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Sample records for checkpoint silencing mechanism

  1. 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

  2. Checkpoint proteins influence telomeric silencing and length maintenance in budding yeast.

    PubMed Central

    Longhese, M P; Paciotti, V; Neecke, H; Lucchini, G

    2000-01-01

    A complex network of surveillance mechanisms, called checkpoints, interrupts cell cycle progression when damage to the genome is detected or when cells fail to complete DNA replication, thus ensuring genetic integrity. In budding yeast, components of the DNA damage checkpoint regulatory network include the RAD9, RAD17, RAD24, MEC3, DDC1, RAD53, and MEC1 genes that are proposed to be involved in different aspects of DNA metabolism. We provide evidence that some DNA damage checkpoint components play a role in maintaining telomere integrity. In fact, rad53 mutants specifically enhance repression of telomere-proximal transcription via the Sir-mediated pathway, suggesting that Rad53 might be required for proper chromatin structure at telomeres. Moreover, Rad53, Mec1, Ddc1, and Rad17 are necessary for telomere length maintenance, since mutations in all of these genes cause a decrease in telomere size. The telomeric shortening in rad53 and mec1 mutants is further enhanced in the absence of SIR genes, suggesting that Rad53/Mec1 and Sir proteins contribute to chromosome end protection by different pathways. The finding that telomere shortening, but not increased telomeric repression of gene expression in rad53 mutants, can be suppressed by increasing dNTP synthetic capacity in these strains suggests that transcriptional silencing and telomere integrity involve separable functions of Rad53. PMID:10924458

  3. 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

  4. Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

    PubMed

    Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

    2017-05-02

    Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

  5. Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

    PubMed Central

    Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

    2017-01-01

    Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. DOI: http://dx.doi.org/10.7554/eLife.25366.001 PMID:28463114

  6. Polo-like kinase-1 regulates kinetochore–microtubule dynamics and spindle checkpoint silencing

    PubMed Central

    Liu, Dan; Davydenko, Olga

    2012-01-01

    Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore–microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore–microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore–microtubule attachments and subsequently silencing the spindle checkpoint. PMID:22908307

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

    PubMed Central

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

    2014-01-01

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

  8. 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.

  9. 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

  10. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    SciTech Connect

    Nakadate, Yusuke; Kodera, Yasuo; Kitamura, Yuka; Tachibana, Taro; Tamura, Tomohide; Koizumi, Fumiaki

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  11. 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.

  12. 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

  13. On the Mechanism of Gene Silencing in Saccharomyces cerevisiae

    PubMed Central

    Steakley, David Lee; Rine, Jasper

    2015-01-01

    Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation. This study provided strong support for the steric occlusion mechanism by the discovery that RNA polymerase of bacteriophage T7 could be substantially blocked from transcribing from its cognate promoter when embedded in silenced chromatin. Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin. The effectiveness of the Sir protein–based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing. PMID:26082137

  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. 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.

  16. Mad2 Checkpoint Gene Silencing Using Epidermal Growth Factor Receptor-Targeted Chitosan Nanoparticles in Non-Small Cell Lung Cancer Model

    PubMed Central

    2015-01-01

    RNA interference has emerged as a powerful strategy in cancer therapy because it allows silencing of specific genes associated with tumor progression and resistance. Mad2 is an essential mitotic checkpoint component required for accurate chromosome segregation during mitosis, and its complete abolition leads to cell death. We have developed an epidermal growth factor receptor (EGFR)-targeted chitosan system for silencing the Mad2 gene as a strategy to efficiently induce cell death in EGFR overexpressing human A549 non-small cell lung cancer cells. Control and EGFR-targeted chitosan nanoparticles loaded with small interfering RNAs (siRNAs) against Mad2 were formulated and characterized for size, charge, morphology, and encapsulation efficiency. Qualitative and quantitative intracellular uptake studies by confocal imaging and flow cytometry, respectively, showed time-dependent enhanced and selective intracellular internalization of EGFR-targeted nanoparticles compared to nontargeted system. Targeted nanoparticles showed nearly complete depletion of Mad2 expression in A549 cells contrasting with the partial depletion in the nontargeted system. Accordingly, Mad2-silencing-induced apoptotic cell death was confirmed by cytotoxicity assay and flow cytometry. Our results demonstrate that EGFR-targeted chitosan loaded with Mad2 siRNAs is a potent delivery system for selective killing of cancer cells. PMID:25256346

  17. Silencers

    NASA Astrophysics Data System (ADS)

    Kurze, U.; Riedel, E.

    Large size silencers are attached to the intake and exhaust of large industrial plants, e.g. forced ventilation systems for mining industry, intake of cooling towers (Fig. 11.1) or flue gas stacks of power plants to protect the neighbourhood from plant noise. Large silencers are also required for ventilation openings of rooms with high internal sound pressure levels, e.g. industrial production halls or subway ventilation ducts.

  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. Mechanisms, applications, and perspectives of antiviral RNA silencing in plants

    PubMed Central

    Garcia-Ruiz, Hernan; Ruiz, Mayra Teresa Garcia; Peralta, Sergio Manuel Gabriel; Gabriel, Cristina Betzabeth Miravel; El-Mounadi, Kautar

    2017-01-01

    Viral diseases of plants cause important economic losses due to reduction in crop quality and quantity to the point of threatening food security in some countries. Given the reduced availability of natural sources, genetic resistance to viruses has been successfully engineered for some plant-virus combinations. A sound understanding of the basic mechanisms governing plant-virus interactions, including antiviral RNA silencing, is the foundation to design better management strategies and biotechnological approaches to engineer and implement antiviral resistance in plants. In this review, we present current molecular models to explain antiviral RNA silencing and its application in basic plant research, biotechnology and genetic engineering. PMID:28890589

  20. Structure and Gene-Silencing Mechanisms of Small Noncoding RNAs

    NASA Astrophysics Data System (ADS)

    Chu, Chia-Ying; Rana, Tariq M.

    Small (19-31-nucleotides) noncoding RNAs were identified in the past 10 years for their distinct function in gene silencing. The best known gene-silencing phenomenon, RNA interference (RNAi), is triggered in a sequence-specific manner by endogenously produced or exogenously introduced small doubled-stranded RNAs. As knowledge of the structure and function of the RNAi machinery has expanded, this phenomenon has become a powerful tool for biochemical research; it has enormous potential for therapeutics. This chapter summarizes significant aspects of three major classes of small noncoding, regulatory RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Here, we focus on the biogenesis of these small RNAs, their structural features and coupled effectors as well as the mechanisms of each small regulatory RNA pathway which reveal fascinating ways by which gene silencing is controlled and fine-tuned at an epigenetic level.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Dual mechanisms regulate the recruitment of spindle assembly checkpoint proteins to the budding yeast kinetochore

    PubMed Central

    Aravamudhan, Pavithra; Chen, Renjie; Roy, Babhrubahan; Sim, Janice; Joglekar, Ajit P.

    2016-01-01

    Recruitment of spindle assembly checkpoint (SAC) proteins by an unattached kinetochore leads to SAC activation. This recruitment is licensed by the Mps1 kinase, which phosphorylates the kinetochore protein Spc105 at one or more of its six MELT repeats. Spc105 then recruits the Bub3-Bub1 and Mad1-Mad2 complexes, which produce the inhibitory signal that arrests cell division. The strength of this signal depends, in part, on the number of Bub3-Bub1 and Mad1-Mad2 molecules that Spc105 recruits. Therefore regulation of this recruitment will influence SAC signaling. To understand this regulation, we established the physiological binding curves that describe the binding of Bub3-Bub1 and Mad1-Mad2 to the budding yeast kinetochore. We find that the binding of both follows the mass action law. Mps1 likely phosphorylates all six MELT repeats of Spc105. However, two mechanisms prevent Spc105 from recruiting six Bub3-Bub1 molecules: low Bub1 abundance and hindrance in the binding of more than one Bub3-Bub1 molecule to the same Spc105. Surprisingly, the kinetochore recruits two Mad1-Mad2 heterotetramers for every Bub3-Bub1 molecule. Finally, at least three MELT repeats per Spc105 are needed for accurate chromosome segregation. These data reveal that kinetochore-intrinsic and -extrinsic mechanisms influence the physiological operation of SAC signaling, potentially to maximize chromosome segregation accuracy. PMID:27170178

  6. Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

    PubMed Central

    Bun, Philippe; Liu, JunJun; Turlier, Hervé; Liu, ZengZhen; Uriot, Karen; Joanny, Jean-François; Coppey-Moisan, Maïté

    2014-01-01

    Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development. PMID:25028874

  7. Structure and Activation Mechanism of the CHK2 DNA Damage Checkpoint Kinase

    SciTech Connect

    Cai, Z.; Chehab, N; Pavletich, N

    2009-01-01

    The CHK2 protein kinase is an important transducer of DNA damage checkpoint signals, and its mutation contributes to hereditary and sporadic cancer. CHK2 activation is triggered by the phosphorylation of Thr68 by the DNA damage-activated ATM kinase. This leads to transient CHK2 dimerization, in part through intermolecular phosphoThr68-FHA domain interactions. Dimerization promotes kinase activation through activation-loop autophosphorylation, but the mechanism of this process has not been clear. The dimeric crystal structure of CHK2, described here, in conjunction with biochemical and mutational data reveals that productive CHK2 dimerization additionally involves intermolecular FHA-kinase domain and FHA-FHA interactions. Ile157, mutated in the Li-Fraumeni cancer-predisposition syndrome, plays a central role in the FHA-kinase domain interface, explaining the lack of dimerization and autophosphorylation of this mutant. In the dimer, the kinase active sites face each other in close proximity, indicating that dimerization may also serve to optimally position the kinase active sites for efficient activation loop transphosphorylation.

  8. 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.

  9. Mechanisms guiding Polycomb activities during gene silencing in Arabidopsis thaliana

    PubMed Central

    He, Chongsheng; Huang, Hai; Xu, Lin

    2013-01-01

    Polycomb group (PcG) proteins act in an evolutionarily conserved epigenetic pathway that regulates chromatin structures in plants and animals, repressing many developmentally important genes by modifying histones. PcG proteins can form at least two multiprotein complexes: Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2, respectively). The functions of Arabidopsis thaliana PRCs have been characterized in multiple stages of development and have diverse roles in response to environmental stimuli. Recently, the mechanism that precisely regulates Arabidopsis PcG activity was extensively studied. In this review, we summarize recent discoveries in the regulations of PcG at the three different layers: the recruitment of PRCs to specific target loci, the polyubiquitination and degradation of PRC2, and the antagonism of PRC2 activity by the Trithorax group proteins. Current knowledge indicates that the powerful activity of the PcG pathway is strictly controlled for specific silencing of target genes during plant development and in response to environmental stimuli. PMID:24312106

  10. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast.

    PubMed

    Iida, Tetsushi; Iida, Naoko; Tsutsui, Yasuhiro; Yamao, Fumiaki; Kobayashi, Takehiko

    2012-10-12

    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(+), the overexpression of ago1(+) alleviated the cell cycle defect in dcr1Δ. 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(+) is dependent on ptr1(+). Nuclear accumulation of poly(A)(+) RNAs was detected in mutants of ago1(+) and ptr1(+), suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. 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.

  12. 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.

  13. Radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy.

    PubMed

    Sharabi, Andrew B; Lim, Michael; DeWeese, Theodore L; Drake, Charles G

    2015-10-01

    Checkpoint blockade immunotherapy has received mainstream attention as a result of striking and durable clinical responses in some patients with metastatic disease and a reasonable response rate in many tumour types. The activity of checkpoint blockade immunotherapy is not restricted to melanoma or lung cancer, and additional indications are expected in the future, with responses already reported in renal cancer, bladder cancer, and Hodgkin's lymphoma among many others. Additionally, the interactions between radiation and the immune system have been investigated, with several studies describing the synergistic effects on local and distant tumour control when radiation therapy is combined with immunotherapy. Clinical enthusiasm for this approach is strengthened by the many ongoing trials combining immunotherapy with definitive and palliative radiation. Herein, we discuss the biological and mechanistic rationale behind combining radiation with checkpoint blockade immunotherapy, with a focus on the preclinical data supporting this potentially synergistic combination. We explore potential hypotheses and important considerations for clinical trial designs. Finally, we reintroduce the notion of radiosensitising immunotherapy, akin to radiosensitising chemotherapy, as a potential definitive therapeutic modality. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Toward a more efficient and scalable checkpoint/restart mechanism in the Community Atmosphere Model

    NASA Astrophysics Data System (ADS)

    Anantharaj, Valentine

    2015-04-01

    The number of cores (both CPU as well as accelerator) in large-scale systems has been increasing rapidly over the past several years. In 2008, there were only 5 systems in the Top500 list that had over 100,000 total cores (including accelerator cores) whereas the number of system with such capability has jumped to 31 in Nov 2014. This growth however has also increased the risk of hardware failure rates, necessitating the implementation of fault tolerance mechanism in applications. The checkpoint and restart (C/R) approach is commonly used to save the state of the application and restart at a later time either after failure or to continue execution of experiments. The implementation of an efficient C/R mechanism will make it more affordable to output the necessary C/R files more frequently. The availability of larger systems (more nodes, memory and cores) has also facilitated the scaling of applications. Nowadays, it is more common to conduct coupled global climate simulation experiments at 1 deg horizontal resolution (atmosphere), often requiring about 103 cores. At the same time, a few climate modeling teams that have access to a dedicated cluster and/or large scale systems are involved in modeling experiments at 0.25 deg horizontal resolution (atmosphere) and 0.1 deg resolution for the ocean. These ultrascale configurations require the order of 104 to 105 cores. It is not only necessary for the numerical algorithms to scale efficiently but the input/output (IO) mechanism must also scale accordingly. An ongoing series of ultrascale climate simulations, using the Titan supercomputer at the Oak Ridge Leadership Computing Facility (ORNL), is based on the spectral element dynamical core of the Community Atmosphere Model (CAM-SE), which is a component of the Community Earth System Model and the DOE Accelerated Climate Model for Energy (ACME). The CAM-SE dynamical core for a 0.25 deg configuration has been shown to scale efficiently across 100,000 cpu cores. At this

  15. Recovery from the DNA Replication Checkpoint.

    PubMed

    Chaudhury, Indrajit; Koepp, Deanna M

    2016-10-28

    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.

  16. 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

  17. Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism.

    PubMed

    Mandriota, Stefano J; Valentijn, Linda J; Lesne, Laurence; Betts, David R; Marino, Denis; Boudal-Khoshbeen, Mary; London, Wendy B; Rougemont, Anne-Laure; Attiyeh, Edward F; Maris, John M; Hogarty, Michael D; Koster, Jan; Molenaar, Jan J; Versteeg, Rogier; Ansari, Marc; Gumy-Pause, Fabienne

    2015-07-30

    Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma.

  18. Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism

    PubMed Central

    Mandriota, Stefano J.; Valentijn, Linda J.; Lesne, Laurence; Betts, David R.; Marino, Denis; Boudal-Khoshbeen, Mary; London, Wendy B.; Rougemont, Anne-Laure; Attiyeh, Edward F.; Maris, John M.; Hogarty, Michael D.; Koster, Jan; Molenaar, Jan J.; Versteeg, Rogier

    2015-01-01

    Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma. PMID:26053094

  19. Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

    SciTech Connect

    Holmfeldt, Per; Sellin, Mikael E.; Gullberg, Martin

    2010-07-15

    Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18{yields}E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.

  20. Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint.

    PubMed

    Holmfeldt, Per; Sellin, Mikael E; Gullberg, Martin

    2010-07-15

    Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18-->E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.

  1. Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

    PubMed Central

    Donnianni, Roberto Antonio; Ferrari, Matteo; Lazzaro, Federico; Clerici, Michela; Tamilselvan Nachimuthu, Benjamin; Plevani, Paolo; Muzi-Falconi, Marco; Pellicioli, Achille

    2010-01-01

    Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends. PMID:20098491

  2. 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.

  3. Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

    PubMed Central

    Mauri, Marta; Kirchner, Marieluise; Aharoni, Reuven; Ciolli Mattioli, Camilla; van den Bruck, David; Gutkovitch, Nadya; Modepalli, Vengamanaidu; Selbach, Matthias; Moran, Yehu; Chekulaeva, Marina

    2017-01-01

    Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria. PMID:27604873

  4. Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution.

    PubMed

    Mauri, Marta; Kirchner, Marieluise; Aharoni, Reuven; Ciolli Mattioli, Camilla; van den Bruck, David; Gutkovitch, Nadya; Modepalli, Vengamanaidu; Selbach, Matthias; Moran, Yehu; Chekulaeva, Marina

    2017-01-25

    Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.

  5. Analysis of absorption and reflection mechanisms in a three-dimensional plate silencer

    NASA Astrophysics Data System (ADS)

    Wang, Chunqi; Huang, Lixi

    2008-06-01

    When a segment of a rigid duct is replaced by a plate backed by a hard-walled cavity, grazing incident sound waves induce plate vibration, hence sound reflection. Based on this mechanism, a broadband plate silencer, which works effectively from low-to-medium frequencies have been developed recently. A typical plate silencer consists of an expansion chamber with two side-branch cavities covered by light but extremely stiff plates. Such a configuration is two-dimensional in nature. In this paper, numerical study is extended to three-dimensional configurations to investigate the potential improvement in sound reflection. Finite element simulation shows that the three-dimensional configurations perform better than the corresponding two-dimensional design, especially in the relatively high frequency region. Further analysis shows that the three-dimensional design gives better plate response at higher axial modes than the simple two-dimensional design. Sound absorption mechanism is also introduced to the plate silencer by adding two dissipative chambers on the two lateral sides of a two-cavity wave reflector, hence a hybrid silencer. Numerical simulation shows that the proposed hybrid silencer is able to achieve a good moderate bandwidth with much reduced total length in comparison with pure absorption design.

  6. Checkpoint inhibition in myeloma.

    PubMed

    Benson, Don M

    2016-12-02

    Historically, attempts at cancer immunotherapy have emphasized strategies designed to stimulate or augment the immune system into action. In the past decade, a complementary approach has developed, that of releasing immune cells from inhibitory restraint. Discoveries in the fundamental biology of how immunity is regulated, how the immune system interfaces with malignancy, and how cancer cells may exploit these processes to evade detection have all been translated into the rapidly growing field of therapeutic immune checkpoint inhibition for cancer. Myeloma is a malignancy associated with significant immune dysfunction imparted both by the disease itself as well as by many of the immunosuppressive therapies that have been used in the past. The growing body of preclinical data regarding immunoregulatory mechanisms that appear active in myeloma has begun to be translated to clinical trials targeting these signaling axes. This review will attempt to summarize the current understanding of the basic biology of several immune checkpoint pathways that may be important in myeloma and provide an up-to-date overview of recent and ongoing clinical trials of immune checkpoint inhibitors in myeloma. Finally, several current challenges and possible future directions of immune checkpoint blockade in myeloma will be reviewed. © 2016 by The American Society of Hematology. All rights reserved.

  7. Genome Reactivation after the Silence in Mitosis: Recapitulating Mechanisms of Development?

    PubMed Central

    Zaret, Kenneth S.

    2014-01-01

    Transcription is silenced during mitosis and re-activated at mitotic exit. The dynamics and identities of “bookmarking” transcription factors and chromatin marks that mediate reactivation often recapitulate that observed during cell identity establishment in development. Thus, features of post-mitotic gene re-activation can provide insights into mechanisms of developmental cell fate establishment. PMID:24780732

  8. A non-canonical site reveals the cooperative mechanisms of microRNA-mediated silencing

    PubMed Central

    Flamand, Mathieu N.; Gan, Hin Hark; Mayya, Vinay K.; Gunsalus, Kristin C.

    2017-01-01

    Abstract Although strong evidence supports the importance of their cooperative interactions, microRNA (miRNA)-binding sites are still largely investigated as functionally independent regulatory units. Here, a survey of alternative 3΄UTR isoforms implicates a non-canonical seedless site in cooperative miRNA-mediated silencing. While required for target mRNA deadenylation and silencing, this site is not sufficient on its own to physically recruit miRISC. Instead, it relies on facilitating interactions with a nearby canonical seed-pairing site to recruit the Argonaute complexes. We further show that cooperation between miRNA target sites is necessary for silencing in vivo in the C. elegans embryo, and for the recruitment of the Ccr4-Not effector complex. Using a structural model of cooperating miRISCs, we identified allosteric determinants of cooperative miRNA-mediated silencing that are required for both embryonic and larval miRNA functions. Our results delineate multiple cooperative mechanisms in miRNA-mediated silencing and further support the consideration of target site cooperation as a fundamental characteristic of miRNA function. PMID:28482037

  9. Underground tests of quantum mechanics. Whispers in the cosmic silence?

    NASA Astrophysics Data System (ADS)

    Curceanu, C.; Bartalucci, S.; Bassi, A.; Bazzi, M.; Bertolucci, S.; Berucci, C.; Bragadireanu, A. M.; Cargnelli, M.; Clozza, A.; De Paolis, L.; Di Matteo, S.; Donadi, S.; Egger, J.-P.; Guaraldo, C.; Iliescu, M.; Laubenstein, M.; Marton, J.; Milotti, E.; Pichler, A.; Pietreanu, D.; Piscicchia, K.; Scordo, A.; Shi, H.; Sirghi, D.; Sirghi, F.; Sperandio, L.; Vazquez Doce, O.; Zmeskal, J.

    2017-08-01

    By performing X-rays measurements in the “cosmic silence” of the underground laboratory of Gran Sasso, LNGS-INFN, we test a basic principle of quantum mechanics: the Pauli Exclusion Principle (PEP), for electrons. We present the achieved results of the VIP experiment and the ongoing VIP2 measurement aiming to gain two orders of magnitude improvement in testing PEP. We also use a similar experimental technique to search for radiation (X and gamma) predicted by continuous spontaneous localization models, which aim to solve the “measurement problem”.

