donna L.; Robbins, Stephen M.; Staeheli, Peter; Forsyth, Peter A.; Alain, Tommy; LaCasse, Eric C.; Korneluk, Robert G.

    2017-01-01

    Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells. PMID:28198370

  9. Tumor-suppressor Genes, Cell Cycle Regulatory Checkpoints, and the Skin

    PubMed Central

    Velez, Ana Maria Abreu; Howard, Michael S.

    2015-01-01

    The cell cycle (or cell-division cycle) is a series of events that take place in a cell, leading to its division and duplication. Cell division requires cell cycle checkpoints (CPs) that are used by the cell to both monitor and regulate the progress of the cell cycle. Tumor-suppressor genes (TSGs) or antioncogenes are genes that protect the cell from a single event or multiple events leading to cancer. When these genes mutate, the cell can progress to a cancerous state. We aimed to perform a narrative review, based on evaluation of the manuscripts published in MEDLINE-indexed journals using the Medical Subject Headings (MeSH) terms “tumor suppressor's genes,” “skin,” and “cell cycle regulatory checkpoints.” We aimed to review the current concepts regarding TSGs, CPs, and their association with selected cutaneous diseases. It is important to take into account that in some cell cycle disorders, multiple genetic abnormalities may occur simultaneously. These abnormalities may include intrachromosomal insertions, unbalanced division products, recombinations, reciprocal deletions, and/or duplication of the inserted segments or genes; thus, these presentations usually involve several genes. Due to their complexity, these disorders require specialized expertise for proper diagnosis, counseling, personal and family support, and genetic studies. Alterations in the TSGs or CP regulators may occur in many benign skin proliferative disorders, neoplastic processes, and genodermatoses. PMID:26110128

  10. The transcription factor E4F1 coordinates CHK1-dependent checkpoint and mitochondrial functions.

    PubMed

    Rodier, Geneviève; Kirsh, Olivier; Baraibar, Martín; Houlès, Thibault; Lacroix, Matthieu; Delpech, Hélène; Hatchi, Elodie; Arnould, Stéphanie; Severac, Dany; Dubois, Emeric; Caramel, Julie; Julien, Eric; Friguet, Bertrand; Le Cam, Laurent; Sardet, Claude

    2015-04-14

    Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells.

  11. Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy

    PubMed Central

    Chen, Qian; Xu, Ligeng; Liang, Chao; Wang, Chao; Peng, Rui; Liu, Zhuang

    2016-01-01

    A therapeutic strategy that can eliminate primary tumours, inhibit metastases, and prevent tumour relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clinically approved components can be used for near-infrared laser-triggered photothermal ablation of primary tumours, generating tumour-associated antigens, which in the presence of R837-containing nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunological responses will be able to attack remaining tumour cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumour models. Furthermore, such strategy offers a strong immunological memory effect, which can provide protection against tumour rechallenging post elimination of their initial tumours. PMID:27767031

  12. High-content fluorescent-based assay for screening activators of DNA damage checkpoint pathways.

    PubMed

    Bin Zhang; Xiubin Gu; Uppalapati, Uma; Ashwell, Mark A; Leggett, David S; Li, Chiang J

    2008-07-01

    Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.

  13. nab-Paclitaxel as a potential partner with checkpoint inhibitors in solid tumors

    PubMed Central

    Soliman, Hatem H

    2017-01-01

    Tumors recognized by the host immune system are associated with better survival. However, the immune system is often suppressed in patients with established tumor burden. Stimulating the immune system to detect and kill tumor cells has been a challenge in cancer therapy for some time. Recently, novel cancer immunotherapies, such as immune checkpoint inhibitors, monoclonal antibodies, and vaccine therapies, have emerged as promising therapeutic approaches for many solid tumors. However, for some tumors, immunotherapy alone has not provided significant benefits, and some may even be fully resistant to immunotherapy. It has been suggested that the immune system may require “priming” before an immunotherapy can elicit an immune response. Although chemotherapies are believed to be immunosuppressive, when given at the right dose and sequence these agents may provide this “priming” effect for the immune system. In addition to direct cytotoxic killing of tumor cells, standard chemotherapeutic agents can elicit immunogenicity through various mechanisms. This review highlights the general immunomodulatory properties of chemotherapy agents. It also provides a rationale for combined therapy with nab-paclitaxel and immune checkpoint inhibitors. Recent clinical trial data with these combination regimens in solid tumors are presented, along with a summary of ongoing trials. PMID:28053544