  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. 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

  12. The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome

    PubMed Central

    Kumari, Daman; Usdin, Karen

    2010-01-01

    Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and the most common known cause of autism. Most cases of FXS result from the expansion of a CGG·CCG repeat in the 5′ UTR of the FMR1 gene that leads to gene silencing. It has previously been shown that silenced alleles are associated with histone H3 dimethylated at lysine 9 (H3K9Me2) and H3 trimethylated at lysine 27 (H3K27Me3), modified histones typical of developmentally repressed genes. We show here that these alleles are also associated with elevated levels of histone H3 trimethylated at lysine 9 (H3K9Me3) and histone H4 trimethylated at lysine 20 (H4K20Me3). All four of these modified histones are present on exon 1 of silenced alleles at levels comparable to that seen on pericentric heterochromatin. The two groups of histone modifications show a different distribution on fragile X alleles: H3K9Me2 and H3K27Me3 have a broad distribution, whereas H3K9Me3 and H4K20Me3 have a more focal distribution with the highest level of these marks being present in the vicinity of the repeat. This suggests that the trigger for gene silencing may be local to the repeat itself and perhaps involves a mechanism similar to that involved in the formation of pericentric heterochromatin. PMID:20843831

  13. The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome.

    PubMed

    Kumari, Daman; Usdin, Karen

    2010-12-01

    Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and the most common known cause of autism. Most cases of FXS result from the expansion of a CGG·CCG repeat in the 5' UTR of the FMR1 gene that leads to gene silencing. It has previously been shown that silenced alleles are associated with histone H3 dimethylated at lysine 9 (H3K9Me2) and H3 trimethylated at lysine 27 (H3K27Me3), modified histones typical of developmentally repressed genes. We show here that these alleles are also associated with elevated levels of histone H3 trimethylated at lysine 9 (H3K9Me3) and histone H4 trimethylated at lysine 20 (H4K20Me3). All four of these modified histones are present on exon 1 of silenced alleles at levels comparable to that seen on pericentric heterochromatin. The two groups of histone modifications show a different distribution on fragile X alleles: H3K9Me2 and H3K27Me3 have a broad distribution, whereas H3K9Me3 and H4K20Me3 have a more focal distribution with the highest level of these marks being present in the vicinity of the repeat. This suggests that the trigger for gene silencing may be local to the repeat itself and perhaps involves a mechanism similar to that involved in the formation of pericentric heterochromatin.

  14. 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

  15. 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.

  16. Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism.

    PubMed Central

    Li, H W; Lucy, A P; Guo, H S; Li, W X; Ji, L H; Wong, S M; Ding, S W

    1999-01-01

    The 2b protein encoded by cucumber mosaic cucumovirus (Cmv2b) acts as an important virulence determinant by suppressing post-transcriptional gene silencing (PTGS), a natural plant defence mechanism against viruses. We report here that the tomato aspermy cucumovirus 2b protein (Tav2b), when expressed from the unrelated tobacco mosaic tobamovirus (TMV) RNA genome, activates strong host resistance responses to TMV in tobacco which are typical of the gene-for-gene disease resistance mechanism. Domain swapping between Cmv2b, which does not elicit these responses, and Tav2b, revealed functional domains in Tav2b critical for triggering virus resistance and hypersensitive cell death. Furthermore, substitution of two amino acids from Tav2b by those found at the same positions in Cmv2b, Lys21-->Val and Arg28-->Ser, abolished the ability to induce hypersensitive cell death and virus resistance. However, in Nicotiana benthamiana, a species related to tobacco, Tav2b functions as a virulence determinant and suppresses PTGS. Thus, a viral suppressor of the host gene silencing defence mechanism is the target of another independent host resistance mechanism. Our results provide new insights into the complex molecular strategies employed by viruses and their hosts for defence, counter-defence and counter counter-defence. PMID:10329615

  17. Mechanism of Action of 2-Aminobenzamide HDAC Inhibitors in Reversing Gene Silencing in Friedreich's Ataxia.

    PubMed

    Soragni, Elisabetta; Chou, C James; Rusche, James R; Gottesfeld, Joel M

    2015-01-01

    The genetic defect in Friedreich's ataxia (FRDA) is the hyperexpansion of a GAA•TTC triplet in the first intron of the FXN gene, encoding the essential mitochondrial protein frataxin. Histone post-translational modifications near the expanded repeats are consistent with heterochromatin formation and consequent FXN gene silencing. Using a newly developed human neuronal cell model, derived from patient-induced pluripotent stem cells, we find that 2-aminobenzamide histone deacetylase (HDAC) inhibitors increase FXN mRNA levels and frataxin protein in FRDA neuronal cells. However, only compounds targeting the class I HDACs 1 and 3 are active in increasing FXN mRNA in these cells. Structural analogs of the active HDAC inhibitors that selectively target either HDAC1 or HDAC3 do not show similar increases in FXN mRNA levels. To understand the mechanism of action of these compounds, we probed the kinetic properties of the active and inactive inhibitors, and found that only compounds that target HDACs 1 and 3 exhibited a slow-on/slow-off mechanism of action for the HDAC enzymes. HDAC1- and HDAC3-selective compounds did not show this activity. Using siRNA methods in the FRDA neuronal cells, we show increases in FXN mRNA upon silencing of either HDACs 1 or 3, suggesting the possibility that inhibition of each of these class I HDACs is necessary for activation of FXN mRNA synthesis, as there appears to be redundancy in the silencing mechanism caused by the GAA•TTC repeats. Moreover, inhibitors must have a long residence time on their target enzymes for this activity. By interrogating microarray data from neuronal cells treated with inhibitors of different specificity, we selected two genes encoding histone macroH2A (H2AFY2) and Polycomb group ring finger 2 (PCGF2) that were specifically down-regulated by the inhibitors targeting HDACs1 and 3 versus the more selective inhibitors for further investigation. Both genes are involved in transcriptional repression and we speculate

  18. Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment.

    PubMed

    Le, Huy Quang; Ghatak, Sushmita; Yeung, Ching-Yan Chloé; Tellkamp, Frederik; Günschmann, Christian; Dieterich, Christoph; Yeroslaviz, Assa; Habermann, Bianca; Pombo, Ana; Niessen, Carien M; Wickström, Sara A

    2016-08-01

    Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

  19. ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing.

    PubMed

    Royo, Hélène; Prosser, Haydn; Ruzankina, Yaroslava; Mahadevaiah, Shantha K; Cloutier, Jeffrey M; Baumann, Marek; Fukuda, Tomoyuki; Höög, Christer; Tóth, Attila; de Rooij, Dirk G; Bradley, Allan; Brown, Eric J; Turner, James M A

    2013-07-01

    In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.

  20. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    SciTech Connect

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  1. 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.

  2. Immunotherapy against endocrine malignancies: immune checkpoint inhibitors lead the way.

    PubMed

    Cunha, Lucas Leite; Marcello, Marjory Alana; Rocha-Santos, Vinicius; Ward, Laura Sterian

    2017-09-11

    Immune checkpoint inhibitors are agents that act by inhibiting the mechanisms of immune escape displayed by various cancers. The success of immune checkpoint inhibitors against several tumors has promoted a new treatment strategy in clinical oncology, and this has encouraged physicians to increase the number of patients who receive the immune checkpoint therapy. In the present article, we review the main concepts regarding immune checkpoint mechanisms and how cancer disrupts them to undergo immune escape. In addition, we describe the most essential concepts related to immune checkpoint inhibitors. We critically review the literature on preclinical and clinical studies of the immune checkpoint inhibitors as a treatment option for thyroid cancer, ovarian carcinoma, pancreatic adenocarcinoma, adrenocortical carcinoma and neuroendocrine tumors. We present the challenges and the opportunities of using immune checkpoint inhibitors against these endocrine malignancies, highlighting the breakthroughs and pitfalls that have recently emerged.

  3. Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses

    PubMed Central

    Lakatos, Lóránt; Szittya, György; Silhavy, Dániel; Burgyán, József

    2004-01-01

    RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19–siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing. PMID:14976549

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

    SciTech Connect

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

    2014-01-01

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

  5. Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death

    PubMed Central

    Nick, Alpa M.; Stone, Rebecca L.; Armaiz-Pena, Guillermo; Ozpolat, Bulent; Tekedereli, Ibrahim; Graybill, Whitney S.; Landen, Charles N.; Villares, Gabriel; Vivas-Mejia, Pablo; Bottsford-Miller, Justin; Kim, Hye Sun; Lee, Ju-Seog; Kim, Soo Mi; Baggerly, Keith A.; Ram, Prahlad T.; Deavers, Michael T.; Coleman, Robert L.; Lopez-Berestein, Gabriel

    2011-01-01

    Background We investigated the clinical and biological significance of p130cas, an important cell signaling molecule, in ovarian carcinoma. Methods Expression of p130cas in ovarian tumors, as assessed by immunohistochemistry, was associated with tumor characteristics and patient survival. The effects of p130cas gene silencing with small interfering RNAs incorporated into neutral nanoliposomes (siRNA-DOPC), alone and in combination with docetaxel, on in vivo tumor growth and on tumor cell proliferation (proliferating cell nuclear antigen) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) were examined in mice bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) or taxane-resistant (HeyA8-MDR) ovarian tumors (n = 10 per group). To determine the specific mechanisms by which p130cas gene silencing abrogates tumor growth, we measured cell viability (MTT assay), apoptosis (fluorescence-activated cell sorting), autophagy (immunoblotting, fluorescence, and transmission electron microscopy), and cell signaling (immunoblotting) in vitro. All statistical tests were two-sided. Results Of 91 ovarian cancer specimens, 70 (76%) had high p130cas expression; and 21 (24%) had low p130cas expression. High p130cas expression was associated with advanced tumor stage (P < .001) and higher residual disease (>1 cm) following primary cytoreduction surgery (P = .007) and inversely associated with overall survival and progression-free survival (median overall survival: high p130cas expression vs low expression, 2.14 vs 9.1 years, difference = 6.96 years, 95% confidence interval = 1.69 to 9.48 years, P < .001; median progression-free survival: high p130cas expression vs low expression, 1.04 vs 2.13 years, difference = 1.09 years, 95% confidence interval = 0.47 to 2.60 years, P = .01). In mice bearing orthotopically implanted HeyA8 or SKOV3ip1 ovarian tumors, treatment with p130cas siRNA-DOPC in combination with docetaxel chemotherapy resulted in the greatest

  6. Why are sobriety checkpoints not widely adopted as an enforcement strategy in the United States?

    PubMed

    Fell, James C; Ferguson, Susan A; Williams, Allan F; Fields, Michele

    2003-11-01

    Sobriety checkpoints have been used by police in the United States for at least the past two decades to enforce impaired driving laws. Research has indicated that sobriety checkpoints are effective in reducing drinking and driving and alcohol-related fatal crashes. Despite this evidence, many police agencies have been unenthusiastic about using checkpoints. Information was collected from all 50 states plus the District of Columbia on the use of sobriety checkpoints. A total of 37 states and the District of Columbia reported conducting sobriety checkpoints at least once or twice during the year. Only 11 states reported that checkpoints were conducted on a weekly basis. Thirteen states do not conduct checkpoints either because of legal or policy issues. More detailed information was collected from five states that conduct checkpoints frequently and matched with information from five similar states that conduct checkpoints infrequently. States with frequent checkpoint programs had several common features such as program themes, support from task forces and citizen activist groups, use of a moderate number of police at the checkpoints, and use of all available funding mechanisms (federal, state, local) to support them. States with infrequent checkpoints claimed a lack of funding and police resources for not conducting more checkpoints, preferred saturation patrols over checkpoints because they were more "productive," and used large numbers of police officers at checkpoints. Ways to overcome perceived barriers to checkpoint use are discussed.

  7. On the robustness of SAC silencing in closed mitosis

    NASA Astrophysics Data System (ADS)

    Ruth, Donovan; Liu, Jian

    Mitosis equally partitions sister chromatids to two daughter cells. This is achieved by properly attaching these chromatids via their kinetochores to microtubules that emanate from the spindle poles. Once the last kinetochore is properly attached, the spindle microtubules pull the sister chromatids apart. Due to the dynamic nature of microtubules, however, kinetochore-microtubule attachment often goes wrong. When this erroneous attachment occurs, it locally activates an ensemble of proteins, called the spindle assembly checkpoint proteins (SAC), which halts the mitotic progression until all the kinetochores are properly attached by spindle microtubules. The timing of SAC silencing thus determines the fidelity of chromosome segregation. We previously established a spatiotemporal model that addresses the robustness of SAC silencing in open mitosis for the first time. Here, we focus on closed mitosis by examining yeast mitosis as a model system. Though much experimental work has been done to study the SAC in cells undergoing closed mitosis, the processes responsible are not well understood. We leverage and extend our previous model to study SAC silencing mechanism in closed mitosis. We show that a robust signal of the SAC protein accumulation at the spindle pole body can be achieved. This signal is a nonlinear increasing function of number of kinetochore-microtubule attachments, and can thus serve as a robust trigger to time the SAC silencing. Together, our mechanism provides a unified framework across species that ensures robust SAC silencing and fidelity of chromosome segregation in mitosis. Intramural research program in NHLBI at NIH.

  8. 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

  9. 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.

  10. RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops.

    PubMed

    Guo, Qigao; Liu, Qing; Smith, Neil A; Liang, Guolu; Wang, Ming-Bo

    2016-12-01

    Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3' UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops.

  11. RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops

    PubMed Central

    Guo, Qigao; Liu, Qing; Smith, Neil A.; Liang, Guolu; Wang, Ming-Bo

    2016-01-01

    Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3’ UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of these RNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops. PMID:28217004

  12. Part II-mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints.

    PubMed

    Aqil, Madeeha; Deliu, Zane; Elseth, Kim M; Shen, Grace; Xue, Jiaping; Radosevich, James A

    2014-03-01

    Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher

  13. Mechanism of SEMA3B gene silencing and clinical significance in glioma.

    PubMed

    Pang, C H; Du, W; Long, J; Song, L J

    2016-03-18

    The aim of the current study was to explore mechanisms of SEMA3B gene expression and its clinical significance in glioma, and provide a theoretical foundation for investigating individualized treatment in glioma. Paraffin-embedded tissues from 43 patients with a confirmed clinical diagnosis of glioma following neurosurgery at the First Affiliated Hospital of Zhengzhou University from December 2013 to April 2014 were selected randomly. An additional three normal brain tissues were obtained following encephalic decompression excision due to acute craniocerebral injury in the same period, which were used as the control group. Immunohistochemical staining for vascular endothelial growth factor was performed on the glioma tissues from the 43 patients. Genomic DNA was extracted for bisulfate conversion and sequencing. SEMA3B was fully expressed in the three normal brain tissues, and incompletely expressed in the 43 glioma tissues, with a lack of expression in 48.8% (21/43) of samples. Moreover, 58% of high-grade gliomas (grade III and IV) lacked SEMA3B expression, which was significantly more than those that lacked expression (20%) in low-grade gliomas (grade I and II), indicating that, as the clinical pathological grade increased, SEMA3B expression decreased. The occurrence and development of malignant tumors is a product of multiple genes and other factors. Here, we provide theoretical basis for glioma development and prognosis involving DNA-methylation driven silencing of SEMA3B, and thus, SEMA3B is a potential target for directed treatments against glioma.

  14. Hydrophobicity of methylated DNA as a possible mechanism for gene silencing

    NASA Astrophysics Data System (ADS)

    Kaur, Parminder; Plochberger, Birgit; Costa, Peter; Cope, Stephanie M.; Vaiana, Sara M.; Lindsay, Stuart

    2012-12-01

    AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged under water. Chromatin reconstituted on unmethylated DNA is less compacted and less sensitive to hydration. These differences must reflect changes in the physical properties of DNA on methylation, but prior studies have not revealed large differences between methylated and unmethylated DNA. Quasi-elastic light scattering studies of solutions of methylated and unmethylated DNA support this view. In contrast, AFM images of molecules at a water/solid interface yield a persistence length that nearly doubles (to 92.5 ± 4 nm) when 9% of the total DNA is methylated. This increase in persistence length is accompanied by a decrease in contour length, suggesting that a significant fraction of the meDNA changes into the stiffer A form as the more hydrophobic meDNA is dehydrated at the interface. This suggests a simple mechanism for gene silencing as the stiffer meDNA is more difficult to remove from nucleosomes.

  15. 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.

  16. 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.

  17. 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.

  18. Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction?

    PubMed

    Baylin, Stephen B; Ohm, Joyce E

    2006-02-01

    Chromatin alterations have been associated with all stages of tumour formation and progression. The best characterized are epigenetically mediated transcriptional-silencing events that are associated with increases in DNA methylation - particularly at promoter regions of genes that regulate important cell functions. Recent evidence indicates that epigenetic changes might 'addict' cancer cells to altered signal-transduction pathways during the early stages of tumour development. Dependence on these pathways for cell proliferation or survival allows them to acquire genetic mutations in the same pathways, providing the cell with selective advantages that promote tumour progression. Strategies to reverse epigenetic gene silencing might therefore be useful in cancer prevention and therapy.

  19. Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms

    PubMed Central

    Ochala, Julien; Gustafson, Ann-Marie; Diez, Monica Llano; Renaud, Guillaume; Li, Meishan; Aare, Sudhakar; Qaisar, Rizwan; Banduseela, Varuna C; Hedström, Yvette; Tang, Xiaorui; Dworkin, Barry; Ford, G Charles; Nair, K Sreekumaran; Perera, Sue; Gautel, Mathias; Larsson, Lars

    2011-01-01

    Abstract The muscle wasting and impaired muscle function in critically ill intensive care unit (ICU) patients delay recovery from the primary disease, and have debilitating consequences that can persist for years after hospital discharge. It is likely that, in addition to pernicious effects of the primary disease, the basic life support procedures of long-term ICU treatment contribute directly to the progressive impairment of muscle function. This study aims at improving our understanding of the mechanisms underlying muscle wasting in ICU patients by using a unique experimental rat ICU model where animals are mechanically ventilated, sedated and pharmacologically paralysed for duration varying between 6 h and 14 days. Results show that the ICU intervention induces a phenotype resembling the severe muscle wasting and paralysis associated with the acute quadriplegic myopathy (AQM) observed in ICU patients, i.e. a preferential loss of myosin, transcriptional down-regulation of myosin synthesis, muscle atrophy and a dramatic decrease in muscle fibre force generation capacity. Detailed analyses of protein degradation pathways show that the ubiquitin proteasome pathway is highly involved in this process. A sequential change in localisation of muscle-specific RING finger proteins 1/2 (MuRF1/2) observed during the experimental period is suggested to play an instrumental role in both transcriptional regulation and protein degradation. We propose that, for those critically ill patients who develop AQM, complete mechanical silencing, due to pharmacological paralysis or sedation, is a critical factor underlying the preferential loss of the molecular motor protein myosin that leads to impaired muscle function or persisting paralysis. PMID:21320889

  20. The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA

    PubMed Central

    Di Santo, Rachael; Aboulhouda, Soufiane; Weinberg, David E

    2016-01-01

    HAC1 encodes a transcription factor that is the central effector of the unfolded protein response (UPR) in budding yeast. When the UPR is inactive, HAC1 mRNA is stored as an unspliced isoform in the cytoplasm and no Hac1 protein is detectable. Intron removal is both necessary and sufficient to relieve the post-transcriptional silencing of HAC1 mRNA, yet the precise mechanism by which the intron prevents Hac1 protein accumulation has remained elusive. Here, we show that a combination of inhibited translation initiation and accelerated protein degradation—both dependent on the intron—prevents the accumulation of Hac1 protein when the UPR is inactive. Functionally, both components of this fail-safe silencing mechanism are required to prevent ectopic production of Hac1 protein and concomitant activation of the UPR. Our results provide a mechanistic understanding of HAC1 regulation and reveal a novel strategy for complete post-transcriptional silencing of a cytoplasmic mRNA. DOI: http://dx.doi.org/10.7554/eLife.20069.001 PMID:27692069

  1. Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons

    PubMed Central

    Shpiz, Sergey; Olovnikov, Ivan; Sergeeva, Anna; Lavrov, Sergey; Abramov, Yuri; Savitsky, Mikhail; Kalmykova, Alla

    2011-01-01

    In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline. PMID:21764773

  2. Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons.

    PubMed

    Shpiz, Sergey; Olovnikov, Ivan; Sergeeva, Anna; Lavrov, Sergey; Abramov, Yuri; Savitsky, Mikhail; Kalmykova, Alla

    2011-11-01

    In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline.

  3. Diverse gene-silencing mechanisms with distinct requirements for RNA polymerase subunits in Zea mays.

    PubMed

    Sloan, Amy E; Sidorenko, Lyudmila; McGinnis, Karen M

    2014-11-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes--mop1-1, Mop2-1, and mop3-1--suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation. Copyright © 2014 by the Genetics Society of America.

  4. Diverse Gene-Silencing Mechanisms with Distinct Requirements for RNA Polymerase Subunits in Zea mays

    PubMed Central

    Sloan, Amy E.; Sidorenko, Lyudmila; McGinnis, Karen M.

    2014-01-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes—mop1-1, Mop2-1, and mop3-1—suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation. PMID:25164883

  5. Silencing MaxiK Activity in Corporal Smooth Muscle Cells Initiates Compensatory Mechanisms to Maintain Calcium Homeostasis

    PubMed Central

    Calenda, Giulia; Suadicani, Sylvia Ottilie; Iglesias, Rodolfo; Spray, David Conover; Melman, Arnold; Davies, Kelvin Paul

    2010-01-01

    Introduction The MaxiK potassium channel is regulated by voltage and intracellular calcium, and plays a critical role in regulating intracellular calcium concentration ([Ca2+]i), which is the ultimate determinant of smooth muscle tone. Tight control of corpus cavernosum smooth muscle (CCSM) tone is critically important and misregulation can result in erectile dysfunction. Aim Because of the tight functional linkage of MaxiK and calcium channel activity, the aim of this study was to determine the effects of silencing and pharmacological inhibition of MaxiK on calcium homeostasis and intercellular calcium signaling in CCSM cells. Methods We compared changes in the basal intracellular [Ca2+]i and parameters defining intercellular calcium wave (ICW) spread in 48 hours MaxiK silenced CCSM cells vs. acute blockade of the channel with iberiotoxin. To analyze changes occurring in gene expression we performed micro-array analysis following MaxiK silencing for 48 hours. Main Outcome Measures Changes in Fura-2 fluorescence intensities were measured to evaluate basal [Ca2+]i levels and ICW parameters. Microarray analysis of global gene expression was performed. Results Forty-eight hours after MaxiK silencing the basal [Ca2+]i, the ICW amplitude and spread among CCSM cells were not markedly different in silenced compared to mock transfected controls, whereas short-term blockade significantly increased basal [Ca2+]i level and amplified Ca2+ signaling among CCSM cells. Micro-array analysis showed that several genes within Ca2+ homeostasis and smooth muscle tone regulation pathways had significantly altered expression. Conclusions Our results indicate that while short-term blockade of the MaxiK channel is associated with an increase in basal [Ca2+]i, Ca2+ homeostasis is restored during the 48 hours period following silencing. We hypothesize that the different pathways regulating [Ca2+]i and CCSM tone are linked through molecular crosstalk and that their coordinated regulation is

  6. A novel mechanism underlying the susceptibility of neuronal cells to nitric oxide: the occurrence and regulation of protein S-nitrosylation is the checkpoint.

    PubMed

    He, J; Wang, T; Wang, P; Han, P; Yin, Q; Chen, C

    2007-09-01

    The susceptibility of neuronal cells to nitric oxide (NO) is a key issue in NO-mediated neurotoxicity. However, the underlying mechanism remains unclear. As a cyclic guanosine monophosphate (cGMP)-independent NO signaling pathway, S-nitrosylation (or S-nitrosation) has been suggested to occur as a post-translational modification in parallel with O-phosphorylation. The underlying mechanism of the involvement of protein S-nitrosylation in the susceptibility of neuronal cells to NO has been little investigated. In this study, we focused on the role of S-nitrosothiols (RSNO) in the susceptibility of a cerebellar cell line R2 to NO. Our results showed the following: (i) S-nitrosoglutathione (GSNO) induced a burst of RSNO in GSH-depleted R2 cells, the majority of which were primarily contributed by the S-nitrosylation of proteins (Pro-SNOs), and was followed by severe neuronal necrosis; (ii) the elevation in the level of Pro-SNOs resulted from a dysfunction of S-nitroglutathione reductase (GSNOR) as a result of its substrate, GSNO, being unavailable in GSH-depleted cells. In the meantime, the suppression of GSNOR increased NO-mediated neurotoxicity in R2 cells, as well as in cerebellar granule neurons; (iii) Our results also demonstrate that the burst of RSNO is the "checkpoint" of cell fate: if RSNO can be reduced to free thiol proteins, cells will survive; if they are further oxidized, cells will die; and (iv) GSH-ethyl ester and Vitamin C protected R2 cells against GSNO neurotoxicity through two distinct mechanisms: by inhibiting the elevation of Pro-SNOs and by reducing Pro-SNOs to free thiol proteins, respectively. A novel mechanism underlying the susceptibility of neuronal cells to NO is proposed and some potential strategies to prevent the NO-mediated neurotoxicity are discussed.

  7. Chicken alpha-globin switching depends on autonomous silencing of the embryonic pi globin gene by epigenetics mechanisms.

    PubMed

    Rincón-Arano, Héctor; Guerrero, Georgina; Valdes-Quezada, Christian; Recillas-Targa, Félix

    2009-10-15

    Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

  8. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  9. 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.

  10. 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.

  11. Hypothyroidism in Cancer Patients on Immune Checkpoint Inhibitors with anti-PD1 Agents: Insights on Underlying Mechanisms.

    PubMed

    Alhusseini, M; Samantray, J

    2017-01-10

    Background: Immune therapy using monoclonal antibodies against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death 1 receptor (PD-1) for various cancers have been reported to cause thyroid dysfunction. Little is known, however, about the underlying pathogenic mechanisms and the course of hypothyroidism that subsequently develops. In this report, we use the change in thyroglobulin and thyroid antibody levels in patients on immune therapy who develop hypothyroidism to better understand its pathogenesis as well as examine the status of hypothyroidism in the long term. Methods: We report a case series of 10 patients who developed hypothyroidism after initiation of immune therapy (either anti-PD-1 alone or in combination with anti-CTLA-4). Available thyroid antibodies including anti-thyroglobulin (anti-Tg), anti-thyroid peroxidase (anti-TPO), and thyroid stimulating immunoglobulin (TSI) were noted during the initial thyroiditis phase as well as the hypothyroid phase. Persistence or remission of hypothyroidism was noted at 6 months. Summary: During the thyroiditis phase, 50% of the patients had elevated Tg titers, 40% had elevated anti-Tg, and 40% had elevated TSI. All of these titers decreased during the hypothyroid phase. Permanent hypothyroidism was noted in 80% of the cases. Conclusion: Hypothyroidism following initiation of immune therapy has immunologic and non-immunologic mediated mechanisms and is likely to be persistent.

  12. Biomarkers for Checkpoint Inhibition.

    PubMed

    Weber, Jeffrey S

    2017-01-01

    The identification of predictive biomarkers for the benefit of cancer immunotherapy is the holy grail of the burgeoning immunotherapy field. Recent work has shown that there are a core of concepts that establish the presence of an immune cell-infiltrate, an inflammatory signature of the tumor microenvironment, and the availability of target antigens defined by mutated neoantigens, as critical for the success of the checkpoint blockade. Genetic analyses have shown that resistance to PD-1 blockade, either innate or adaptive, may be due to existing or de novo mutations in signaling pathways critical for T-cell function in a modest proportion of cases. Major hurdles in the field that remain to be overcome are the difficulty of obtaining tumor biopsies for biomarker assessment, the heterogeneity of biomarker expression within tumors and within different tumors from the same patient, and the inducibility of some biomarkers by disease-related processes. Although assessment of peripheral blood or serum biomarkers would be ideal, few data suggest that they would reliably predict outcome with checkpoint blockade. Ultimately, some amalgamated biomarker that includes tumor and host factors will be required to predict which patients are likely to benefit from, or be resistant to, the effects of checkpoint inhibition.

  13. MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms

    PubMed Central

    Heair, Hannah M.; Kemper, Austin G.; Roy, Bhaskar; Lopes, Helena B.; Rashid, Harunur; Clarke, John C.; Afreen, Lubana K.; Ferraz, Emanuela P.; Kim, Eddy; Javed, Amjad; Beloti, Marcio M.; MacDougall, Mary

    2015-01-01

    Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. PMID:26124283

  14. A genome-wide analysis of the RNA-guided silencing pathway in coffee reveals insights into its regulatory mechanisms.

    PubMed

    Noronha Fernandes-Brum, Christiane; Marinho Rezende, Pâmela; Cherubino Ribeiro, Thales Henrique; Ricon de Oliveira, Raphael; Cunha de Sousa Cardoso, Thaís; Rodrigues do Amaral, Laurence; de Souza Gomes, Matheus; Chalfun-Junior, Antonio

    2017-01-01

    microRNAs (miRNAs) are derived from self-complementary hairpin structures, while small-interfering RNAs (siRNAs) are derived from double-stranded RNA (dsRNA) or hairpin precursors. The core mechanism of sRNA production involves DICER-like (DCL) in processing the smallRNAs (sRNAs) and ARGONAUTE (AGO) as effectors of silencing, and siRNA biogenesis also involves action of RNA-Dependent RNA Polymerase (RDR), Pol IV and Pol V in biogenesis. Several other proteins interact with the core proteins to guide sRNA biogenesis, action, and turnover. We aimed to unravel the components and functions of the RNA-guided silencing pathway in a non-model plant species of worldwide economic relevance. The sRNA-guided silencing complex members have been identified in the Coffea canephora genome, and they have been characterized at the structural, functional, and evolutionary levels by computational analyses. Eleven AGO proteins, nine DCL proteins (which include a DCL1-like protein that was not previously annotated), and eight RDR proteins were identified. Another 48 proteins implicated in smallRNA (sRNA) pathways were also identified. Furthermore, we identified 235 miRNA precursors and 317 mature miRNAs from 113 MIR families, and we characterized ccp-MIR156, ccp-MIR172, and ccp-MIR390. Target prediction and gene ontology analyses of 2239 putative targets showed that significant pathways in coffee are targeted by miRNAs. We provide evidence of the expansion of the loci related to sRNA pathways, insights into the activities of these proteins by domain and catalytic site analyses, and gene expression analysis. The number of MIR loci and their targeted pathways highlight the importance of miRNAs in coffee. We identified several roles of sRNAs in C. canephora, which offers substantial insight into better understanding the transcriptional and post-transcriptional regulation of this major crop.

  15. 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

  16. Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing.

    PubMed

    TenHarmsel, A; Austin, R J; Savenelli, N; Biggin, M D

    1993-05-01

    In this study, we examined how the Drosophila developmental control gene even-skipped (eve) represses transcription. Tissue culture cells were used to show that eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Different portions of eve were fused to a heterologous DNA binding domain to show that three adjacent regions of eve contribute to silencing. There appear to be two mechanisms by which eve protein represses transcription. In this study, we used in vitro transcription and DNA binding experiments to provide evidence for one of these mechanisms. Repression in vitro correlates with binding of eve protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other eve molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA, and the data suggest that this intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the zeste transcription factor. Binding of eve protein is shown to exclude binding by zeste protein. These data suggest a mechanism for silencing whereby a repressor protein would be targeted to DNA by a high-affinity element, which itself does not overlap activator elements. Cooperative binding of further repressor molecules to distant low-affinity sites, and competition with activators bound at these sites lead to repression at a distance.

  17. Histone methyltransferases MES-4 and MET-1 promote meiotic checkpoint activation in Caenorhabditis elegans.

    PubMed

    Lamelza, Piero; Bhalla, Needhi

    2012-01-01

    Chromosomes that fail to synapse during meiosis become enriched for chromatin marks associated with heterochromatin assembly. This response, called meiotic silencing of unsynapsed or unpaired chromatin (MSUC), is conserved from fungi to mammals. In Caenorhabditis elegans, unsynapsed chromosomes also activate a meiotic checkpoint that monitors synapsis. The synapsis checkpoint signal is dependent on cis-acting loci called Pairing Centers (PCs). How PCs signal to activate the synapsis checkpoint is currently unknown. We show that a chromosomal duplication with PC activity is sufficient to activate the synapsis checkpoint and that it undergoes heterochromatin assembly less readily than a duplication of a non-PC region, suggesting that the chromatin state of these loci is important for checkpoint function. Consistent with this hypothesis, MES-4 and MET-1, chromatin-modifying enzymes associated with transcriptional activity, are required for the synapsis checkpoint. In addition, a duplication with PC activity undergoes heterochromatin assembly when mes-4 activity is reduced. MES-4 function is required specifically for the X chromosome, while MES-4 and MET-1 act redundantly to monitor autosomal synapsis. We propose that MES-4 and MET-1 antagonize heterochromatin assembly at PCs of unsynapsed chromosomes by promoting a transcriptionally permissive chromatin environment that is required for meiotic checkpoint function. Moreover, we suggest that different genetic requirements to monitor the behavior of sex chromosomes and autosomes allow for the lone unsynapsed X present in male germlines to be shielded from inappropriate checkpoint activation.

  18. Histone Methyltransferases MES-4 and MET-1 Promote Meiotic Checkpoint Activation in Caenorhabditis elegans

    PubMed Central

    Lamelza, Piero; Bhalla, Needhi

    2012-01-01

    Chromosomes that fail to synapse during meiosis become enriched for chromatin marks associated with heterochromatin assembly. This response, called meiotic silencing of unsynapsed or unpaired chromatin (MSUC), is conserved from fungi to mammals. In Caenorhabditis elegans, unsynapsed chromosomes also activate a meiotic checkpoint that monitors synapsis. The synapsis checkpoint signal is dependent on cis-acting loci called Pairing Centers (PCs). How PCs signal to activate the synapsis checkpoint is currently unknown. We show that a chromosomal duplication with PC activity is sufficient to activate the synapsis checkpoint and that it undergoes heterochromatin assembly less readily than a duplication of a non-PC region, suggesting that the chromatin state of these loci is important for checkpoint function. Consistent with this hypothesis, MES-4 and MET-1, chromatin-modifying enzymes associated with transcriptional activity, are required for the synapsis checkpoint. In addition, a duplication with PC activity undergoes heterochromatin assembly when mes-4 activity is reduced. MES-4 function is required specifically for the X chromosome, while MES-4 and MET-1 act redundantly to monitor autosomal synapsis. We propose that MES-4 and MET-1 antagonize heterochromatin assembly at PCs of unsynapsed chromosomes by promoting a transcriptionally permissive chromatin environment that is required for meiotic checkpoint function. Moreover, we suggest that different genetic requirements to monitor the behavior of sex chromosomes and autosomes allow for the lone unsynapsed X present in male germlines to be shielded from inappropriate checkpoint activation. PMID:23166523

  19. 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.

  20. 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.

  1. C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms.

    PubMed

    Duan, Guowen; Saint, Robert B; Helliwell, Chris A; Behm, Carolyn A; Wang, Ming-Bo; Waterhouse, Peter M; Gordon, Karl H J

    2013-04-01

    Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.

  2. 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

  3. Epigenetic silencing in transgenic plants

    PubMed Central

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. PMID:26442010

  4. Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco.

    PubMed

    Alamillo, Josefa M; Saénz, Pilar; García, Juan Antonio

    2006-10-01

    Plum pox virus (PPV) is able to replicate in inoculated leaves of Nicotiana tabacum, but is defective in systemic movement in this host. However, PPV produces a systemic infection in transgenic tobacco expressing the silencing suppressor P1/HC-Pro from tobacco etch virus (TEV). In this work we show that PPV is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (NahG) that degrades salicylic acid (SA). Replication and accumulation of PPV is higher in the locally infected leaves of plants deficient in SA or expressing TEV P1/HC-Pro silencing suppressor. Accumulation of viral derived small RNAs was reduced in the NahG transgenic plants, suggesting that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco. Besides, expression of SA-mediated defense transcripts, such as those of pathogenesis-related (PR) proteins PR-1 and PR-2 or alternative oxidase-1, as well as that of the putative RNA-dependent RNA polymerase NtRDR1, is induced in response to PPV infection, and the expression patterns of these defense transcripts are altered in the TEV P1/HC-Pro transgenic plants. Long-distance movement of PPV is highly enhanced in NahG x P1/HC-Pro double-transgenic plants and systemic symptoms in these plants reveal that the expression of an RNA-silencing suppressor and the lack of SA produce additive but distinct effects. Our results suggest that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco, and that silencing suppressors, such as P1/HC-Pro, also alter the SA-mediated defense. Both an RNA-silencing and an SA-mediated defense mechanism could act together to limit PPV infection.

  5. Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924.

    PubMed

    Blank, Jonathan L; Liu, Xiaozhen J; Cosmopoulos, Katherine; Bouck, David C; Garcia, Khristofer; Bernard, Hugues; Tayber, Olga; Hather, Greg; Liu, Ray; Narayanan, Usha; Milhollen, Michael A; Lightcap, Eric S

    2013-01-01

    MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

  6. Requirements for Linux Checkpoint/Restart

    SciTech Connect

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

    2002-02-26

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

  7. 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

  8. 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.

  9. 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

  10. Prospect for immune checkpoint blockade: dynamic and comprehensive monitorings pave the way.

    PubMed

    Wang, Weili; Liu, Jie; He, Yijing; McLeod, Howard L

    2017-08-01

    Immune checkpoint blockade, which releases the brake of the immune system to enhance anticancer immune response, stands out in the cancer immunotherapy field due to their remarkable and long-lasting effect. However, the overall response rate for currently approved immune checkpoint inhibitors is only about 10-40%. We have summarized three major components, which are the presence of checkpoints, the immune-activation mechanism and the immune-inhibitory mechanism, containing six factors to describe the cancer-immune interaction dynamically and comprehensively, which shed light on promising biomarkers in immune checkpoint therapy.

  11. Methylthioadenosine phosphorylase gene is silenced by promoter hypermethylation in human lymphoma cell line DHL-9: another mechanism of enzyme deficiency.

    PubMed

    Ishii, Masaaki; Nakazawa, Keiko; Wada, Hideo; Nishioka, Junji; Nakatani, Kaname; Yamada, Yasuaki; Kamihira, Shimeru; Kusunoki, Masato; Nobori, Tsutomu

    2005-04-01

    Methylthioadenosine phosphorylase (MTAP) involved in the metabolism of purine and polyamine has been known to be deficient in a variety of tumors. Although this enzyme deficiency was reportedly caused by partial or total deletion of the MTAP gene, human MTAP-deficient lymphoma cell line DHL-9 has the intact MTAP gene. In order to determine the mechanism of MTAP deficiency in DHL-9, we carried out methylation-specific PCR analysis of sodium bisulfite-treated genomic DNA followed by DNA sequence analysis. Following incubation with various concentrations of 5-Aza-2'-deoxycytidine, DHL-9 cells were subjected to RT-PCR and an immunoblot analysis for MTAP expression. MTAP promoter in DHL-9 cells was methylated at cytosine of all CpG dinucleotides analyzed. Moreover, 5-Aza-2'-deoxycytidine treatment induced DHL-9 cells to express MTAP mRNA and protein. Taken together, MTAP deficiency in DHL-9 was caused by transcriptional silencing due to promoter methylation. Promoter methylation of the MTAP gene was also found in DNA samples from adult T-cell leukemia patients. These results indicated that promoter hypermethylation is another mechanism of MTAP deficiency in human malignancy. Thus, immunological diagnostics will be needed for an accurate evaluation of MTAP expression at the protein level.

  12. [Reversal of resistance to adriamycin in human breast cancer cell line MCF-7/ADM by silencing AEG-1 gene and its mechanism].

    PubMed

    Yuan, Lei; Shi, Ran-Ran; Rao, Shu-Mei; Song, Jin-Ling; Cui, Ming-Chen

    2014-10-25

    The aim of this study was to investigate the effects of AEG-1 gene silencing on the chemoresistance of human breast cancer cell line MCF-7/ADM and its possible mechanism. MCF-7/ADM cells were incubated in the medium containing adriamycin (ADM). The recombinant pLKO.1-shAEG-1 plasmid was constructed to silence AEG-1 expression in human breast cancer MCF-7/ADM cells. MTT assay was employed to detect the anti-tumor effect of ADM on MCF-7/ADM cells, and IC50 value of ADM was calculated according to MTT. Flow cytometry was used to determine the apoptosis. Western blot was used to analyze the expression levels of AEG-1, p-Akt, p-MDM2, p-Bad, p53 and MDR1. The result showed MCF-7/ADM had a significantly higher expression level of AEG-1 compared with that of MCF-7 (P < 0.05), however, the expression of AEG-1 was decreased after AEG-1 gene silencing. The IC50 value of ADM in shAEG-1 group was significantly lower than that in shcontrol group. AEG-1 gene silencing induced cell apoptosis and enhanced the pro-apoptotic effect of ADM on MCF-7/ADM cells. After AEG-1 gene silencing, the phosphorylation of Akt, MDM2 and Bad was inhibited (P < 0.05), the protein levels of p53 and MDR1 were up-regulated (P < 0.05) and down-regulated (P < 0.05) respectively, compared with control. In conclusion, the results suggest that AEG-1 gene silencing can reverse the ADM resistance in human breast cancer cell line MCF-7/ADM by means of inducing apoptosis and down-regulating the protein level of MDR1.

  13. Combined Radiation Therapy and Immune Checkpoint Blockade Therapy for Breast Cancer.

    PubMed

    Hu, Zishuo I; Ho, Alice Y; McArthur, Heather L

    2017-09-01

    Treatment with checkpoint inhibitors has shown durable responses in a number of solid tumors, including melanoma, lung, and renal cell carcinoma. However, most breast cancers are resistant to monotherapy with checkpoint inhibitors. Radiation therapy (RT) has been shown to have a number of immunostimulatory effects, including priming the immune system, recruiting immune cells to the tumor environment, and altering the immunosuppressive effects of the tumor microenvironment. RT therefore represents a promising adjuvant therapy to checkpoint blockade in breast cancer. We review the data from the checkpoint blockade studies on breast cancer reported to date, the mechanisms by which RT potentiates immune responses, the preclinical and clinical data of checkpoint blockade and RT combinations, and the landscape of current clinical trials of RT and immune checkpoint inhibitor combinations in breast cancer. Clinical trials with checkpoint blockade therapy have demonstrated response rates of up to 19% in breast cancer, and many of the responses are durable. Preclinical data indicate that RT combined with checkpoint inhibition synergizes not only to enhance antitumor efficacy but also to induce responses outside of the radiation field. Thus multiple clinical trials are currently investigating the combination of checkpoint inhibition with RT. The use of combination strategies that incorporate chemotherapy and/or local strategies such as RT may be needed to augment responses to immune therapy in breast cancer. Preclinical and clinical results show that RT in combination with checkpoint blockade may be a promising therapeutic option in breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

    PubMed Central

    Wang, Sidney H.; Elgin, Sarah C. R.

    2011-01-01

    Transposon control is a critical process during reproduction. The PIWI family proteins can play a key role, using a piRNA-mediated slicing mechanism to suppress transposon activity posttranscriptionally. In Drosophila melanogaster, Piwi is predominantly localized in the nucleus and has been implicated in heterochromatin formation. Here, we use female germ-line–specific depletion to study Piwi function. This depletion of Piwi leads to infertility and to axis specification defects in the developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Germ-line Piwi does not appear to be required for piRNA production. Instead, Piwi requires Aubergine (and presumably secondary piRNA) for proper localization. A subset of transposons that show significant overexpression in germ-line Piwi-depleted ovaries exhibit a corresponding loss of HP1a and H3K9me2. Germ-line HP1a depletion also leads to a loss of transposon silencing, demonstrating the functional requirement for HP1a enrichment at these loci. Considering our results and those of others, we infer that germ-line Piwi functions downstream of piRNA production to promote silencing of some transposons via recruitment of HP1a. Thus, in addition to its better-known function in posttranscriptional silencing, piRNA also appears to function in a targeting mechanism for heterochromatin formation mediated by Piwi. PMID:22160707

  15. Checkpoint immunotherapy in head and neck cancers.

    PubMed

    Zolkind, Paul; Uppaluri, Ravindra

    2017-08-23

    Checkpoint inhibitors have recently gained FDA approval for the treatment of cisplatin-resistant recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) by outperforming standard of care chemotherapy and inducing durable responses in a subset of patients. These monoclonal antibodies unleash the patient's own immune system to target cancer cells. HNSCC is a good target for these agents as there is ample evidence of active immunosurveillance in the head and neck and a number of immune evasion mechanisms by which HNSCCs form progressive disease including via the PD-1/PD-L1 axis. As HNSCCs typically possess a moderately high mutation burden, they should express numerous mutation-derived antigen targets for immune detection. However, with response rates less than 20% in clinical trials, there is a need for biomarkers to screen patients as well as clinical trials evaluating novel combinations to improve outcomes. The aim of this review is to provide historical and mechanistic context for the use of checkpoint inhibitors in head and neck cancer and provide a perspective on the role of novel checkpoints, biomarkers, and combination therapies that are evolving in the near term for patients with HNSCC.

  16. A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo.

    PubMed

    Encalada, Sandra E; Willis, John; Lyczak, Rebecca; Bowerman, Bruce

    2005-03-01

    During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

  17. Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

    PubMed

    Ugarte-Uribe, Begoña; Grijalvo, Santiago; Pertíñez, Samuel Núñez; Busto, Jon V; Martín, César; Alagia, Adele; Goñi, Félix M; Eritja, Ramón; Alkorta, Itziar

    2017-01-01

    The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

  18. Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats.

    PubMed

    Starlard-Davenport, Athena; Tryndyak, Volodymyr P; James, Smitha R; Karpf, Adam R; Latendresse, John R; Beland, Frederick A; Pogribny, Igor P

    2010-03-01

    Breast cancer, the most common malignancy in women, emerges through a multistep process, encompassing the progressive sequential evolution of morphologically distinct stages from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma and metastasis. The success of treatment of breast cancer could be greatly improved by the detection at early stages of cancer. In the present study, we investigated the underlying molecular mechanisms involved in breast carcinogenesis in Augustus and Copenhagen-Irish female rats, a cross between the ACI strains, induced by continuous exposure to 17beta-estradiol. The results of our study demonstrate that early stages of estrogen-induced breast carcinogenesis are characterized by altered global DNA methylation, aberrant expression of proteins responsible for the proper maintenance of DNA methylation pattern and epigenetic silencing of the critical Rassf1a (Ras-association domain family 1, isoform A) tumor suppressor gene. Interestingly, transcriptional repression of the Rassf1a gene in mammary glands during early stages of breast carcinogenesis was associated with an increase in trimethylation of histones H3 lysine 9 and H3 lysine 27 and de novo CpG island methylation and at the Rassf1a promoter and first exon. In conclusion, we demonstrate that epigenetic alterations precede formation of preneoplastic lesions indicating the significance of epigenetic events in induction of oncogenic pathways in early stages of carcinogenesis.