  14. Role of Clinical Pharmacology in the Development and Approval of Immunotherapies Targeting Immune Checkpoints.

    PubMed

    Rahman, A

    2016-12-01

    Immune surveillance plays a critical role in preventing the development and progression of cancer. Immune modulators, such as interferon-gamma or interleukin-2, have been a part of the cancer treatment armament over the past few decades. However, new understandings regarding the role of the costimulatory and coinhibitory molecules associated with T-cells and antigen-presenting cells as well as tumor necrosis factor receptors and ligands have ushered the new era of immunotherapy for cancer treatment. We now know that primary cancer cells evade screening by the innate immune system, proliferate, and form metastases by upregulating immune inhibitory pathways referred to as immune checkpoints. The recent development of therapies that target immune checkpoints, such as cytotoxic T lymphocyte antigen 4, programmed cell death 1, programmed cell death ligand 1, indoleamine 2,3-dioxygenase, T-cell immunoglobulin and mucin domain 3, and lymphocyte activation gene 3 precisely target the immune system and give new hope for treating various types of cancer. In select marker-enriched populations, immunotherapies provide high response rates as well as durable responses in terms of progression-free survival and overall survival. Numerous factors, such as patient's immune system, the expression of targets on both immune and cancer cells, maintenance of an effective drug exposure, and tolerability to these agents may play a role in this unique observation.

  15. Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses.

    PubMed

    Liu, Jun; Yuan, Ying; Chen, Wenna; Putra, Juan; Suriawinata, Arief A; Schenk, Austin D; Miller, Halli E; Guleria, Indira; Barth, Richard J; Huang, Yina H; Wang, Li

    2015-05-26

    V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a negative immune-checkpoint protein that suppresses T-cell responses. To determine whether VISTA synergizes with another immune-checkpoint, programmed death 1 (PD-1), this study characterizes the immune responses in VISTA-deficient, PD-1-deficient (KO) mice and VISTA/PD-1 double KO mice. Chronic inflammation and spontaneous activation of T cells were observed in both single KO mice, demonstrating their nonredundancy. However, the VISTA/PD-1 double KO mice exhibited significantly higher levels of these phenotypes than the single KO mice. When bred onto the 2D2 T-cell receptor transgenic mice, which are predisposed to development of inflammatory autoimmune disease in the CNS, the level of disease penetrance was significantly enhanced in the double KO mice compared with in the single KO mice. Consistently, the magnitude of T-cell response toward foreign antigens was synergistically higher in the VISTA/PD-1 double KO mice. A combinatorial blockade using monoclonal antibodies specific for VISTA and PD-L1 achieved optimal tumor-clearing therapeutic efficacy. In conclusion, our study demonstrates the nonredundant role of VISTA that is distinct from the PD-1/PD-L1 pathway in controlling T-cell activation. These findings provide the rationale to concurrently target VISTA and PD-1 pathways for treating T-cell-regulated diseases such as cancer.

  16. Checkpoint modulation--A new way to direct the immune system against renal cell carcinoma.

    PubMed

    Bedke, Jens; Kruck, Stephan; Gakis, Georgios; Stenzl, Arnulf; Goebell, Peter J

    2015-01-01

    The introduction of targeted therapies like the tyrosine kinase (TKI) and mammalian target of rapamycin (mTOR) inhibitors has improved patients' survival in general. Nevertheless the prognosis remains limited. Therapies with a new mode of action are urgently warranted, especially those who would provoke long-term responders or long-lasting complete remissions as observed with unspecific immunotherapy with the cytokines interleukin-2 and interferon-α. In the recent years a deeper understanding of the underlying immunology of T cell activation led to the development of checkpoint inhibitors, which are mainly monocloncal antibodies and which enhances the presence of the co-stimulatory signals needed for T cell activation or priming. This review discusses the clinical data and ongoing studies available for the inhibition of the PD-1 (CD279) and CTLA-4 (CD152) axis in mRCC. In addition, potential future immunological targets are discussed. This approach of T-cell activation or re-activation by immunological checkpoint inhibition holds the inherent promise to directly affect the tumor cell and thereby to potentially cure a subset of patients with mRCC.