  19. 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.

  20. Lazy checkpoint coordination for bounding rollback propagation

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. Kent

    1992-01-01

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

  1. Lazy checkpoint coordination for bounding rollback propagation

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  2. RARβ Promoter Methylation as an Epigenetic Mechanism of Gene Silencing in Non-small Cell Lung Cancer.

    PubMed

    Dutkowska, A; Antczak, A; Pastuszak-Lewandoska, D; Migdalska-Sek, M; Czarnecka, K H; Górski, P; Kordiak, J; Nawrot, E; Brzeziańska-Lasota, E

    2016-01-01

    The retinoid acid receptor-p (RARβ) gene is one of the tumor suppressor genes (TSGs), which is frequently deleted or epigenetically silenced at an early stage of tumor progression. In this study we investigated the promoter methylation and expression status of the RARβ gene in 60 surgically resected non-small cell lung cancer (NSCLC) tissue samples and 60 corresponding unchanged lung tissue samples, using methylation-specific PCR and real-time-polymerase chain reaction (qPCR) techniques. We correlated the results with the pathological features of tumors and clinical characteristics of patients. qPCR analysis detected a significantly lower RARβ expression in the patients with adenocarcinoma (AC) and large cell carcinoma (LCC) than in those with squamous cell carcinoma (SCC) (AC vs. SCC, p = 0.032; AC and LCC vs. SCC, p = 0.0 13). Additionally, significantly lower expression of the RARβ gene was revealed in the patients with non-squamous cell cancer with a history of smoking assessed as pack-years (PY < 40 vs. PY ≥ 40, p = 0.045). Regarding RARβ promoter methylation, we found significant differences in the methylation index in the SCC group when considering pTNM staging; with higher index values in T1a + T1b compared with T2a + T2b and T3 + T4 groups (p = 0.024). There was no correlation between the methylation status and expression level of the RARβ gene, which suggests that other molecular mechanisms influence the RARβ expression in NSCLC patients. In conclusion, different expression of the RARβ gene in SCC and NSCC makes the RARβ gene a valuable diagnostic marker for differentiating the NSCLC subtypes.

  3. 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.

  4. Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

    PubMed Central

    Wood, Matthew D

    2010-01-01

    The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. the checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. the ira1Δ ira2Δ recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery and implicates the Ras signaling pathway as an important regulator of mitotic events. PMID:20716966

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

    PubMed Central

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

    2015-01-01

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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Mathematical model of the morphogenesis checkpoint in budding yeast

    PubMed Central

    Ciliberto, Andrea; Novak, Bela; Tyson, John J.

    2003-01-01

    The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next. PMID:14691135

  12. Mathematical model of the morphogenesis checkpoint in budding yeast.

    PubMed

    Ciliberto, Andrea; Novak, Bela; Tyson, John J

    2003-12-22

    The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.

  13. 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.

  14. Viral manipulation of DNA repair and cell cycle checkpoints

    PubMed Central

    Chaurushiya, Mira S.; Weitzman, Matthew D.

    2009-01-01

    Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are exquisitely coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation. PMID:19473887

  15. Do checkpoint inhibitors rely on gut microbiota to fight cancer?

    PubMed

    Firwana, Belal; Avaritt, Nathan; Shields, Bradley; Ravilla, Rahul; Makhoul, Issam; Hutchins, Laura; Tackett, Alan J; Mahmoud, Fade

    2017-01-01

    The field of gut microbiota is of growing interest, especially in the recent discoveries of its interaction with host immune responses, which when disrupted, can further alter immunity. It also plays a role in cancer development, its microenvironment and response to anticancer therapeutics. Several recently published experimental studies had explored the efficacy of modifying microbiota to enhance the response of checkpoint inhibitors, suggesting its beneficial function in cancer management and potential to be targeted as a therapeutic agent to enhance efficacy of checkpoint inhibitors. Here we review available evidence, mechanisms and hypotheses of its use to enhance cancer response.

  16. Non-volatile memory for checkpoint storage

    SciTech Connect

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

    2014-07-22

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

  17. 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.

  18. MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.

    PubMed

    Ichijima, Yosuke; Ichijima, Misako; Lou, Zhenkun; Nussenzweig, André; Camerini-Otero, R Daniel; Chen, Junjie; Andreassen, Paul R; Namekawa, Satoshi H

    2011-05-01

    Chromosome-wide inactivation is an epigenetic signature of sex chromosomes. The mechanism by which the chromosome-wide domain is recognized and gene silencing is induced remains unclear. Here we identify an essential mechanism underlying the recognition of the chromosome-wide domain in the male germline. We show that mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX), defines the chromosome-wide domain, initiates meiotic sex chromosome inactivation (MSCI), and leads to XY body formation. Importantly, MSCI consists of two genetically separable steps. The first step is the MDC1-independent recognition of the unsynapsed axis by DNA damage response (DDR) factors such as ataxia telangiectasia and Rad3-related (ATR), TOPBP1, and γH2AX. The second step is the MDC1-dependent chromosome-wide spreading of DDR factors to the entire chromatin. Furthermore, we demonstrate that, in somatic cells, MDC1-dependent amplification of the γH2AX signal occurs following replicative stress and is associated with transcriptional silencing. We propose that a common DDR pathway underlies both MSCI and the response of somatic cells to replicative stress. These results establish that the DDR pathway centered on MDC1 triggers epigenetic silencing of sex chromosomes in germ cells.

  19. Antiviral silencing in animals.

    PubMed

    Li, Hong-Wei; Ding, Shou-Wei

    2005-10-31

    RNA silencing or RNA interference (RNAi) refers to the small RNA-guided gene silencing mechanism conserved in a wide range of eukaryotic organisms from plants to mammals. As part of this special issue on the biology, mechanisms and applications of RNAi, here we review the recent advances on defining a role of RNAi in the responses of invertebrate and vertebrate animals to virus infection. Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been identified. Assays used for the identification of viral suppressors and possible biological functions of both viral miRNAs and suppressors are discussed. We propose that herpes viral miRNAs may act as specificity factors to initiate heterochromatin assembly of the latent viral DNA genome in the nucleus.

  20. Switching of dominant retrotransposon silencing strategies from posttranscriptional to transcriptional mechanisms during male germ-cell development in mice

    PubMed Central

    Inoue, Kota; Fukuda, Kei; Sasaki, Hiroyuki

    2017-01-01

    Mammalian genomes harbor millions of retrotransposon copies, some of which are transpositionally active. In mouse prospermatogonia, PIWI-interacting small RNAs (piRNAs) combat retrotransposon activity to maintain the genomic integrity. The piRNA system destroys retrotransposon-derived RNAs and guides de novo DNA methylation at some retrotransposon promoters. However, it remains unclear whether DNA methylation contributes to retrotransposon silencing in prospermatogonia. We have performed comprehensive studies of DNA methylation and polyA(+) RNAs (transcriptome) in developing male germ cells from Pld6/Mitopld and Dnmt3l knockout mice, which are defective in piRNA biogenesis and de novo DNA methylation, respectively. The Dnmt3l mutation greatly reduced DNA methylation levels at most retrotransposons, but its impact on their RNA abundance was limited in prospermatogonia. In Pld6 mutant germ cells, although only a few retrotransposons exhibited reduced DNA methylation, many showed increased expression at the RNA level. More detailed analysis of RNA sequencing, nascent RNA quantification, profiling of cleaved RNA ends, and the results obtained from double knockout mice suggest that PLD6 works mainly at the posttranscriptional level. The increase in retrotransposon expression was larger in Pld6 mutants than it was in Dnmt3l mutants, suggesting that RNA degradation by the piRNA system plays a more important role than does DNA methylation in prospermatogonia. However, DNA methylation had a long-term effect: hypomethylation caused by the Pld6 or Dnmt3l mutation resulted in increased retrotransposon expression in meiotic spermatocytes. Thus, posttranscriptional silencing plays an important role in the early stage of germ cell development, then transcriptional silencing becomes important in later stages. In addition, intergenic and intronic retrotransposon sequences, in particular those containing the antisense L1 promoters, drove ectopic expression of nearby genes in both

  1. RNA silencing and plant viral diseases.

    PubMed

    Wang, Ming-Bo; Masuta, Chikara; Smith, Neil A; Shimura, Hanako

    2012-10-01

    RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.

  2. 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.

  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. 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.

  5. 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.

  6. Advances in plant gene silencing methods.

    PubMed

    Pandey, Prachi; Senthil-Kumar, Muthappa; Mysore, Kirankumar S

    2015-01-01

    Understanding molecular mechanisms of transcriptional and posttranscriptional gene silencing pathways in plants over the past decades has led to development of tools and methods for silencing a target gene in various plant species. In this review chapter, both the recent understanding of molecular basis of gene silencing pathways and advances in various widely used gene silencing methods are compiled. We also discuss the salient features of the different methods like RNA interference (RNAi) and virus-induced gene silencing (VIGS) and highlight their advantages and disadvantages. Gene silencing technology is constantly progressing as reflected by rapidly emerging new methods. A succinct discussion on the recently developed methods like microRNA-mediated virus-induced gene silencing (MIR-VIGS) and microRNA-induced gene silencing (MIGS) is also provided. One major bottleneck in gene silencing approaches has been the associated off-target silencing. The other hurdle has been the lack of a universal approach that can be applied to all plants. For example, we face hurdles like incompatibility of VIGS vectors with the host and inability to use MIGS for plant species which are not easily transformable. However, the overwhelming research in this direction reflects the scope for overcoming the short comings of gene silencing technology.

  7. Reducing space overhead for independendent checkpointing

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  8. Don't forget the lyrics! Spatiotemporal dynamics of neural mechanisms spontaneously evoked by gaps of silence in familiar and newly learned songs.

    PubMed

    Gabriel, Damien; Wong, Thian Chiew; Nicolier, Magali; Giustiniani, Julie; Mignot, Coralie; Noiret, Nicolas; Monnin, Julie; Magnin, Eloi; Pazart, Lionel; Moulin, Thierry; Haffen, Emmanuel; Vandel, Pierre

    2016-07-01

    The vast majority of people experience musical imagery, the sensation of reliving a song in absence of any external stimulation. Internal perception of a song can be deliberate and effortful, but also may occur involuntarily and spontaneously. Moreover, musical imagery is also involuntarily used for automatically completing missing parts of music or lyrics from a familiar song. The aim of our study was to explore the onset of musical imagery dynamics that leads to the automatic completion of missing lyrics. High-density electroencephalography was used to record the cerebral activity of twenty healthy volunteers while they were passively listening to unfamiliar songs, very familiar songs, and songs previously listened to for two weeks. Silent gaps inserted into these songs elicited a series of neural activations encompassing perceptual, attentional and cognitive mechanisms (range 100-500ms). Familiarity and learning effects emerged as early as 100ms and lasted 400ms after silence occurred. Although participants reported more easily mentally imagining lyrics in familiar rather than passively learnt songs, the onset of neural mechanisms and the power spectrum underlying musical imagery were similar for both types of songs. This study offers new insights into the musical imagery dynamics evoked by gaps of silence and on the role of familiarity and learning processes in the generation of these dynamics. The automatic and effortless method presented here is a potentially useful tool to understand failure in the familiarity and learning processes of pathological populations. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. 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.

  10. RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa.

    PubMed

    Chicas, Agustin; Cogoni, Carlo; Macino, Giuseppe

    2004-01-01

    RNA interference (RNAi) can silence genes at the transcriptional level by targeting locus-specific Lys9H3 methylation or at the post-transcriptional level by targeting mRNA degradation. Here we have cloned and sequenced genomic regions methylated in Lys9H3 in Neurospora crassa to test the requirements for components of the RNAi pathway in this modification. We find that 90% of clones map to repeated sequences and relics of transposons that have undergone repeat-induced point mutations (RIP). We find siRNAs derived from transposon relics indicating that the RNAi machinery targets these regions. This is confirmed by the fact that the presence of these siRNAs depends on components of the RNAi pathway such as the RdRP (QDE-1), the putative RecQ helicase (QDE-3) and the two Dicer enzymes. We show that Lys9H3 methylation of RIP sequences is not affected in mutants of the RNAi pathway indicating that the RNAi machinery is not involved in transcriptional gene silencing in Neurospora. We find that RIP regions are transcribed and that the transcript level increases in the mutants of the RNAi pathway. These data suggest that the biological function of the Neurospora RNAi machinery is to control transposon relics and repeated sequences by targeting degradation of transcripts derived from these regions.

  11. RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa

    PubMed Central

    Chicas, Agustin; Cogoni, Carlo; Macino, Giuseppe

    2004-01-01

    RNA interference (RNAi) can silence genes at the transcriptional level by targeting locus-specific Lys9H3 methylation or at the post-transcriptional level by targeting mRNA degradation. Here we have cloned and sequenced genomic regions methylated in Lys9H3 in Neurospora crassa to test the requirements for components of the RNAi pathway in this modification. We find that 90% of clones map to repeated sequences and relics of transposons that have undergone repeat-induced point mutations (RIP). We find siRNAs derived from transposon relics indicating that the RNAi machinery targets these regions. This is confirmed by the fact that the presence of these siRNAs depends on components of the RNAi pathway such as the RdRP (QDE-1), the putative RecQ helicase (QDE-3) and the two Dicer enzymes. We show that Lys9H3 methylation of RIP sequences is not affected in mutants of the RNAi pathway indicating that the RNAi machinery is not involved in transcriptional gene silencing in Neurospora. We find that RIP regions are transcribed and that the transcript level increases in the mutants of the RNAi pathway. These data suggest that the biological function of the Neurospora RNAi machinery is to control transposon relics and repeated sequences by targeting degradation of transcripts derived from these regions. PMID:15302921

  12. Practising Silence in Teaching

    ERIC Educational Resources Information Center

    Forrest, Michelle

    2013-01-01

    The concept "silence" has diametrically opposed meanings; it connotes peace and contemplation as well as death and oblivion. Silence can also be considered a practice. There is keeping the rule of silence to still the mind and find inner truth, as well as forcibly silencing in the sense of subjugating another to one's own purposes.…

  13. Practising Silence in Teaching

    ERIC Educational Resources Information Center

    Forrest, Michelle

    2013-01-01

    The concept "silence" has diametrically opposed meanings; it connotes peace and contemplation as well as death and oblivion. Silence can also be considered a practice. There is keeping the rule of silence to still the mind and find inner truth, as well as forcibly silencing in the sense of subjugating another to one's own purposes.…

  14. 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.

  15. The Dominant Inhibitory Chalcone Synthase Allele C2-Idf (Inhibitor diffuse) From Zea mays (L.) Acts via an Endogenous RNA Silencing Mechanism

    PubMed Central

    Della Vedova, Chris B.; Lorbiecke, René; Kirsch, Helene; Schulte, Michael B.; Scheets, Kay; Borchert, Lutz M.; Scheffler, Brian E.; Wienand, Udo; Cone, Karen C.; Birchler, James A.

    2005-01-01

    The flavonoid pigment pathway in plants has been used as a model system for studying gene regulatory mechanisms. C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in this biosynthetic pathway of maize. Homozygous C2-Idf plants show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. To explore the nature of this effect, the C2-Idf allele was cloned. The gene structure of the C2-Idf haplotype differs substantially from that of the normal c2 gene in that three copies are present. Two of these are located in close proximity to each other in a head-to-head orientation and the third is closely linked. Previous experiments showed that the lower level of pigmentation in heterozygotes is correlated with reduced enzyme activity and low steady-state mRNA levels. We found that c2 transcription occurs in nuclei of C2-Idf/C2 heterozygotes, but mRNA does not accumulate, suggesting that the inhibition is mediated by RNA silencing. Infection of C2-Idf/C2 heterozygotes with viruses that carry suppressors of RNA silencing relieved the phenotypic inhibition, restoring pigment production and mRNA levels. Finally, we detected small interfering RNAs (siRNAs) in plants carrying C2-Idf, but not in plants homozygous for the wild-type C2 allele. Together, our results indicate that the inhibitory effect of C2-Idf occurs through RNA silencing. PMID:15956664

  16. 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

  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. Modeling dual pathways for the metazoan spindle assembly checkpoint

    PubMed Central

    Sear, Richard P.; Howard, Martin

    2006-01-01

    Using computational modeling, we investigate mechanisms of signal transduction. We focus on the spindle assembly checkpoint, where a single unattached kinetochore is able to signal to prevent cell cycle progression. The inhibitory signal switches off rapidly once spindle microtubules have attached to all kinetochores. This requirement tightly constrains the possible mechanisms. Here we investigate two possible mechanisms for spindle checkpoint operation in metazoan cells, both supported by recent experiments. The first involves the free diffusion and sequestration of cell cycle regulators. This mechanism is severely constrained both by experimental fluorescence recovery data and by the large volumes involved in open mitosis in metazoan cells. By using a simple mathematical analysis and computer simulation, we find that this mechanism can generate the inhibition found in experiment but likely requires a two-stage signal amplification cascade. The second mechanism involves spatial gradients of a short-lived inhibitory signal that propagates first by diffusion but then primarily by active transport along spindle microtubules. We propose that both mechanisms may be operative in the metazoan spindle assembly checkpoint, with either able to trigger anaphase onset even without support from the other pathway. PMID:17065324

  19. Modeling dual pathways for the metazoan spindle assembly checkpoint.

    PubMed

    Sear, Richard P; Howard, Martin

    2006-11-07

    Using computational modeling, we investigate mechanisms of signal transduction. We focus on the spindle assembly checkpoint, where a single unattached kinetochore is able to signal to prevent cell cycle progression. The inhibitory signal switches off rapidly once spindle microtubules have attached to all kinetochores. This requirement tightly constrains the possible mechanisms. Here we investigate two possible mechanisms for spindle checkpoint operation in metazoan cells, both supported by recent experiments. The first involves the free diffusion and sequestration of cell cycle regulators. This mechanism is severely constrained both by experimental fluorescence recovery data and by the large volumes involved in open mitosis in metazoan cells. By using a simple mathematical analysis and computer simulation, we find that this mechanism can generate the inhibition found in experiment but likely requires a two-stage signal amplification cascade. The second mechanism involves spatial gradients of a short-lived inhibitory signal that propagates first by diffusion but then primarily by active transport along spindle microtubules. We propose that both mechanisms may be operative in the metazoan spindle assembly checkpoint, with either able to trigger anaphase onset even without support from the other pathway.

  20. 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.

  1. Checkpoint Inhibitors and Their Application in Breast Cancer

    PubMed Central

    Bedognetti, Davide; Maccalli, Cristina; Bader, Salha B.J. Al; Marincola, Francesco M.; Seliger, Barbara

    2016-01-01

    Summary Immune checkpoints are crucial for the maintenance of self-tolerance and for the modulation of immune responses in order to minimize tissue damage. Tumor cells take advantage of these mechanisms to evade immune recognition. A significant proportion of tumors, including breast cancers, can express co-inhibitory molecules that are important formediating the escape from T cell-mediated immune surveillance. The interaction of inhibitory receptors with their ligands can be blocked by specific molecules. Monoclonal antibodies (mAbs) directed against the cytotoxic T lymphocyte-associated antigen-4 (CTLA4) and, more recently, against the programmed cell death protein 1 (PD1), have been approved for the therapy of melanoma (anti-CTLA4 and anti-PD1 mAbs) and non-small cell lung cancer (anti-PD1 mAbs). Moreover, inhibition of PD1 signaling has shown extremely promising signs of activity in breast cancer. An increasing number of molecules directed against other immune checkpoints are currently under clinical development. In this review, we summarize the evidence supporting the implementation of checkpoint inhibition in breast cancer by reviewing in detail data on PD-L1 expression and its regulation. In addition, opportunities to boost anti-tumor immunity in breast cancer with checkpoint inhibitor-based immunotherapies alone and in combination with other treatment options will be discussed. PMID:27239172

  2. 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

  3. Checkpoint Inhibition in Hodgkin Lymphoma: Saving the Best for Last?

    PubMed

    Lin, Richard J; Diefenbach, Catherine S

    2016-10-15

    Hodgkin lymphoma is a unique disease entity characterized by a low number of neoplastic tumor cells surrounded by an inflammatory microenvironment composed of dysfunctional immune cells. Recent molecular and genetic studies have revealed that upregulation of the immune checkpoint pathway programmed death 1/programmed death ligand 1 is a key oncogenic driver of Hodgkin lymphoma. Corroborating these mechanistic studies, early-phase clinical trials using the checkpoint inhibitors nivolumab and pembrolizumab in treatment regimens for relapsed and/or refractory Hodgkin lymphoma have demonstrated impressive response rates, a promising durability of response, and a favorable side-effect profile. Given its targeted mechanism of action, acceptable safety, and clinically meaningful activity, the checkpoint inhibitor nivolumab was recently approved by the US Food and Drug Administration as therapy for classical Hodgkin lymphoma that has relapsed or progressed after autologous stem cell transplantation (ASCT) and post-ASCT consolidation therapy with brentuximab vedotin. In this article we review the scientific rationale, preclinical evidence, and most recent clinical data for the use of checkpoint inhibitor therapy in patients with relapsed Hodgkin lymphoma.

  4. 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

  5. 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

  6. Alternative DNA Damage Checkpoint Pathways in Eukaryotes

    DTIC Science & Technology

    2000-04-01

    checkpoint pathway in Saccharomyces cerevisiae. Our hypothesis is that CHES1 does so by activating an alternative DNA damage-induced checkpoint pathway. The...difficulties, therefore we also tried the candidate gene and the yeast 2-hybrid approaches but with no success. In this report, we proposed alternative

  7. Replication Checkpoint: Tuning and Coordination of Replication Forks in S Phase

    PubMed Central

    Hustedt, Nicole; Gasser, Susan M.; Shimada, Kenji

    2013-01-01

    Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. During S phase, cells also provide a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication forks. The DNA replication checkpoint is crucial for maintaining genome stability, because replication forks become vulnerable to collapse when they encounter obstacles such as nucleotide adducts, nicks, RNA-DNA hybrids, or stable protein-DNA complexes. These can be exogenously induced or can arise from endogenous cellular activity. Here, we summarize the initiation and transduction of the replication checkpoint as well as its targets, which coordinate cell cycle events and DNA replication fork stability. PMID:24705211

  8. 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

  9. Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA

    PubMed Central

    2017-01-01

    In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage. PMID:28806726

  10. 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.

  11. RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

    PubMed Central

    Sifuentes-Romero, Itzel; Merchant-Larios, Horacio; Milton, Sarah L.; Moreno-Mendoza, Norma; Díaz-Hernández, Verónica; García-Gasca, Alejandra

    2013-01-01

    The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination. PMID:24705165

  12. Traffic into silence: endomembranes and post-transcriptional RNA silencing

    PubMed Central

    Kim, Yun Ju; Maizel, Alexis; Chen, Xuemei

    2014-01-01

    microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small RNAs that repress gene expression at the post-transcriptional level in plants and animals. Small RNAs guide Argonaute-containing RNA-induced silencing complexes to target RNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Although our knowledge of small RNA biogenesis, turnover, and mechanisms of action has dramatically expanded in the past decade, the subcellular location of small RNA-mediated RNA silencing still needs to be defined. In contrast to the prevalent presumption that RNA silencing occurs in the cytosol, emerging evidence reveals connections between the endomembrane system and small RNA activities in plants and animals. Here, we summarize the work that uncovered this link between small RNAs and endomembrane compartments and present an overview of the involvement of the endomembrane system in various aspects of RNA silencing. We propose that the endomembrane system is an integral component of RNA silencing that has been long overlooked and predict that a marriage between cell biology and RNA biology holds the key to a full understanding of post-transcriptional gene regulation by small RNAs. PMID:24668229

  13. 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.

  14. 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.

  15. Local infiltration of high- and low-molecular-weight RNA from silenced sunflower (Helianthus annuus L.) plants triggers post-transcriptional gene silencing in non-silenced plants.

    PubMed

    Hewezi, Tarek; Alibert, Gilbert; Kallerhoff, Jean

    2005-01-01

    Using grafting procedures, we have characterized post-transcriptional gene silencing (PTGS) in transgenic sunflower expressing beta-glucuronidase (GUS) activity. Silencing was observed as early as 2 weeks after grafting of non-silenced scions on to silenced rootstock. Transmission of the systemic signal occurs solely from stock to scion, is independent of the physiological age of the rootstock and is not heritable. Furthermore, we report, for the first time in plants, an easy and low-cost method of activating RNA silencing by infiltration of purified RNA from silenced plants. Local application of total RNA derived from silenced sunflower plants to leaves of non-silenced plants induces PTGS in newly developed leaves above the point of infiltration, as shown by reduced GUS activity and mRNA levels. Silenced plants contain 21-23-nucleotide RNAs hybridizing to transgene target sequences, in contrast with leaves of non-silenced plants. However, de novo production of GUS-specific short RNA in non-silenced plants can be activated by leaf infiltration of low-molecular-weight RNAs isolated from leaves of silenced plants. Significant levels were detected as early as 2 weeks after infiltration, peaked at 3 weeks and declined 5 weeks after infiltration. Our results provide evidence that RNA infiltration in sunflower induces transient silencing and is not transmitted to offspring. This approach could be of major use in dissecting the mechanisms involved in PTGS.

  16. Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy.

    PubMed

    Peng, Dongjun; Kryczek, Ilona; Nagarsheth, Nisha; Zhao, Lili; Wei, Shuang; Wang, Weimin; Sun, Yuqing; Zhao, Ende; Vatan, Linda; Szeliga, Wojciech; Kotarski, Jan; Tarkowski, Rafał; Dou, Yali; Cho, Kathleen; Hensley-Alford, Sharon; Munkarah, Adnan; Liu, Rebecca; Zou, Weiping

    2015-11-12

    Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism. However, its role in cancer immunopathology and immunotherapy is poorly understood. Using human ovarian cancers as our model, here we show that enhancer of zeste homologue 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) and DNA methyltransferase 1 (DNMT1)-mediated DNA methylation repress the tumour production of T helper 1 (TH1)-type chemokines CXCL9 and CXCL10, and subsequently determine effector T-cell trafficking to the tumour microenvironment. Treatment with epigenetic modulators removes the repression and increases effector T-cell tumour infiltration, slows down tumour progression, and improves the therapeutic efficacy of programmed death-ligand 1 (PD-L1; also known as B7-H1) checkpoint blockade and adoptive T-cell transfusion in tumour-bearing mice. Moreover, tumour EZH2 and DNMT1 are negatively associated with tumour-infiltrating CD8(+) T cells and patient outcome. Thus, epigenetic silencing of TH1-type chemokines is a novel immune-evasion mechanism of tumours. Selective epigenetic reprogramming alters the T-cell landscape in cancer and may enhance the clinical efficacy of cancer therapy.

  17. Integrated Circuits: How Transcriptional Silencing and Counter-Silencing Facilitate Bacterial Evolution

    PubMed Central

    Will, W. Ryan; Navarre, William W.; Fang, Ferric C.

    2014-01-01

    Horizontal gene transfer is a major contributor to bacterial evolution and diversity. For a bacterial cell to utilize newly-acquired traits such as virulence and antibiotic resistance, new genes must be integrated into the existing regulatory circuitry to allow appropriate expression. Xenogeneic silencing of horizontally-acquired genes by H-NS or other nucleoid-associated proteins avoids adventitious expression and can be relieved by other DNA-binding counter-silencing proteins in an environmentally- and physiologically-responsive manner. Biochemical and genetic analyses have recently demonstrated that counter-silencing can occur at a variety of promoter architectures, in contrast to classical transcriptional activation. Disruption of H-NS nucleoprotein filaments by DNA bending is a suggested mechanism by which silencing can be relieved. This review discusses recent advances in our understanding of the mechanisms and importance of xenogeneic silencing and counter-silencing in the successful integration of horizontally-acquired genes into regulatory networks. PMID:25461567

  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. "Schneebeli" and "Birger" silencers

    NASA Technical Reports Server (NTRS)

    Dollfus, Charles

    1923-01-01

    The Schneebeli silencer is made entirely of soft sheet steel. It is connected with the engine for receiving the exhaust gases, and consists of two parts: the silencer proper and a conical exit. The Birger silencer, made by the Ad Astra Company in Switzerland, is based on the principle of rapid cooling of the exhaust gases.

  20. Hitchcock's Melodramatic Silence.

    ERIC Educational Resources Information Center

    Hemmeter, Thomas

    1996-01-01

    Argues that the filmwork of Alfred Hitchcock shows his manipulation of melodramatic silence in that his films demonstrate a link between silence and truth. Concludes that in the simultaneous longing for and denial of the power of film silence lies the modernist complexity of Hitchcock's films that suggests the uses of melodramatic language in a…

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

    PubMed

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

    2016-10-01

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

  2. 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.

  3. 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

  4. 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

  5. Long range epigenetic silencing is a trans-species mechanism that results in cancer specific deregulation by overriding the chromatin domains of normal cells.

    PubMed

    Forn, Marta; Muñoz, Mar; Tauriello, Daniele V F; Merlos-Suárez, Anna; Rodilla, Verónica; Bigas, Anna; Batlle, Eduard; Jordà, Mireia; Peinado, Miguel A

    2013-12-01

    DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unknown whether there is a coordinated regulation of the genes embedded in these regions in normal cells and in early stages of tumor progression. To better characterize the molecular events associated with the regulation and remodeling of these regions we analyzed two regions undergoing LRES in human colon cancer in the mouse model. We demonstrate that LRES also occurs in murine cancer in vivo and mimics the molecular features of the human phenomenon, namely, downregulation of gene expression, acquisition of inactive histone marks, and DNA hypermethylation of specific CpG islands. The genes embedded in these regions showed a dynamic and autonomous regulation during mouse intestinal cell differentiation, indicating that, in the framework considered here, the coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign adenomas in Apc(Min/+) mice showed overexpression of most of the genes affected by LRES in cancer, which suggests that the repressive remodeling of the region is a late event. Chromatin immunoprecipitation analysis of the transcriptional insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain boundaries as compared with normal cells. Malignant regression of cancer cells by in vitro differentiation resulted in partial reversion of LRES and gain of CTCF binding. We conclude that genes in LRES regions are plastically regulated in cell differentiation and hyperproliferation, but are constrained to a coordinated repression by abolishing boundaries and the autonomous regulation of chromatin domains in cancer cells.

  6. The same, only different - DNA damage checkpoints and their reversal throughout the cell cycle.

    PubMed

    Shaltiel, Indra A; Krenning, Lenno; Bruinsma, Wytse; Medema, René H

    2015-02-15

    Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made. © 2015. Published by The Company of Biologists Ltd.

  7. Topoisomerase II Inhibitors and Poisons, and the Influence of Cell Cycle Checkpoints.

    PubMed

    D Arcy, Nicholas; Gabrielli, Brian

    2017-01-01

    Interactions between the decatenation checkpoint and Topoisomerase II (TopoII) are vital for maintaining integrity of the genome. Agents that target this enzyme have been in clinical use in cancer therapy for over 30 years with great success. The types of compounds that have been developed to target TopoII are broadly divided into poisons and catalytic inhibitors. The TopoII poisons are in clinical use as anti-cancer therapies, although in common to most chemotherapeutic agents, they display considerable normal tissue toxicity. Inhibition of the TopoIIb isoform has been implicated in this cytotoxicity. Response to TopoII active agents is determined by several factors, but cell cycle checkpoints play a large role in sensitivity and resistance. The G2/M phase checkpoints are of particular importance in considering the effectiveness of these drugs and are reviewed in this article. Functionality of the ATM dependent decatenation checkpoint may represent a new avenue for selective cancer therapy. Here we review the function of TopoII, the anti-cancer mechanisms and limitations of current catalytic inhibitors and poisons, and their influence on cell cycle checkpoints. We will also assess potential new mechanisms for targeting this enzyme to limit normal tissue toxicity, and how the cell cycle checkpoint triggered by these drugs may provide an alternative and possibly better target for novel therapies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. 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

  9. 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

  10. 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

  11. [Effect of RNA Interference-silenced TAK1 on Kasumi-1 cell Proliferation Inhibition Induced by As2O3 and Its Mechanism].

    PubMed

    Liu, Sha; Yuan, Fang-Fang; Mi, Rui-Hua; Wang, Xiao-Jiao; Fan, Rui-Hua; Wei, Xu-Dong

    2017-04-01

    To explore the effect of transforming growth factor-β activated kinase-1(TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by As2O3 and its mechanism. The experiments were divided into 4 groups, including control group(Kasumi-1 cells treated with non-specific siRNA), TAK1 specific siRNA treated group (Kasumi 1 treated with TAK specific siRNA), As2O3 treated group (Kasumi 1 cells treated with As2O3) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus As2O3). The proliferation inhibition rate of Kasami 1 cells was detected by CCK-8 method, the apoptotic rate of cells was detected by flow eytometry, the expressions of TAK1, phosphorylated c-Jun N-terminal kinase(p-JNK) and apoptosis-related proteins were detected by Western blot. As2O3 could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 µmol/L with IC50 of (3.79±0.36) µmol/L at 24 h, and also inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 10 µmol/L with IC50 of (2.38±0.17) µmol/L at 48 h, but then the inhibitory effect reached plateau. After treating Kasumi-1 cells with TAK1 siRNA and 3.5 µmol/L As2O3 for 24 h, the proliferation inhibition rate was (10.86±1.64)% and (49.80±2.19)%, meanwhile the apoptosis rate was (8.47±0.75)% and (24.78±2.14)%, all significantly higher than those in control group (P<0.05, P<0.01). The proliferation inhibition rate and apoptosis rate of the combined treated group were significantly higher than that in control and single treated groups (P<0.05, P<0.01), TAK1 silencing and 3.5 µmol/L of As2O3 could decrease the expression of TAK1, p-JNK, c-Fos, c-Jun and BCL-2 in different degrees, and increase the expression levels of BAX and the activated (cleaved) caspase-3, 9 with statistically significant differences as compared with control group (P< 0.05). When Kasumi-1 cells were treated with TAK1 specific siRNA plus As2O3 for 24 h, protein

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Characteristics of post-transcriptional gene silencing.

    PubMed

    Chicas, A; Macino, G

    2001-11-01

    A number of gene silencing phenomena that inactivate genes at the post-transcriptional level have been identified. Due to its potential for studying gene function, post-transcriptional gene silencing (PTGS) has become an intense area of research. In this review we describe the different means of inducing PTGS and discuss the possible biological roles of these artificially induced phenomena. We also discuss other features of PTGS such as the mechanism of mRNA degradation, the nature of the silencing signal and the mechanism of PTGS inhibition by viral proteins.

  18. Characteristics of post-transcriptional gene silencing

    PubMed Central

    Chicas, Agustin; Macino, Giuseppe

    2001-01-01

    A number of gene silencing phenomena that inactivate genes at the post-transcriptional level have been identified. Due to its potential for studying gene function, post-transcriptional gene silencing (PTGS) has become an intense area of research. In this review we describe the different means of inducing PTGS and discuss the possible biological roles of these artificially induced phenomena. We also discuss other features of PTGS such as the mechanism of mRNA degradation, the nature of the silencing signal and the mechanism of PTGS inhibition by viral proteins. PMID:11713190

  19. Plant RNA silencing in viral defence.

    PubMed

    Pantaleo, Vitantonio

    2011-01-01

    RNA silencing is described in plants and insects as a defence mechanism against foreign nucleic acids, such as invading viruses. The RNA silencing-based antiviral defence involves the production of virus-derived small interfering RNAs and their association to effector proteins, which together drive the sequence specific inactivation of viruses. The entire process of antiviral defence 'borrows' several plant factors involved in other specialized RNA silencing endogenous pathways. Different viruses use variable strategies to infect different host plants, which render the antiviral RNA silencing a complex phenomenon far to be completely clarified. This chapter reports current advances in understanding the main steps of the plant's RNA-silencing response to viral invasion and discusses some of the key questions still to be answered.

  20. Centromere-associated protein E: a motor that puts the brakes on the mitotic checkpoint.

    PubMed

    Wood, Kenneth W; Chua, Penelope; Sutton, David; Jackson, Jeffrey R

    2008-12-01

    Cell cycle checkpoints have long been recognized as important nodes for regulating cell proliferation and maintaining genomic integrity. These checkpoints are often altered in cancer and represent promising points for therapeutic intervention. Until recently, direct targeting of the mitotic checkpoint has been an untapped area for cancer drug discovery. Regulation of the mitotic checkpoint is complex, but many of the critical players have been identified and functionally characterized. A substantial number of these proteins can be localized to the kinetochore, a structure located at the centromeric region of each mitotic chromosome. The kinetochore mediates chromosome attachment to spindle microtubules and subsequent chromosome movement. The mitotic checkpoint monitors microtubule attachment and chromosome position on the mitotic spindle, inhibiting progression into anaphase until proper attachment and metaphase positioning is achieved. Centromere-associated protein E is a kinesin microtubule motor protein that plays an essential role in integrating the mechanics of microtubule-chromosome interactions with mitotic checkpoint signaling, and has emerged as a novel target for cancer therapy.

  1. 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

  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. 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.

  5. 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.

  6. 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

  7. Silence Amenity Engineering

    NASA Astrophysics Data System (ADS)

    Fujita, Hajime

    Engineering civilization brought convenient and comfortable life to us. However, some environmental problems such as various pollutions have also been developed with it. Acoustical noise is one of the major problems in modern life. Noise is generated from a noise source and propagates through transmitting medium such as the air and eventually reaches a receiver, usually a human being. The noise problem can be avoided, therefore, if one of those three elements in the noise problem is removed completely. In actual case, engineers are looking for most efficient way combining the controls for these three elements. In this article, basic characteristics of noise is reviewed briefly at first, then sound field analysis to predict sound transmission is discussed Aerodynamic noise is one of the major problems in silence amenity engineering today. Basic concept of the aerodynamic noise generation mechanism is discussed in detail with applications to turbo-machinery and high speed train noise control technology.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway

    PubMed Central

    Domínguez-Calderón, Alaide; Ávila-Flores, Antonia; Ponce, Arturo; López-Bayghen, Esther; Calderón-Salinas, José-Víctor; Luis Reyes, José; Chávez-Munguía, Bibiana; Segovia, José; Angulo, Carla; Ramírez, Leticia; Gallego-Gutiérrez, Helios; Alarcón, Lourdes; Martín-Tapia, Dolores; Bautista-García, Pablo; González-Mariscal, Lorenza

    2016-01-01

    Renal compensatory hypertrophy (RCH) restores normal kidney function after disease or loss of kidney tissue and is characterized by an increase in organ size due to cell enlargement and not to cell proliferation. In MDCK renal epithelial cells, silencing of the tight junction protein zona occludens 2 (ZO-2 KD) induces cell hypertrophy by two mechanisms: prolonging the time that cells spend at the G1 phase of the cell cycle due to an increase in cyclin D1 level, and augmenting the rate of protein synthesis. The latter is triggered by the nuclear accumulation and increased transcriptional activity of Yes-associated protein (YAP), the main target of the Hippo pathway, which results in decreased expression of phosphatase and tensin homologue. This in turn increased the level of phosphatidylinositol (3,4,5)-triphosphate, which transactivates the Akt/mammalian target of rapamycin pathway, leading to activation of the kinase S6K1 and increased synthesis of proteins and cell size. In agreement, in a rat model of uninephrectomy, RCH is accompanied by decreased expression of ZO-2 and nuclear expression of YAP. Our results reveal a novel role of ZO-2 as a modulator of cell size. PMID:27009203

  13. Fetal hemoglobin silencing in humans

    PubMed Central

    Oneal, Patricia A.; Gantt, Nicole M.; Schwartz, Joseph D.; Bhanu, Natarajan V.; Lee, Y. Terry; Moroney, John W.; Reed, Christopher H.; Schechter, Alan N.; Luban, Naomi L. C.; Miller, Jeffery L.

    2006-01-01

    Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies. PMID:16735596

  14. Attenuation of G1 checkpoint function by the non-genotoxic carcinogen phenobarbital.

    PubMed

    Gonzales, A J; Christensen, J G; Preston, R J; Goldsworthy, T L; Tlsty, T D; Fox, T R

    1998-07-01

    Non-genotoxic chemical carcinogens are capable of inducing tumors in rodents without interacting with or directly altering the genetic material. Since a preponderance of evidence suggests that cancer results from the accumulation of genetic alterations, the mechanisms by which many non-genotoxic carcinogens induce genotoxic events remain unclear. The present study investigated whether the mitogenic, non-genotoxic carcinogen phenobarbital (PB) could alter cell-cycle checkpoint controls, thereby indirectly leading to the accumulation of genetic damage. Initial studies involved characterizing cell-cycle checkpoint responses to DNA damage in freshly isolated B6C3F1 mouse hepatocytes. These cells responded to bleomycin-induced DNA damage by arresting in G1 and G2. Cell-cycle arrest was coupled with p53 protein induction; however, p21WAF1 protein levels remained unchanged. Studies that utilized hepatocytes isolated from C57BL p53-/- mice showed that the DNA damage-induced G1 cell-cycle arrest was dependent on p53 function, but cell-cycle arrest in G2 was not affected by loss of p53. PB was able to delay and attenuate the G1 checkpoint response without altering G2 checkpoint function. A reduction in p53 protein, but not transcript levels, was observed in hepatocytes exposed to PB. Additionally, PB delayed and attenuated p53 protein induction during DNA damage, which suggests that changes in the p53 protein may be contributing to the attenuated G1 checkpoint response caused by PB. Altered G1 checkpoint function represents an epigenetic mechanism by which phenobarbital may prevent the detection and repair of DNA damage and indirectly increase the frequency of genotoxic events above that occurring spontaneously. Abrogation of checkpoint controls may, thus, play an important mechanistic role in mitogenic, non-genotoxic chemical carcinogenesis.

  15. Mechanism of fiber carcinogenesis: from reactive radical species to silencing of the beta igH3 gene.

    PubMed

    Hei, Tom K; Xu, An; Huang, Sarah X; Zhao, Yongliang

    2006-11-01

    Although the U.S. Environmental Protection Agency has restricted the industrial use of regulated forms of asbestos in the United States since the early 1970s, environmental exposure to asbestos remains a health concern in the United States and is a significant health issue among developing countries. Exposure to asbestos is associated with chronic pulmonary diseases and cancer of the lung, pleura, and peritoneum. The mechanism of fiber carcinogenesis is far from clear and is likely to be complex, depending on fiber dimensions, surface properties, and physical durability. The induction of reactive oxygen and nitrogen species upon phagocytosis of fibers plays an important role in fiber genotoxicity. The beta igH3, a secreted protein induced by the transforming growth factor-beta and essential for cell adhesion, is downregulated in asbestos-induced tumorigenic human bronchial epithelial cells. Ectopic expression of the beta igH3 gene abrogates the tumorigenic phenotype and suggests that the gene plays a causal role in fiber carcinogenesis. A better understanding of the carcinogenic mechanism of asbestos and other mineral fibers will provide useful information on interventional and preventive measures for asbestos-mediated diseases such as human pleural and peritoneal mesotheliomas.

  16. CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.

    PubMed

    Kashima, Lisa; Idogawa, Masashi; Mita, Hiroaki; Shitashige, Miki; Yamada, Tesshi; Ogi, Kazuhiro; Suzuki, Hiromu; Toyota, Minoru; Ariga, Hiroyoshi; Sasaki, Yasushi; Tokino, Takashi

    2012-04-13

    The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.

  17. [Therapeutic Cancer Vaccine and Immune Checkpoint Inhibitor].

    PubMed

    Mimura, Kousaku; Kono, Koji

    2017-09-01

    Therapeutic cancer vaccine enhances a specific immune response against tumor cells in vivo, resulting in exertion of antitumor effects. On the other hand, immune checkpoint inhibitors promote the induction of tumor-specific T cells and also enhance the cytotoxic abilityof these T cells in tumor microenvironment. There is a possibilitythat immune checkpoint inhibitors enhance tumor immune responses induced bytherapeutic cancer vaccine, and it is expected that additive or synergistic effects will be obtained bythe combination of them. Moreover, according to previous reports, we should use an immune checkpoint inhibitor to enhance the cytotoxic ability of tumor-specific T cells as the combination for therapeutic cancer vaccine. Furthermore, the combination of a specific antibodyagainst newlyidentified co-inhibitoryreceptors (Lag-3, Tim-3, TIGIT, etc)and a therapeutic cancer vaccine is also one of newlyexpected treatments in the future.

  18. 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.