  17. Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X rays.

    PubMed

    Hirayama, R; Uzawa, A; Matsumoto, Y; Noguchi, M; Kase, Y; Takase, N; Ito, A; Koike, S; Ando, K; Okayasu, R; Furusawa, Y

    2011-02-01

    We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV µm(-1) carbon ions and X rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X rays was 1.68±0.31, and this value was not changed after 1 h rejoining time (1.40±0.26). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.

  18. Design and experimental research of a novel inchworm type piezo-driven rotary actuator with the changeable clamping radius.

    PubMed

    Zhao, Hongwei; Fu, Lu; Ren, Luquan; Huang, Hu; Fan, Zunqiang; Li, Jianping; Qu, Han

    2013-01-01

    In this paper, a novel piezo-driven rotary actuator with the changeable clamping radius is developed based on the inchworm principle. This actuator mainly utilizes three piezoelectric actuators, a flexible gripper, a clamping block, and a rotor to achieve large stroke rotation with high resolution. The design process of the flexible gripper consisting of the driving unit and the clamping unit is described. Lever-type mechanisms were used to amplify the micro clamping displacements. The amplifying factor and parasitic displacement of the lever-type mechanism in the clamping unit was analyzed theoretically and experimentally. In order to investigate the rotation characteristics of the actuator, a series of experiments was carried out. Experimental results indicate that the actuator can rotate at a speed of 77,488 μrad/s with a driving frequency of 167 Hz. The rotation resolution and maximum load torque of the actuator are 0.25 μrad and 37 N mm, respectively. The gripper is movable along the z direction based on an elevating platform, and the clamping radius can change from 10.6 mm to 25 mm. Experimental results confirm that the actuator can achieve different rotation speeds by changing the clamping radius.

  19. Estimates on the eigenvalues of the clamped plate problem on domains in Euclidean spaces

    NASA Astrophysics Data System (ADS)

    Yolcu, Selma Yıldırım; Yolcu, Türkay

    2013-04-01

    The purpose of this article is two-fold. First, we obtain some certain bounds for the sums of (positive and negative) powers of the eigenvalues of the clamped plate problem of the Dirichlet bi-Laplacian operator Δ 2|_{{D}}, restricted to a bounded domain {D}subset {{R}}^d with d ⩾ 2. Second, we establish lower bounds for the sums of eigenvalues of Δ ^2|_{{D}} sharper than the bounds recently obtained by Cheng and Wei ["A lower bound for eigenvalues of a clamped plate problem," Calculus Var. Partial Differ. Equ. 42(3-4), 579-590 (2011)], 10.1007/s00526-011-0399-6. All these estimates are sharp in the sense of Weyl asymptotics.

  20. Robotic surgery and hemostatic agents in partial nephrectomy: a high rate of success without vascular clamping.

    PubMed

    Morelli, Luca; Morelli, John; Palmeri, Matteo; D'Isidoro, Cristiano; Kauffmann, Emanuele Federico; Tartaglia, Dario; Caprili, Giovanni; Pisano, Roberta; Guadagni, Simone; Di Franco, Gregorio; Di Candio, Giulio; Mosca, Franco

    2015-09-01

    Robot-assisted partial nephrectomy has been proposed as a technique to overcome technical challenges of laparoscopic partial nephrectomy. We prospectively collected and analyzed data from 31 patients who underwent robotic partial nephrectomy with systematic use of hemostatic agents, between February 2009 and October 2014. Thirty-three renal tumors were treated in 31 patients. There were no conversions to open surgery, intraoperative complications, or blood transfusions. The mean size of the resected tumors was 27 mm (median 20 mm, range 5-40 mm). Twenty-seven of 33 lesions (82%) did not require vascular clamping and therefore were treated in the absence of ischemia. All margins were negative. The high partial nephrectomy success rate without vascular clamping suggests that robotic nephron-sparing surgery with systematic use of hemostatic agents may be a safe, effective method to completely avoid ischemia in the treatment of selected renal masses.