  19. 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

  20. 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

  1. Association of Autoimmune Encephalitis With Combined Immune Checkpoint Inhibitor Treatment for Metastatic Cancer.

    PubMed

    Williams, Tanya J; Benavides, David R; Patrice, Kelly-Ann; Dalmau, Josep O; de Ávila, Alexandre Leon Ribeiro; Le, Dung T; Lipson, Evan J; Probasco, John C; Mowry, Ellen M

    2016-08-01

    Paraneoplastic encephalitides usually precede a diagnosis of cancer and are often refractory to immunosuppressive therapy. Conversely, autoimmune encephalitides are reversible conditions that can occur in the presence or absence of cancer. To report the induction of autoimmune encephalitis in 2 patients after treatment of metastatic cancer with a combination of the immune checkpoint inhibitors nivolumab and ipilimumab. A retrospective case study was conducted of the clinical and management course of 2 patients with progressive, treatment-refractory metastatic cancer who were treated with a single dose each (concomitantly) of the immune checkpoint inhibitors nivolumab, 1 mg/kg, and ipilimumab, 3 mg/kg. Nivolumab and ipilimumab. The clinical response to immunosuppressive therapy in suspected autoimmune encephalitis in the setting of immune checkpoint inhibitor use. Autoantibody testing confirmed identification of anti-N-methyl-D-aspartate receptor antibodies in the cerebrospinal fluid of 1 patient. Withdrawal of immune checkpoint inhibitors and initiation of immunosuppressive therapy, consisting of intravenous methylprednisolone sodium succinate equivalent to 1000 mg of methylprednisolone for 5 days, 0.4 mg/kg/d of intravenous immunoglobulin for 5 days, and 2 doses of rituximab, 1000 mg, in 1 patient and oral prednisone, 60 mg/d, in the other patient, resulted in improved neurologic symptoms. Immune checkpoint inhibition may favor the development of immune responses against neuronal antigens, leading to autoimmune encephalitis. Early recognition and treatment of autoimmune encephalitis in patients receiving immune checkpoint blockade therapy will likely be essential for maximizing clinical recovery and minimizing the effect of drug-related toxic effects. The mechanisms by which immune checkpoint inhibition may contribute to autoimmune encephalitis require further study.

  2. Silencing transposable elements in the Drosophila germline.

    PubMed

    Yang, Fu; Xi, Rongwen

    2017-02-01

    Transposable elements or transposons are DNA pieces that can move around within the genome and are, therefore, potential threat to genome stability and faithful transmission of the genetic information in the germline. Accordingly, self-defense mechanisms have evolved in the metazoan germline to silence transposons, and the primary mechanism requires the germline-specific non-coding small RNAs, named Piwi-interacting RNA (piRNAs), which are in complex with Argonaute family of PIWI proteins (the piRNA-RISC complexes), to silence transposons. piRNA-mediated transposon silencing occurs at both transcriptional and post-transcriptional levels. With the advantages of genetic manipulation and advances of sequencing technology, much progress has been made on the molecular mechanisms of piRNA-mediated transposon silencing in Drosophila melanogaster, which will be the focus of this review. Because piRNA-mediated transposon silencing is evolutionarily conserved in metazoan, model organisms, such as Drosophila, will continue to be served as pioneer systems towards the complete understanding of transposon silencing in the metazoan germline.

  3. 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.

  4. Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing[OPEN

    PubMed Central

    Fultz, Dalen

    2017-01-01

    The propagation of epigenetic marks has received a great deal of attention, yet the initiation of epigenetic silencing of a new transgene, virus, or transposable element (TE) remains enigmatic. The overlapping and simultaneous function of multiple silencing mechanisms has obscured this area of investigation. Here, we revealed two broad mechanisms that can initiate silencing independently: identity-based and expression-dependent silencing. We found that identity-based silencing is targeted by 21- to 22-nucleotide or 24-nucleotide small interfering RNAs (siRNAs) generated from previously silenced regions of the genome. By transforming exogenous TEs into Arabidopsis thaliana, we circumvented identity-based silencing, allowing us to isolate and investigate the molecular mechanism of expression-dependent silencing. We found that several siRNA-generating mechanisms all trigger de novo expression-dependent RNA-directed DNA methylation (RdDM) through RNA Polymerase V. In addition, while full-length TEs quickly progress beyond RdDM to heterochromatin formation and the final maintenance methylation state, TE fragments stall at the RdDM phase. Lastly, we found that transformation into a mutant genotype followed by introgression into the wild type does not result in the same level of silencing as direct transformation into the wild type. This demonstrates that the plant genotype during a narrow window of time at TE insertion (or transgene transformation) is key for establishing the transgenerational extent of epigenetic silencing. PMID:28193737

  5. 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

  6. SNAIL1 combines competitive displacement of ASCL2 and epigenetic mechanisms to rapidly silence the EPHB3 tumor suppressor in colorectal cancer.

    PubMed

    Rönsch, Kerstin; Jägle, Sabine; Rose, Katja; Seidl, Maximilian; Baumgartner, Francis; Freihen, Vivien; Yousaf, Afsheen; Metzger, Eric; Lassmann, Silke; Schüle, Roland; Zeiser, Robert; Michoel, Tom; Hecht, Andreas

    2015-02-01

    EPHB3 is a critical cellular guidance factor in the intestinal epithelium and an important tumor suppressor in colorectal cancer (CRC) whose expression is frequently lost at the adenoma-carcinoma transition when tumor cells become invasive. The molecular mechanisms underlying EPHB3 silencing are incompletely understood. Here we show that EPHB3 expression is anti-correlated with inducers of epithelial-mesenchymal transition (EMT) in primary tumors and CRC cells. In vitro, SNAIL1 and SNAIL2, but not ZEB1, repress EPHB3 reporter constructs and compete with the stem cell factor ASCL2 for binding to an E-box motif. At the endogenous EPHB3 locus, SNAIL1 triggers the displacement of ASCL2, p300 and the Wnt pathway effector TCF7L2 and engages corepressor complexes containing HDACs and the histone demethylase LSD1 to collapse active chromatin structure, resulting in rapid downregulation of EPHB3. Beyond its impact on EPHB3, SNAIL1 deregulates markers of intestinal identity and stemness and in vitro forces CRC cells to undergo EMT with altered morphology, increased motility and invasiveness. In xenotransplants, SNAIL1 expression abrogated tumor cell palisading and led to focal loss of tumor encapsulation and the appearance of areas with tumor cells displaying a migratory phenotype. These changes were accompanied by loss of EPHB3 and CDH1 expression. Intriguingly, SNAIL1-induced phenotypic changes of CRC cells are significantly impaired by sustained EPHB3 expression both in vitro and in vivo. Altogether, our results identify EPHB3 as a novel target of SNAIL1 and suggest that disabling EPHB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae.

    PubMed

    Daniel, Jewel A; Keyes, Brice E; Ng, Yvonne P Y; Freeman, C Onyi; Burke, Daniel J

    2006-01-01

    The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.

  8. 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…

  9. Transcriptional gene silencing in humans

    PubMed Central

    Weinberg, Marc S.; Morris, Kevin V.

    2016-01-01

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  10. Nickel and Epigenetic Gene Silencing

    PubMed Central

    Sun, Hong; Shamy, Magdy; Costa, Max

    2013-01-01

    Insoluble nickel compounds are well-established human carcinogens. Occupational exposure to these compounds leads to increased incidence of lung and nasal cancer in nickel refinery workers. Apart from its weak mutagenic activity and hypoxia mimicking effect there is mounting experimental evidence indicating that epigenetic alteration plays an important role in nickel-induced carcinogenesis. Multiple epigenetic mechanisms have been identified to mediate nickel-induced gene silencing. Nickel ion is able to induce heterochromatinization by binding to DNA-histone complexes and initiating chromatin condensation. The enzymes required for establishing or removing epigenetic marks can be targeted by nickel, leading to altered DNA methylation and histone modification landscapes. The current review will focus on the epigenetic changes that contribute to nickel-induced gene silencing. PMID:24705264

  11. Aberrantly Silenced Promoters Retain a Persistent Memory of the Silenced State After Long-Term Reactivation

    PubMed Central

    Oyer, Jon A.; Yates, Phillip A.; Godsey, Sarah; Turker, Mitchell S.

    2010-01-01

    A hallmark of aberrant DNA methylation-associated silencing is reversibility. However, long-term stability of reactivated promoters has not been explored. To examine this issue, spontaneous reactivant clones were isolated from mouse embryonal carcinoma cells bearing aberrantly silenced Aprt alleles and re-silencing frequencies were determined as long as three months after reactivation occurred. Despite continuous selection for expression of the reactivated Aprt alleles, exceptionally high spontaneous re-silencing frequencies were observed. A DNA methylation analysis demonstrated retention of sporadic methylation of CpG sites in a protected region of the Aprt promoter in many reactivant alleles suggesting a role for these methylated sites in the re-silencing process. In contrast, a chromatin immunoprecipitation (ChIP) analysis for methyl-H3K4, acetyl-H3K9, and dimethyl-H3K9 levels failed to reveal a specific histone modification that could explain high frequency re-silencing. These results demonstrate that aberrantly silenced and reactivated promoters retain a persistent memory of having undergone the silencing process and suggest the failure to eliminate all CpG methylation as a potential contributing mechanism. PMID:21035468

  12. 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

  13. Initial characterization of a low-molecular-weight factor enhancing the checkpoint response.

    PubMed

    Fan, Xiaoxiang; Cheong, Nge; Iliakis, George

    2010-10-01

    In higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cell cycle, delays induced in G(2) are longer and frequently correlate with resistance to killing by radiation. Therefore, modulation of the G(2) checkpoint offers a means to modulate cellular radiosensitivity. Although compounds are known that reduce the G(2) checkpoint and act as radiosensitizers, compounds enhancing this checkpoint have not been reported. Here we summarize evidence for a factor with such properties. We show that a highly radioresistant rat embryo fibroblast (REF) cell line displays a strong G(2) checkpoint partly as a result of a factor excreted into the growth medium by nonirradiated cells. Various tests indicate that this G(2)-arrest modulating activity (GAMA) is a small molecule showing detectable retention only after passing through filters with a molecular weight cutoff limit of less than 1,000 Da. GAMA is heat stable and resistant to treatment with proteases or nucleases. Electroelution tests show that GAMA is uncharged at neutral pH, a result that is in agreement with the observed failure to bind S- or Q-Sepharose. Investigations on the mechanism of GAMA function indicate ligand-receptor interactions and allow the classification of cells as producers, responders or both. Compounds with properties such as those of GAMA bridge intercellular communication with the DNA damage response and may function as radioprotectors.

  14. 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.

  15. Immune and molecular correlates in melanoma treated with immune checkpoint blockade.

    PubMed

    Byrne, Elizabeth H; Fisher, David E

    2017-06-01

    Immunotherapy for metastatic melanoma has a decades-long history, and the relatively recent use of checkpoint inhibitors has revolutionized treatment. Durable and sometimes complete remission of metastatic melanoma is now achievable in some patients who receive checkpoint-blocking therapy. However, it is unclear why some patients fare better than others. This review highlights several molecular indicators of response to checkpoint inhibition in metastatic melanoma, focusing on tumor programmed death ligand 1 expression, major histocompatibility complex class I expression, mutational load in the tumor, and T-cell infiltration into the tumor. In addition, clinical correlates of response, notably vitiligo and other immune-related adverse events, can potentially shed light on the mechanisms by which checkpoint blockade may achieve such great success, particularly in melanoma. The authors propose that microphthalmia-associated transcription factor-a key regulator of melanocyte survival, melanin production, and melanoma transformation-produces a molecular landscape in melanocytes and melanoma cells that can make melanomas particularly susceptible to checkpoint blockade and also can result in immune attack on normal melanocytes. Cancer 2017;123:2143-53. © 2017 American Cancer Society. © 2017 American Cancer Society.

  16. Alternative DNA Damage Checkpoint Pathways in Eukaryotes

    DTIC Science & Technology

    2002-04-01

    data suggest that Ches1 functions in an alternative checkpoint pathway. Our goals are to identify genes constituting this pathway, to isolate the...human counterparts, and to compare their structure and activity in normal and cancer tissues. In order to identify the genes involved in the alternative

  17. Hyper-methylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells.

    PubMed

    Manjegowda, Mohan C; Gupta, Paridhi Singhal; Limaye, Anil M

    2017-05-30

    potential mechanism of GPER1 silencing in breast tumors.

  18. Endocrine side effects induced by immune checkpoint inhibitors.

    PubMed

    Corsello, Salvatore Maria; Barnabei, Agnese; Marchetti, Paolo; De Vecchis, Liana; Salvatori, Roberto; Torino, Francesco

    2013-04-01

    In recent years, progress has been made in cancer immunotherapy by the development of drugs acting as modulators of immune checkpoint proteins, such as the cytotoxic T-lymphocyte antigen-4 (CTLA4) and programmed death-1 (PD-1), two co-inhibitory receptors that are expressed on T cells upon activation. These molecules play crucial roles in maintaining immune homeostasis by down-regulating T-cell signaling, thereby preventing unbridled T-cell proliferation while maintaining tolerance to self-antigens, such as tumor-associated antigens. CTLA4 blockade through systemic administration of the CTLA4-blocking antibody ipilimumab was shown to confer significant survival benefit and prolonged stable disease in patients affected by advanced cutaneous melanoma. Other immune checkpoint inhibitors are under clinical evaluation. However, immune checkpoint blockade can lead to the breaking of immune self-tolerance, thereby inducing a novel syndrome of autoimmune/autoinflammatory side effects, designated as "immune-related adverse events," mainly including rash, colitis, hepatitis, and endocrinopathies. We searched the medical literature using the words "hypophysitis," "hypopituitarism," "thyroid," "adrenal insufficiency," and "endocrine adverse events" in association with "immune checkpoint inhibitors," "ipilimumab," "tremelimumab," "PD-1," and "PD-1-L." The spectrum of endocrine disease experienced by patients treated with ipilimumab includes most commonly hypophysitis, more rarely thyroid disease or abnormalities in thyroid function tests, and occasionally primary adrenal insufficiency. Hypophysitis has emerged as a distinctive side effect of CTLA4-blocking antibodies, establishing a new form of autoimmune pituitary disease. This condition, if not promptly recognized, may be life-threatening (due to secondary hypoadrenalism). Hypopituitarism caused by these agents is rarely reversible, and prolonged or lifelong substitutive hormonal treatment is often required. The precise

  19. The replication checkpoint prevents two types of fork collapse without regulating replisome stability

    PubMed Central

    Dungrawala, Huzefa; Rose, Kristie L.; Bhat, Kamakoti P.; Mohni, Kareem N.; Glick, Gloria G.; Couch, Frank B.; Cortez, David

    2015-01-01

    Summary The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators. PMID:26365379

  20. 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

  1. Combination of immune checkpoint inhibitors and radiotherapy: Review of the literature.

    PubMed

    Sindoni, Alessandro; Minutoli, Fabio; Ascenti, Giorgio; Pergolizzi, Stefano

    2017-05-01

    Literature experiences in cancer treatment usually deal with either targeting the tumour cell or the immune system, which often fail to reach the curative purposes in many solid tumours. On the other hand, one mechanism of radiation-induced tumour control is the activation of the adaptive immune system by tumour antigen release following radiotherapy. So, combining radiation therapy with immune checkpoint blockade treatment at the same time may represent a way to stimulate the adaptive immune system, with further amplification of immune responses reached through systemic immune checkpoint blockade. Until now, only few studies deal with the association of immune checkpoint blockade treatment and radiotherapy. In this review, we evaluate this association, highlighting this possibility as a new strategy to improve outcome in cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Developmental Checkpoints and Feedback Circuits Time Insect Maturation

    PubMed Central

    Rewitz, Kim F.; Yamanaka, Naoki; O’Connor, Michael B.

    2014-01-01

    The transition from juvenile to adult is a fundamental process that allows animals to allocate resource toward reproduction after completing a certain amount of growth. In insects, growth to a species-specific target size induces pulses of the steroid hormone ecdysone that triggers metamorphosis and reproductive maturation. The past few years have seen significant progress in understanding the interplay of mechanisms that coordinate timing of ecdysone production and release. These studies show that the neuroendocrine system monitors complex size-related and nutritional signals, as well as external cues, to time production and release of ecdysone. Based on results discussed here, we suggest that developmental progression to adulthood is controlled by checkpoints that regulate the genetic timing program enabling it to adapt to different environmental conditions. These checkpoints utilize a number of signaling pathways to modulate ecdysone production in the prothoracic gland. Release of ecdysone activates an autonomous cascade of both feedforward and feedback signals that determine the duration of the ecdysone pulse at each developmental transitions. Conservation of the genetic mechanisms that coordinate the juvenile-adult transition suggests that insights from the fruit fly Drosophila will provide a framework for future investigation of developmental timing in metazoans. PMID:23347514

  7. 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.

  8. 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.

  9. Compiler-Enhanced Incremental Checkpointing for OpenMP Applications

    SciTech Connect

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

    2008-01-21

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

  10. 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.

  11. RNAi induced gene silencing in crop improvement.

    PubMed

    Sinha, Subodh Kumar

    2010-12-01

    The RNA silencing is one of the innovative and efficient molecular biology tools to harness the down-regulation of expression of gene(s) specifically. To accomplish such selective modification of gene expression of a particular trait, homology dependent gene silencing uses a stunning variety of gene silencing viz. co-suppression, post-transcriptional gene silencing, virus-induced gene silencing etc. This family of diverse molecular phenomena has a common exciting feature of gene silencing which is collectively called RNA interference abbreviated to as RNAi. This molecular phenomenon has become a focal point of plant biology and medical research throughout the world. As a result, this technology has turned out to be a powerful tool in understanding the function of individual gene and has ultimately led to the tremendous use in crop improvement. This review article illustrates the application of RNAi in a broad area of crop improvement where this technology has been successfully used. It also provides historical perspective of RNAi discovery and its contemporary phenomena, mechanism of RNAi pathway.

  12. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

  13. An electronically tunable duct silencer using dielectric elastomer actuators.

    PubMed

    Lu, Zhenbo; Godaba, Hareesh; Cui, Yongdong; Foo, Choon Chiang; Debiasi, Marco; Zhu, Jian

    2015-09-01

    A duct silencer with tunable acoustic characteristics is presented in this paper. Dielectric elastomer, a smart material with lightweight, high elastic energy density and large deformation under high direct current/alternating current voltages, was used to fabricate this duct silencer. The acoustic performances and tunable mechanisms of this duct silencer were experimentally investigated. It was found that all the resonance peaks of this duct silencer could be adjusted using external control signals without any additional mechanical part. The physics of the tunable mechanism is further discussed based on the electro-mechanical interactions using finite element analysis. The present promising results also provide insight into the appropriateness of the duct silencer for possible use as next generation acoustic treatment device to replace the traditional acoustic treatment.

  14. 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

  15. 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.

  16. 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

  17. [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.

  18. Extending the Binomial Checkpointing Technique for Resilience

    SciTech Connect

    Walther, Andrea; Narayanan, Sri Hari Krishna

    2016-10-10

    In terms of computing time, adjoint methods offer a very attractive alternative to compute gradient information, re- quired, e.g., for optimization purposes. However, together with this very favorable temporal complexity result comes a memory requirement that is in essence proportional with the operation count of the underlying function, e.g., if algo- rithmic differentiation is used to provide the adjoints. For this reason, checkpointing approaches in many variants have become popular. This paper analyzes an extension of the so-called binomial approach to cover also possible failures of the computing systems. Such a measure of precaution is of special interest for massive parallel simulations and adjoint calculations where the mean time between failure of the large scale computing system is smaller than the time needed to complete the calculation of the adjoint information. We de- scribe the extensions of standard checkpointing approaches required for such resilience, provide a corresponding imple- mentation and discuss numerical results.

  19. 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.

  20. Conceiving silence: infertility as discursive contradiction in Ireland.

    PubMed

    Allison, Jill

    2011-03-01

    This article examines the production and reproduction of silence around infertility in Ireland. Based on narratives collected during 18 months of fieldwork, this article locates the contradictory role of silence in both the private experiences of individuals faced with a difficulty conceiving and in institutions constituted as mechanisms of public support. For many people who experience infertility, silence is rooted in the social stigma associated with reproductive failure or sexual inadequacy. Silence protects privacy while at the same time foreclosing both challenges to assumptions that fertility is the norm and any counterdiscourse to the heteronormative, profamily society in Ireland. I show how the reproduction of silence about infertility is a legacy of Ireland's history, reproductive politics, and the cultural idiom of choice. I argue that support networks and Internet bulletin boards on websites create opportunities to dialogue in silence, reproducing isolation rather than creating public discourse.

  1. Mammalian Homologs of Yeast Checkpoint Genes

    DTIC Science & Technology

    2001-07-01

    this interaction fo DNA damage-dependent regulation of Chk2 is discussed. 14. SUBJECT TERMS 15. NUMBER OF PAGES Breast Cancer , Checkpoint control...and how it regulates targets. This work is directly relevant to breast cancer , since it is now clear that Chk2 is an intermediary linking DNA...Li- Fraumeni syndrome, which predisposes to breast cancer and other cancers [6]. Connection with Approved Technical Objectives. Technical Objective 1

  2. 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.

  3. 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.

  4. Optimal viral strategies for bypassing RNA silencing.

    PubMed

    Rodrigo, Guillermo; Carrera, Javier; Jaramillo, Alfonso; Elena, Santiago F

    2011-02-06

    The RNA silencing pathway constitutes a defence mechanism highly conserved in eukaryotes, especially in plants, where the underlying working principle relies on the repressive action triggered by the intracellular presence of double-stranded RNAs. This immune system performs a post-transcriptional suppression of aberrant mRNAs or viral RNAs by small interfering RNAs (siRNAs) that are directed towards their target in a sequence-specific manner. However, viruses have evolved strategies to escape from silencing surveillance while promoting their own replication. Several viruses encode suppressor proteins that interact with different elements of the RNA silencing pathway and block it. The different suppressors are not phylogenetically nor structurally related and also differ in their mechanism of action. Here, we adopt a model-driven forward-engineering approach to understand the evolution of suppressor proteins and, in particular, why viral suppressors preferentially target some components of the silencing pathway. We analysed three strategies characterized by different design principles: replication in the absence of a suppressor, suppressors targeting the first protein component of the pathway and suppressors targeting the siRNAs. Our results shed light on the question of whether a virus must opt for devoting more time into transcription or into translation and on which would be the optimal step of the silencing pathway to be targeted by suppressors. In addition, we discussed the evolutionary implications of such designing principles.

  5. 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.

  6. 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.

  7. 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.

  8. Current Status of Immune Checkpoint Inhibitors in Gastrointestinal Cancers.

    PubMed

    Kim, Bum Jun; Jang, Hyun Joo; Kim, Hyeong Su; Kim, Jung Han

    2017-01-01

    The expansion of our understanding of tumor immunity and the recent success of new cancer immunotherapy has reignited the hope that we can treat cancer effectively with immunotherapeutic approaches. Immune checkpoint inhibitors have shown significant efficacy in the treatment of some solid and hematologic malignancies. Encouraged by recent success in some other types of malignancies, many clinical trials are ongoing to evaluate the efficacy of immune checkpoint inhibitors in gastrointestinal (GI) malignancies. In this review, we briefly discuss theoretical background and current status of immune checkpoint inhibitors in GI cancers. We summarize the key studies and present the ongoing clinical trials involving immune checkpoint inhibitors in GI cancers.

  9. On Observing Student Silence

    ERIC Educational Resources Information Center

    Amundrud, Thomas

    2011-01-01

    This article uses conversation analysis (CA) to look at how students in an advanced EGAP (English for general academic purposes) course discussion test create and manage the silence of a group member during the 7-min session. This is combined with a personal narrative inquiry, coinspired by autoethnography, on the author's participation in the…

  10. The Gift of Silence

    ERIC Educational Resources Information Center

    Haskins, Cathleen

    2011-01-01

    Slowing down, quieting the mind and body, and experiencing silence nourishes the spirit. Montessori educators are mandated to cultivate not just the intellect but the whole child. They recognize that nurturing the spirit of the child is part of what makes this form of education work so well. This article discusses the benefits of stillness and…

  11. The Gift of Silence

    ERIC Educational Resources Information Center

    Haskins, Cathleen

    2011-01-01

    Slowing down, quieting the mind and body, and experiencing silence nourishes the spirit. Montessori educators are mandated to cultivate not just the intellect but the whole child. They recognize that nurturing the spirit of the child is part of what makes this form of education work so well. This article discusses the benefits of stillness and…

  12. On Observing Student Silence

    ERIC Educational Resources Information Center

    Amundrud, Thomas

    2011-01-01

    This article uses conversation analysis (CA) to look at how students in an advanced EGAP (English for general academic purposes) course discussion test create and manage the silence of a group member during the 7-min session. This is combined with a personal narrative inquiry, coinspired by autoethnography, on the author's participation in the…

  13. Transgenerational analysis of transcriptional silencing in zebrafish

    PubMed Central

    Akitake, Courtney M.; Macurak, Michelle; Halpern, Marnie E.; Goll, Mary G.

    2011-01-01

    The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation. PMID:21223961

  14. Screening and identification of virus-encoded RNA silencing suppressors.

    PubMed

    Karjee, Sumona; Islam, Mohammad Nurul; Mukherjee, Sunil K

    2008-01-01

    RNA silencing, including RNA interference, is a novel method of gene regulation and one of the potent host-defense mechanisms against the viruses. In the course of evolution, the viruses have encoded proteins with the potential to suppress the host RNA silencing mechanism as a counterdefense strategy. The virus-encoded RNA silencing suppressors (RSSs) can serve as important biological tools to dissect the detailed RNA silencing pathways and also to evolve the antiviral strategies. Screening and identification of the RSSs are indeed of utmost significance in the field of plant biotechnology. We describe two Green Fluorescent Protein (GFP) reporter-based plant assay systems that rely on two different principles, namely reversal of silencing and enhancement of rolling circle replication (RCR) of geminiviral replicon. These proof-of-concept examples and assay systems could be used to screen various plant, animal, and insect viral ORFs for identification of the RSS activities.

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

    DOE PAGES

    Bhatt, Shweta; Gupta, Manoj K.; Khamaisi, Mogher; ...

    2015-08-04

    The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated inmore » Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. Lastly, we propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.« less

  16. Robust cell size checkpoint from spatiotemporal positive feedback loop in fission yeast.

    PubMed

    Yan, Jie; Ni, Xin; Yang, Ling

    2013-01-01

    Cells must maintain appropriate cell size during proliferation. Size control may be regulated by a size checkpoint that couples cell size to cell division. Biological experimental data suggests that the cell size is coupled to the cell cycle in two ways: the rates of protein synthesis and the cell polarity protein kinase Pom1 provide spatial information that is used to regulate mitosis inhibitor Wee1. Here a mathematical model involving these spatiotemporal regulations was developed and used to explore the mechanisms underlying the size checkpoint in fission yeast. Bifurcation analysis shows that when the spatiotemporal regulation is coupled to the positive feedback loops (active Cdc2 promotes its activator, Cdc25, and suppress its inhibitor, Wee1), the mitosis-promoting factor (MPF) exhibits a bistable steady-state relationship with the cell size. The switch-like response from the positive feedback loops naturally generates the cell size checkpoint. Further analysis indicated that the spatial regulation provided by Pom1 enhances the robustness of the size checkpoint in fission yeast. This was consistent with experimental data.

  17. Endocrine-related adverse events associated with immune checkpoint blockade and expert insights on their management.

    PubMed

    Sznol, Mario; Postow, Michael A; Davies, Marianne J; Pavlick, Anna C; Plimack, Elizabeth R; Shaheen, Montaser; Veloski, Colleen; Robert, Caroline

    2017-07-01

    Agents that modulate immune checkpoint proteins, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have become a mainstay in cancer treatment. The clinical benefit afforded by immune checkpoint inhibitors can be accompanied by immune-related adverse events (irAE) that affect the skin, gastrointestinal tract, liver, and endocrine system. The types of irAEs associated with immune checkpoint inhibitors are generally consistent across tumor types. Immune-related endocrine events can affect the pituitary, thyroid, and adrenal glands, as well as other downstream target organs. These events are unique when compared with other irAEs because the manifestations are often irreversible. Immune-related endocrine events are typically grade 1/2 in severity and often present with non-specific symptoms, making them difficult to diagnose. The mechanisms underlying immune-related target organ damage in select individuals remain mostly undefined. Management includes close patient monitoring, appropriate laboratory testing for endocrine function, replacement of hormones, and consultation with an endocrinologist when appropriate. An awareness of the symptoms and management of immune-related endocrine events may aid in the safe and appropriate use of immune checkpoint inhibitors in clinical practice. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  18. The mitotic checkpoint complex (MCC): looking back and forth after 15 years

    PubMed Central

    Liu, Song-Tao; Zhang, Hang

    2017-01-01

    The mitotic checkpoint is a specialized signal transduction pathway that contributes to the fidelity of chromosome segregation. The signaling of the checkpoint originates from defective kinetochore-microtubule interactions and leads to formation of the mitotic checkpoint complex (MCC), a highly potent inhibitor of the Anaphase Promoting Complex/Cyclosome (APC/C)—the E3 ubiquitin ligase essential for anaphase onset. Many important questions concerning the MCC and its interaction with APC/C have been intensively investigated and debated in the past 15 years, such as the exact composition of the MCC, how it is assembled during a cell cycle, how it inhibits APC/C, and how the MCC is disassembled to allow APC/C activation. These efforts have culminated in recently reported structure models for human MCC:APC/C supra-complexes at near-atomic resolution that shed light on multiple aspects of the mitotic checkpoint mechanisms. However, confusing statements regarding the MCC are still scattered in the literature, making it difficult for students and scientists alike to obtain a clear picture of MCC composition, structure, function and dynamics. This review will comb through some of the most popular concepts or misconceptions about the MCC, discuss our current understandings, present a synthesized model on regulation of CDC20 ubiquitination, and suggest a few future endeavors and cautions for next phase of MCC research.

  19. Spindle assembly checkpoint regulation of chromosome segregation in mammalian oocytes.

    PubMed

    Polanski, Zbigniew

    2013-01-01

    The spindle assembly checkpoint (SAC) is a surveillance mechanism that monitors the quality of the spindle during division and blocks anaphase entry in the presence of anomalies that could result in erroneous segregation of the chromosomes. Because human aneuploidy is mainly linked to the erroneous segregation of genetic material in oocytes, the issue of the effectiveness of the SAC in female meiosis is especially important. The present review summarises our understanding of the SAC control of mammalian oocyte meiosis, including its possible impact on the incidence of embryonic aneuploidy. Owing to the peculiarities of cell cycle control in female meiosis, the integration of the SAC within such a specific environment results in several unusual situations, which are also discussed.

  20. 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.

  1. Silence: a double-edged sword.

    PubMed

    Bunkers, Sandra Schmidt

    2013-01-01

    The author in this column discusses the concept silence and the contradictions present with silence. Silence can be experienced in the following life patterns: silence as a pattern of surrendering to moments of awakening; silence as a pattern of bearing witness to life story; and, silence as a pattern of betrayal of trust. Each of these patterns is explored with their relevance for nursing.

  2. Two different mitotic checkpoint inhibitors of the anaphase-promoting complex/cyclosome antagonize the action of the activator Cdc20

    PubMed Central

    Eytan, Esther; Braunstein, Ilana; Ganoth, Dvora; Teichner, Adar; Hittle, James C.; Yen, Tim J.; Hershko, Avram

    2008-01-01

    The mitotic checkpoint system ensures the fidelity of chromosome segregation by preventing the completion of mitosis in the presence of any misaligned chromosome. When activated, it blocks the initiation of anaphase by inhibiting the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Little is known about the biochemical mechanisms by which this system inhibits APC/C, except for the existence of a mitotic checkpoint complex (MCC) inhibitor of APC/C composed of the APC/C activator Cdc20 associated with the checkpoint proteins Mad2, BubR1, and Bub3. We have been studying the mechanisms of the mitotic checkpoint system in extracts that reproduce its downstream events. We found that inhibitory factors are associated with APC/C in the checkpoint-arrested state, which can be recovered from immunoprecipitates. Only a part of the inhibitory activity was caused by MCC [Braunstein I, Miniowitz S, Moshe Y, Hershko A (2007) Proc Natl Acad Sci USA 104:4870–4875]. Here, we show that during exit from checkpoint, rapid disassembly of MCC takes place while APC/C is still inactive. This observation suggested the possible involvement of multiple factors in the regulation of APC/C by the mitotic checkpoint. We have separated a previously unknown inhibitor of APC/C from MCC. This inhibitor, called mitotic checkpoint factor 2 (MCF2), is associated with APC/C only in the checkpoint-arrested state. The inhibition of APC/C by both MCF2 and MCC was decreased at high concentrations of Cdc20. We propose that both MCF2 and MCC inhibit APC/C by antagonizing Cdc20, possibly by interaction with the Cdc20-binding site of APC/C. PMID:18591651

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. Chromodomain protein Swi6-mediated role of DNA polymerase alpha in establishment of silencing in fission Yeast.

    PubMed

    Ahmed, S; Saini, S; Arora, S; Singh, J

    2001-12-21

    Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, we analyzed several temperature-sensitive mutants of DNA polymerase alpha. We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase alpha in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol alpha and is delocalized away from the silent mat loci in the mutant. Our results demonstrate a role of DNA polymerase alpha in the establishment of silencing. We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.

  8. Postreplication gaps at UV lesions are signals for checkpoint activation

    PubMed Central

    Callegari, A. John; Clark, Emily; Pneuman, Amanda; Kelly, Thomas J.

    2010-01-01

    Exposure of eukaryotic cells to UV light induces a checkpoint response that delays cell-cycle progression after cells enter S phase. It has been hypothesized that this checkpoint response provides time for repair by signaling the presence of structures generated when the replication fork encounters UV-induced DNA damage. To gain insight into the nature of the signaling structures, we used time-lapse microscopy to determine the effects of deficiencies in translesion DNA polymerases on the checkpoint response of the fission yeast Schizosaccharomyces pombe. We found that disruption of the genes encoding translesion DNA polymerases Polκ and Polη significantly prolonged the checkpoint response, indicating that the substrates of these enzymes are signals for checkpoint activation. Surprisingly, we found no evidence that the translesion polymerases Rev1 and Polζ repair structures that are recognized by the checkpoint despite their role in maintaining viability after UV irradiation. Quantitative flow cytometry revealed that cells lacking translesion polymerases replicate UV-damaged DNA at the same rate at WT cells, indicating that the enhanced checkpoint response of cells lacking Polκ and Polη is not the result of stalled replication forks. These observations support a model in which postreplication DNA gaps with unrepaired UV lesions in the template strand act both as substrates for translesion polymerases and as signals for checkpoint activation. PMID:20404181

  9. 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

  10. Homology-dependent gene silencing and host defense in plants.

    PubMed

    Matzke, Marjori A; Aufsatz, Werner; Kanno, Tatsuo; Mette, M Florian; Matzke, Antonius J M

    2002-01-01

    Analyses of transgene silencing phenomena in plants and other organisms have revealed the existence of epigenetic silencing mechanisms that are based on recognition of nucleic acid sequence homology at either the DNA or RNA level. Common triggers of homology-dependent gene silencing include inverted DNA repeats and double-stranded RNA, a versatile silencing molecule that can induce both degradation of homologous RNA in the cytoplasm and methylation of homologous DNA sequences in the nucleus. Inverted repeats might be frequently associated with silencing because they can potentially interact in cis and in trans to trigger DNA methylation via homologous DNA pairing, or they can be transcribed to produce double-stranded RNA. Homology-dependent gene silencing mechanisms are ideally suited for countering natural parasitic sequences such as transposable elements and viruses, which are usually present in multiple copies and/or produce double-stranded RNA during replication. These silencing mechanisms can thus be regarded as host defense strategies to foreign or invasive nucleic acids. The high content of transposable elements and, in some cases, endogenous viruses in many plant genomes suggests that host defenses do not always prevail over invasive sequences. During evolution, slightly faulty genome defense responses probably allowed transposable elements and viral sequences to accumulate gradually in host chromosomes and to invade host genes. Possible beneficial consequences of this "foreign" DNA buildup include the establishment of genome defense-derived epigenetic control mechanisms for regulating host gene expression and acquired hereditary immunity to some viruses.

  11. The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects.

    PubMed Central

    Craven, R J; Petes, T D

    2001-01-01

    Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family. Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing. We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p. We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis. Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells. Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p. PMID:11333225

  12. 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

  13. APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

    PubMed Central

    Willis, Jeremy; Patel, Yogin; Lentz, Barry L.; Yan, Shan

    2013-01-01

    The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H2O2-damaged chromatin. APE2 3′-phosphodiesterase and 3′-5′ exonuclease activity is essential for single-stranded DNA generation in the 3′–5′ direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1–dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response. PMID:23754435

  14. [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!

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Telomeric Trans-Silencing: An Epigenetic Repression Combining RNA Silencing and Heterochromatin Formation

    PubMed Central

    Josse, Thibaut; Teysset, Laure; Todeschini, Anne-Laure; Sidor, Clara M; Anxolabéhère, Dominique; Ronsseray, Stéphane

    2007-01-01

    The study of P-element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. Here, we report that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. We show that this silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3–7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway. PMID:17941712

  20. Rethinking the Day of Silence

    ERIC Educational Resources Information Center

    Murphy, Adriana

    2013-01-01

    Back in 2006, 7th and 8th graders at Green Acres, the K-8 independent school where the author taught in suburban Maryland, participated in the Day of Silence. The Day of Silence is a national event: Students across the country take a one-day pledge of silence to show that they want to make schools safe for all students, regardless of their sexual…

  1. Rethinking the Day of Silence

    ERIC Educational Resources Information Center

    Murphy, Adriana

    2013-01-01

    Back in 2006, 7th and 8th graders at Green Acres, the K-8 independent school where the author taught in suburban Maryland, participated in the Day of Silence. The Day of Silence is a national event: Students across the country take a one-day pledge of silence to show that they want to make schools safe for all students, regardless of their sexual…

  2. 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

  3. 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

  4. Fulminant Myocarditis with Combination Immune Checkpoint Blockade

    PubMed Central

    Johnson, Douglas B.; Balko, Justin M.; Compton, Margaret L.; Chalkias, Spyridon; Gorham, Joshua; Xu, Yaomin; Hicks, Mellissa; Puzanov, Igor; Alexander, Matthew R.; Bloomer, Tyler L.; Becker, Jason; Slosky, David A.; Phillips, Elizabeth J.; Pilkinton, Mark A.; Craig-Owens, Laura; Kola, Nina; Plautz, Gregory; Reshef, Daniel S.; Deutsch, Jonathan S.; Deering, Raquel P.; Olenchock, Benjamin A.; Lichtman, Andrew H.; Roden, Dan M.; Seidman, Christine E.; Koralnik, Igor J.; Seidman, Jonathan G.; Hoffman, Robert D.; Taube, Janis M.; Diaz, Luis A.; Anders, Robert A.; Sosman, Jeffrey A.; Moslehi, Javid J.

    2016-01-01

    Summary Immune checkpoint inhibitors significantly improve clinical outcomes in numerous malignancies, but high-grade immune-related adverse events can occur, particularly with combination immunotherapy. Herein, we report two melanoma patients who developed fatal myocarditis following treatment with ipilimumab and nivolumab. Both patients developed myositis with rhabdomyolysis, early progressive and refractory cardiac electrical instability, and myocarditis with robust T-cell and macrophage infiltrates. Selective clonal T-cell populations infiltrating the myocardium were identical to those present in tumor and skeletal muscle. Pharmacovigilance data revealed that myocarditis occurred in 0.27% of patients treated with ipilimumab/nivolumab, suggesting this is a rare, potentially fatal, T-cell-driven drug reaction. PMID:27806233

  5. Applying gene silencing technology to contraception

    PubMed Central

    Dissen, Gregory A.; Lomniczi, Alejandro; Boudreau, Ryan L.; Chen, Yong Hong; Davidson, Beverly L.; Ojeda, Sergio R.

    2013-01-01

    Contents Population control of feral animals is often difficult, as it can be dangerous for the animals, labor intensive, and expensive. Therefore, a useful tool for control of animal populations would be a nonsurgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to noninvasively sterilize cats and dogs. For this approach to succeed it has to meet several conditions: The target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include: a) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction, and b) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy. PMID:23279544

  6. "Listening Silence" and Its Discursive Effects

    ERIC Educational Resources Information Center

    Applebaum, Barbara

    2016-01-01

    While researchers have studied how white silence protects white innocence and white ignorance, in this essay Barbara Applebaum explores a form of white silence that she refers to as "listening silence" in which silence protects white innocence but does not necessarily promote resistance to learning. White listening silence can appear to…

  7. "Listening Silence" and Its Discursive Effects

    ERIC Educational Resources Information Center

    Applebaum, Barbara

    2016-01-01

    While researchers have studied how white silence protects white innocence and white ignorance, in this essay Barbara Applebaum explores a form of white silence that she refers to as "listening silence" in which silence protects white innocence but does not necessarily promote resistance to learning. White listening silence can appear to…

  8. 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.

  9. 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.

  10. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Edwin Hubble's Silence

    NASA Astrophysics Data System (ADS)

    Lago, D.

    2013-04-01

    In late 1928 Edwin Hubble was right in the middle of using V. M. Slipher's redshift data to prove that the universe is expanding, when Hubble's boss, George Hale, directed him to drop everything and rush to the Grand Canyon and test it as a possible site for Hale's planned 200-inch telescope. On his way, Hubble stopped at Lowell Observatory and met with V. M. Slipher. The letters both men wrote about this visit suggest that Hubble never said a word about his being in the middle of using Slipher's research to transform the universe. At the least, this silence is symbolic of the silence with which astronomical history has often treated Slipher's work. A survey of the historical literature suggests several reasons for this. Theorists and observers in astronomy (and other sciences) have long had different perspectives about how science works, and those who place more importance on theory have tended to credit the idea of the expanding universe to the theorists. Also, many sources indicate that Edwin Hubble was not a modest man or generous about sharing credit.

  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. DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD

    PubMed Central

    Lim, Jong-Won; Snider, Lauren; Yao, Zizhen; Tawil, Rabi; Van Der Maarel, Silvère M.; Rigo, Frank; Bennett, C. Frank; Filippova, Galina N.; Tapscott, Stephen J.

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD. PMID:26041815

  14. DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD.

    PubMed

    Lim, Jong-Won; Snider, Lauren; Yao, Zizhen; Tawil, Rabi; Van Der Maarel, Silvère M; Rigo, Frank; Bennett, C Frank; Filippova, Galina N; Tapscott, Stephen J

    2015-09-01

    Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints.

    PubMed

    Mu, Jung-Jung; Wang, Yi; Luo, Hao; Leng, Mei; Zhang, Jinglan; Yang, Tao; Besusso, Dario; Jung, Sung Yun; Qin, Jun

    2007-06-15

    ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

  16. A new look at NHTSA's evaluation of the 1984 Charlottesville Sobriety Checkpoint Program: implications for current checkpoint issues.

    PubMed

    Voas, Robert B

    2008-03-01

    Currently, the implementation of sobriety checkpoint programs, which have been demonstrated to be effective in reducing alcohol-related crashes, is limited by the belief that they require large consignments of police officers and result in few arrests. However, one of the earliest evaluations of a checkpoint program in Charlottesville, Virginia, demonstrated that effective checkpoints could be mounted in which police officers made as many arrests as officers on regular patrols. That study was printed by the NHTSA but was not published in a peer-reviewed journal. Because of its significance to current issues in the staffing of and procedures for checkpoint operations, this article reanalyzes the results of that study and describes the procedures implemented in checkpoints. A before-and-after control design was used to measure the change in nighttime crashes from three baseline years to the program year. Two analyses were conducted: the first on the percentage of all crashes occurring at night in the test city--Charlottesville--and the second on the percentage of all nighttime crashes in the state of Virginia that occurred in the test city. In addition, three waves of random-digit-dialing telephone surveys were conducted: one before and two during the checkpoint program in the test city, and the comparison city, Blacksburg. Finally, the number of impaired-driving arrests per officer hour at the checkpoints was compared with the number of arrests per hour by officers on regular patrol and the effect on arrests of the use of passive sensors was determined. The monthly percentage of nighttime crashes in Charlottesville was reduced by 17% (p = 000) in relation to the baseline level. The percentage of nighttime crashes in the state of Virginia that occurred in Charlottesville was reduced by 11% (p = .013) from baseline levels. Drivers arrested at checkpoints had lower BACs than those arrested by the regular patrols; however, the conviction rates were the same. The arrest

  17. Silence, an Eye of Knowledge

    ERIC Educational Resources Information Center

    Aghamohammadi, Mehdi

    2017-01-01

    One of the conspicuous features of the twentieth-century West was silence. This idea could be supported by examining reflections of Ludwig Wittgenstein, Fritz Mauthner, John Cage, Samuel Beckett, Ihab Hassan, Franz Kafka, Wassily Kandinsky, Jean-Paul Sartre, Virginia Woolf, Wolfgang Iser, Jacques Derrida, and Pierre Macherey. To me, silence is not…

  18. Organizational Silence in Sports Employees

    ERIC Educational Resources Information Center

    Bastug, Gulsum; Pala, Adem; Yilmaz, Taner; Duyan, Mehdi; Gunel, Ilker

    2016-01-01

    Organizational silence can be defined as a way of behaviour belonging to men and women employees in the organization exhibited without reflecting their feelings, ideas, concerns and suggestions related with their workplaces, works for which they are responsible or other activities of the organization. In the period of organizational silence,…

  19. 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

  20. 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

  1. 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

  2. Identification of Late Larval Stage Developmental Checkpoints in Caenorhabditis elegans Regulated by Insulin/IGF and Steroid Hormone Signaling Pathways

    PubMed Central

    Schindler, Adam J.; Baugh, L. Ryan; Sherwood, David R.

    2014-01-01

    Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint. PMID:24945623

  3. The Evolving Role of Immune Checkpoint Inhibitors in Cancer Treatment

    PubMed Central

    Chow, Laura Q.M.

    2015-01-01

    Traditional treatment modalities for advanced cancer (radiotherapy, chemotherapy, or targeted agents) act directly on tumors to inhibit or destroy them. Along with surgery, these modalities are predominantly palliative, with toxicity and only modest improvements in survival in patients with advanced solid tumors. Accordingly, long-term survival rates for most patients with advanced cancer remain low, thus there is a need for cancer treatments with favorable benefit and toxicity profiles that can potentially result in long-term survival. The immune system plays a critical role in the recognition and eradication of tumor cells (“immune surveillance”), and immunotherapies based on this concept have been used for decades with some success against a few tumor types; however, most immunotherapies were limited by a lack of either substantial efficacy or specificity, resulting in toxicity. We now have a greater understanding of the complex interactions between the immune system and tumors and have identified key molecules that govern these interactions. This information has revitalized the interest in immunotherapy as an evolving treatment modality using immunotherapeutics designed to overcome the mechanisms exploited by tumors to evade immune destruction. Immunotherapies have potentially complementary mechanisms of action that may allow them to be combined with other immunotherapeutics, chemotherapy, targeted therapy, or other traditional therapies. This review discusses the concepts and data behind immunotherapies, with a focus on the checkpoint inhibitors and their responses, toxicities, and potential for long-term survival, and explores promising single-agent and combination therapies in development. Implications for Practice: Immunotherapy is an evolving treatment approach based on the role of the immune system in eradicating cancer. An example of an immunotherapeutic is ipilimumab, an antibody that blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4) to augment

  4. Identification of Novel Mitotic Checkpoint Genes in Breast Cancer

    DTIC Science & Technology

    2002-05-01

    chromosomal instability to alterations in genes and proteins that regulate the mitotic spindle checkpoint. We first characterized the degree of chromosomal...chromosome attachment to mitotic spindle microtubules . Genetic events disrupting the mitotic spindle checkpoint may allow cells to proliferate with... Regulates onset of anaphase via monitoring S. cerevisiae, spans 1382 bp MAD2A 25 kDa kinetochore/ spindle attachments at metaphase homolog-like 1 693

  5. Publicized sobriety checkpoint programs: a community guide systematic review.

    PubMed

    Bergen, Gwen; Pitan, Adesola; Qu, Shuli; Shults, Ruth A; Chattopadhyay, Sajal K; Elder, Randy W; Sleet, David A; Coleman, Heidi L; Compton, Richard P; Nichols, James L; Clymer, John M; Calvert, William B

    2014-05-01

    Publicized sobriety checkpoint programs deter alcohol-impaired driving by stopping drivers systematically to assess their alcohol impairment. Sobriety checkpoints were recommended in 2001 by the Community Preventive Services Task Force for reducing alcohol-impaired driving, based on strong evidence of effectiveness. Since the 2001 review, attention to alcohol-impaired driving as a U.S. public health problem has decreased. This systematic review was conducted to determine if available evidence supports the effectiveness of publicized sobriety checkpoint programs in reducing alcohol-impaired driving, given the current context. The economic costs and benefits of the intervention were also assessed. This review focused on studies that evaluated the effects of publicized sobriety checkpoint programs on alcohol-involved crash fatalities. Using Community Guide methods, a systematic search was conducted for studies published between July 2000 and March 2012 that assessed the effectiveness of publicized sobriety checkpoint programs. Fourteen evaluations of selective breath testing and one of random breath testing checkpoints met the inclusion criteria for the systematic review, conducted in 2012. Ten evaluations assessed the effects of publicized sobriety checkpoint programs on alcohol-involved crash fatalities, finding a median reduction of 8.9% in this crash type (interquartile interval=-16.5%, -3.5%). Five economic evaluations showed benefit-cost ratios ranging from 2:1 to 57:1. The number of studies, magnitude of effect, and consistency of findings indicate strong evidence of the effectiveness of publicized sobriety checkpoint programs in reducing alcohol-involved crash fatalities. Economic evidence shows that these programs also have the potential for substantial cost savings. Published by Elsevier Inc.

  6. 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

  7. Neuron-restrictive Silencer Factor (NRSF) Represses Cocaine- and Amphetamine-regulated Transcript (CART) Transcription and Antagonizes cAMP-response Element-binding Protein Signaling through a Dual NRSE Mechanism*

    PubMed Central

    Zhang, Jing; Wang, Sihan; Yuan, Lin; Yang, Yinxiang; Zhang, Bowen; Liu, Qingbin; Chen, Lin; Yue, Wen; Li, Yanhua; Pei, Xuetao

    2012-01-01

    Cocaine- and amphetamine-regulated transcript (CART) peptide plays a pivotal role in neuroprotection against stroke-related brain injury. However, the regulatory mechanism on CART transcription, especially the repression mechanism, is not fully understood. Here, we show that the transcriptional repressor neuron-restrictive silencer elements (NRSF, also known as REST) represses CART expression through direct binding to two NRSF-binding elements (NRSEs) in the CART promoter and intron 1 (named pNRSE and iNRSE, respectively). EMSA show that NRSF binds to pNRSE and iNRSE directly in vitro. ChIP assays show that NRSF recruits differential co-repressor complexes including CoREST and HDAC1 to these NRSEs. The presence of both NRSEs is required for efficient repression of CART transcription as indicated by reporter gene assays. NRSF overexpression antagonizes forskolin-mediated up-regulation of CART mRNA and protein. Ischemia insult triggered by oxygen-glucose deprivation (OGD) enhances NRSF mRNA levels and then NRSF antagonizes the CREB signaling on CART activation, leading to augmented cell death. Depletion of NRSF in combination with forskolin treatment increases neuronal survival after ischemic insult. These findings reveal a novel dual NRSE mechanism by which NRSF represses CART expression and suggest that NRSF may serve as a therapeutic target for stroke treatment. PMID:23086924

  8. The eerie silence

    NASA Astrophysics Data System (ADS)

    Davies, Paul

    2010-03-01

    Whether or not we are alone in the universe is one of the great outstanding questions of existence. For thousands of years it was restricted to the realm of philosophy and theology, but 50 years ago it became part of science. In April 1960 a young US astronomer, Frank Drake, began using a radio telescope to investigate whether signals from an extraterrestrial community might be coming our way. Known as the Search for Extraterrestrial Intelligence, or SETI, it has grown into a major international enterprise, involving scientific institutions in several countries. Apart from a few oddities, however, all that the radio astronomers have encountered is an eerie silence. So is humankind the only technological civilization in the universe after all? Or might we be looking for the wrong thing in the wrong place at the wrong time?

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. Endocrine dysfunction following immune checkpoint inhibitor therapy.

    PubMed

    Konda, Bhavana; Nabhan, Fadi; Shah, Manisha H

    2017-10-01

    Immune checkpoint inhibitors (ICI) represent an important milestone in the modern era of antineoplastic therapy and have ushered optimism amongst oncologists and patients alike. These agents, however, are associated with significant potential toxicities, the importance of which cannot be overstated. The clinical presentation, diagnosis, and management strategies of immune-related endocrinopathies associated with ICI use are described in this case-based review. An increasing number of ICI have shown promise in the management of various malignancies in the recent years. These include cytotoxic T lymphocyte antigen-4 inhibitors, programmed cell death 1 (PD-1) antibodies, and PD-ligand 1 (PD-L1) antibodies. Several endocrinopathies, including hypophysitis, thyroid dysfunction, hyperglycemia, and primary adrenal insufficiency, have been associated with the use of these agents. Toxicities may range from mild transient laboratory abnormalities to potentially life-threatening ones, warranting immediate therapeutic intervention. Combination ICI therapies may be associated with a greater risk of endocrine dysfunction when compared with monotherapy. The clinical presentation and laboratory assessment of these patients often pose a diagnostic challenge as they may be confused by the symptoms related to their underlying malignancy or potential associated acute illnesses. ICI use is associated with serious endocrinopathies that may have a nonspecific initial presentation. A constant eye for these symptoms and a systematic approach to diagnosis are essential for prompt initiation of therapy and prevention of significant complications.

  16. Induction of a caffeine-sensitive S-phase cell cycle checkpoint by psoralen plus ultraviolet A radiation.

    PubMed

    Joerges, Christoph; Kuntze, Inge; Herzinger, Thomas; Herzinge, Thomas

    2003-09-18

    Induction of interstrand crosslinks (ICLs) in chromosomal DNA is considered a major reason for the antiproliferative effect of psoralen plus ultraviolet A (PUVA). It is unclear as to whether PUVA-induced cell cycle arrest is caused by ICLs mechanically stalling replication forks or by triggering cell cycle checkpoints. Cell cycle checkpoints serve to maintain genomic stability by halting cell cycle progression to prevent replication of damaged DNA templates or segregation of broken chromosomes. Here, we show that HaCaT keratinocytes treated with PUVA arrest with S-phase DNA content. Cells that had completed DNA replication were not perturbed by PUVA and passed through mitosis. Cells treated with PUVA during G1-phase continued traversing G1 until arresting in early S-phase. PUVA induced rapid phosphorylation of the Chk1 checkpoint kinase at Ser345 and a concomitant decrease in Cdc25A levels. Chk1 phosphorylation, decrease of Cdc25 A levels and S-phase arrest were abolished by caffeine, demonstrating that active checkpoint signaling rather than passive mechanical blockage by ICLs causes the PUVA-induced replication arrest. Overexpression of Cdc25A only partially overrode the S-phase arrest, suggesting that additional signaling events implement PUVA-induced S-phase arrest.

  17. Fission Yeast Apc15 Stabilizes MCC-Cdc20-APC/C Complexes, Ensuring Efficient Cdc20 Ubiquitination and Checkpoint Arrest.

    PubMed

    May, Karen M; Paldi, Flora; Hardwick, Kevin G

    2017-03-28

    During mitosis, cells must segregate the replicated copies of their genome to their daughter cells with extremely high fidelity. Segregation errors lead to an abnormal chromosome number (aneuploidy), which typically results in disease or cell death [1]. Chromosome segregation and anaphase onset are initiated through the action of the multi-subunit E3 ubiquitin ligase known as the anaphase-promoting complex or cyclosome (APC/C [2]). The APC/C is inhibited by the spindle checkpoint in the presence of kinetochore attachment defects [3, 4]. Here we demonstrate that two non-essential APC/C subunits (Apc14 and Apc15) regulate association of spindle checkpoint proteins, in the form of the mitotic checkpoint complex (MCC), with the APC/C. apc14Δ mutants display increased MCC association with the APC/C and are unable to silence the checkpoint efficiently. Conversely, apc15Δ mutants display reduced association between the MCC and APC/C, are defective in poly-ubiquitination of Cdc20, and are checkpoint defective. In vitro reconstitution studies have shown that human MCC-APC/C can contain two molecules of Cdc20 [5-7]. Using a yeast strain expressing two Cdc20 genes with different epitope tags, we show by co-immunoprecipitation that this is true in vivo. MCC binding to the second molecule of Cdc20 is mediated via the C-terminal KEN box in Mad3. Somewhat surprisingly, complexes containing both molecules of Cdc20 accumulate in apc15Δ cells, and the implications of this observation are discussed.

  18. Evolutionary expansion of a regulatory network by counter-silencing

    PubMed Central

    Will, William R.; Bale, Denise H.; Reid, Philip J.; Libby, Stephen J.; Fang, Ferric C.

    2014-01-01

    Horizontal gene transfer plays a major role in bacterial evolution. Successful acquisition of new genes requires their incorporation into existing regulatory networks. This study compares the regulation of conserved genes in the PhoPQ regulon of Salmonella enterica serovar Typhimurium with that of PhoPQ-regulated horizontally-acquired genes, which are silenced by the histone-like protein H-NS. We demonstrate that PhoP up-regulates conserved and horizontally-acquired genes by distinct mechanisms. Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally-acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing. Biochemical analyses show that a horizontally-acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodeling of the H-NS nucleoprotein filament and the subsequent restoration of open complex formation as the central mechanism of counter-silencing. Our results indicate that counter-silencing is favored in the regulatory integration of newly-acquired genes because it is able to accommodate multiple promoter architectures. PMID:25348042

  19. 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.

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

    PubMed Central

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

    2015-01-01

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

  1. 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.

  2. 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

  3. Transient knock down of checkpoint kinase 1 in hematopoietic progenitors is linked to bone marrow toxicity.

    PubMed

    Hu, Wenyue; Zong, Qing; John-Baptiste, Annette; Jessen, Bart

    2011-07-28

    Checkpoint kinase 1 (Chk1) is required for both intra-S phase and G2/M checkpoints in cell cycle, and plays critical roles in maintaining genomic stability and transducing DNA damage response. Chk1 deficiency has been shown to inhibit T-cell differentiation and resulted in severe anemia in a Chk1 heterozygous mouse model. To date, there has been a good correlation between Chk1 inhibition and in vitro bone marrow toxicity among small molecule inhibitors. To better understand the role of Chk1 in hematopoiesis, we conducted transient Chk1 gene silencing in human bone marrow progenitor cells using siRNA and electroporation. At 48h post electroporation, approximately 70% inhibition of Chk1 was confirmed using real-time RT-PCR and immunoblotting, which resulted in more than 60% reduction in cell count when compared to the non-specific siRNA control on day 6 post-electroporation. This result was confirmed using a colony forming unit assay, where reduced number in both erythroid and granulocyte colonies was observed with Chk1 siRNA treatment. The Chk1 gene inhibition in bone marrow progenitor cells resulted in significant induction of apoptosis, but not cell cycle arrest, as assessed using flow cytometry. In this study an effective method to knock down a gene of interest was established in hard-to-transfect hematopoietic stem cells. Furthermore, our results support a direct role of Chk1 in maintaining normal hematopoiesis in the bone marrow. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  4. Viral suppressors of RNA silencing in Wheat mosaic virus (WMoV)

    USDA-ARS?s Scientific Manuscript database

    RNA silencing is the most effective antiviral adaptive defense mechanism mounted in higher plants to combat viral infection and proliferation. However, viruses have developed a variety of efficient counter-defense mechanisms by suppression of RNA silencing (VSR) in order to successfully impede the h...

  5. Silence and the Notion of the Commons.

    ERIC Educational Resources Information Center

    Franklin, Ursula

    1994-01-01

    Stresses the value of silence, the right to have silence, and how technology has manipulated the sound environment and therefore taken silence out of common availability. Discusses noise pollution and the manipulative use of sound for private gain. Suggests taking action to restore the right to silence. (LP)

  6. Silence and the Notion of the Commons.

    ERIC Educational Resources Information Center

    Franklin, Ursula

    1994-01-01

    Stresses the value of silence, the right to have silence, and how technology has manipulated the sound environment and therefore taken silence out of common availability. Discusses noise pollution and the manipulative use of sound for private gain. Suggests taking action to restore the right to silence. (LP)

  7. How to use intentional silence.

    PubMed

    Kemerer, Douglas

    2016-09-07

    Rationale and key points This article explains intentional silence, which can provide a therapeutic nursing presence that demonstrates compassion and respect for the patient. » Intentional silence can enhance the therapeutic relationship between the nurse and patient. » Intentional silence can be used to reduce the patient's emotional lability by ensuring that they feel listened to. » It is important that nurses are able to respond therapeutically to rhetorical statements and/or those concerning serious or severe clinical circumstances. » The nurse can use intentional silence to support the patient in acknowledging, processing and reflecting on changes in their health. Reflective activity 'How to' articles can help to update your practice and ensure it remains evidence based. Apply this article to your practice. Reflect on and write a short account of: 1. How this article will change your practice. 2. How you could use this article to educate your colleagues. Subscribers can upload their reflective accounts at rcni.com/portfolio.

  8. Chloroplast division checkpoint in eukaryotic algae

    PubMed Central

    Sumiya, Nobuko; Fujiwara, Takayuki; Era, Atsuko; Miyagishima, Shin-ya

    2016-01-01

    Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase–specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle. PMID:27837024

  9. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

    NASA Astrophysics Data System (ADS)

    Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

    2004-11-01

    Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

  10. Requirement for the phospho-H2AX binding module of Crb2 in double-strand break targeting and checkpoint activation.

    PubMed

    Sanders, Steven L; Arida, Ahmad R; Phan, Funita P

    2010-10-01

    Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.

  11. Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

    PubMed

    Antonov, A S; Munn, D H; Kolodgie, F D; Virmani, R; Gerrity, R G

    1997-06-15

    Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

  12. Activation of the S-Phase Checkpoint Inhibits Degradation of the F-Box Protein Dia2 ▿

    PubMed Central

    Kile, Andrew C.; Koepp, Deanna M.

    2010-01-01

    A stable genome is critical to cell viability and proliferation. During DNA replication, the S-phase checkpoint pathway responds to replication stress. In budding yeast, the chromatin-bound F-box protein Dia2 is required to maintain genomic stability and may help replication complexes overcome sites of damaged DNA and natural fragile regions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases. We show here that Dia2 is itself targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint pathway inhibits Dia2 protein degradation. S-phase checkpoint mutants fail to stabilize Dia2 in response to replication stress. Deletion of DIA2 from these checkpoint mutants exacerbates their sensitivity to hydroxyurea, suggesting that stabilization of Dia2 contributes to the replication stress response. Unlike the case for other F-box proteins, deletion of the F-box domain in Dia2 does not stabilize the protein. Rather, an N-terminal domain that is also required for nuclear localization is necessary for degradation. When a strong nuclear localization signal (NLS) is added to dia2 mutants lacking this domain, the Dia2 protein is both stable and nuclear. Together, our results suggest that Dia2 protein turnover does not involve an autocatalytic mechanism and that Dia2 proteolysis is inhibited by activation of the replication stress response. PMID:19858292

  13. Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required For Mitotic ATM Activation and the Spindle Checkpoint

    PubMed Central

    Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T.C.; Fu, Li; Xu, Bo

    2011-01-01

    Summary The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically-activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis. PMID:22099307

  14. Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint.

    PubMed

    Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T C; Fu, Li; Xu, Bo

    2011-11-18

    The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Experimental studies of a drumlike silencer

    NASA Astrophysics Data System (ADS)

    Choy, Y. S.; Huang, Lixi

    2002-11-01

    The theoretical finding of the broadband performance of a reactive silencer is validated experimentally. The silencer consists of two highly stretched membranes lining part of the duct and backed by two long and shallow cavities. The test rig was built with a small square duct of 5 cm in dimension, and each cavity is 5 cm deep and 25 cm long. Two types of metal foils, stainless steel and copper, were used, and the lowest membrane-to-air mass ratio was 1.3. A transmission loss in excess of 10 dB was achieved over more than one octave band. For one configuration close to the optimal parameters, the predicted ratio of the frequency band limits is 2.47, while the experiment gave 2.35. Three spectral peaks were found in the stopband, as predicted, but the peaks were broader than prediction, indicating the presence of significant sound energy dissipation mechanisms. Comparison with theoretical simulation shows that the cavity damping dominates over membrane friction. Tests using heavier membranes and membrane with different levels of tension also agree with predictions. Issues of practical implementation of the concept as a flow-through silencer are also addressed. copyright 2002 Acoustical Society of America.

  16. Sterigmatocystin-induced checkpoint adaptation depends on Chk1 in immortalized human gastric epithelial cells in vitro.

    PubMed

    Jiang, Xiujuan; Wang, Juan; Xing, Lingxiao; Shen, Haitao; Lian, Weiguang; Yi, Li; Zhang, Donghui; Yang, Haiyan; Liu, Jianghui; Zhang, Xianghong

    2017-01-01

    Sterigmatocystin (ST) is a common contaminant detected in food and animal feed that has been recognized as a possible human carcinogen. Our previous studies demonstrate that ST causes DNA damage and subsequently triggers cell cycle arrest in G2 and apoptosis in immortalized human gastric epithelial cells (GES-1). Recently, studies have shown that in certain contexts, cells with DNA damage may escape checkpoint arrest and enter mitosis without repairing the damage. The term for this process is "checkpoint adaptation," and it increases the risk of unstable genome propagation, which may contribute to carcinogenesis. Thus, we aimed to investigate whether checkpoint adaptation occurs in GES-1 cells treated with ST and explored the underlying molecular mechanisms that contribute to this phenotype. In this study, we found that ST treatment for 24 h in GES-1 cells led to an initial G2 arrest; however, a fraction of GES-1 cells became large and rounded, and the number of p-H3-positive cells increased sharply after ST treatment for 48 h. Moreover, collection of the large and rounded cells by mechanical shake-off revealed that the majority of these large cells were found in the mitotic phase of the cell cycle. Importantly, we found that these rounded cells entered mitosis despite damaged DNA and that a small subset of this cell population survived and continued to propagate. These results suggest that ST induces an initial G2 arrest that is subsequently followed by G2 phase checkpoint adaptation, which may potentially promote genomic instability and result in tumorigenesis. Furthermore, we showed that activation of Chk1 contributes to the G2 arrest in GES-1 cells that are treated with ST for 24 h and that prolonged treatment of cells with ST for 48 h led to a decrease in the total protein and phosphorylation levels of Chk1 in mitotic cells, indicating that checkpoint adaptation may be driven by inactivation of Chk1. Knockdown studies confirmed that cells entered mitosis

  17. 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.

  18. 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

  19. 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.

  20. 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

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

    PubMed

    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. Copyright© Ferrata Storti Foundation.

  2. 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

  3. 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.

  4. Detailed Structural-Functional Analysis of the Krüppel-like Factor 16 (KLF16) Transcription Factor Reveals Novel Mechanisms for Silencing Sp/KLF Sites Involved in Metabolism and Endocrinology*

    PubMed Central

    Daftary, Gaurang S.; Lomberk, Gwen A.; Buttar, Navtej S.; Allen, Thomas W.; Grzenda, Adrienne; Zhang, Jinsan; Zheng, Ye; Mathison, Angela J.; Gada, Ravi P.; Calvo, Ezequiel; Iovanna, Juan L.; Billadeau, Daniel D.; Prendergast, Franklyn G.; Urrutia, Raul

    2012-01-01

    Krüppel-like factor (KLF) proteins have elicited significant attention due to their emerging key role in metabolic and endocrine diseases. Here, we extend this knowledge through the biochemical characterization of KLF16, unveiling novel mechanisms regulating expression of genes involved in reproductive endocrinology. We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes). KLF16 also regulated the expression of several genes essential for metabolic and endocrine processes in sex steroid-sensitive uterine cells. Mechanistically, we determined that KLF16 possesses an activation domain that couples to histone acetyltransferase-mediated pathways, as well as a repression domain that interacts with the histone deacetylase chromatin-remodeling system via all three Sin3 isoforms, suggesting a higher level of plasticity in chromatin cofactor selection. Molecular modeling combined with molecular dynamic simulations of the Sin3a-KLF16 complex revealed important insights into how this interaction occurs at an atomic resolution level, predicting that phosphorylation of Tyr-10 may modulate KLF16 function. Phosphorylation of KLF16 was confirmed by in vivo 32P incorporation and controlled by a Y10F site-directed mutant. Inhibition of Src-type tyrosine kinase signaling as well as the nonphosphorylatable Y10F mutation disrupted KLF16-mediated gene silencing, demonstrating that its function is regulatable rather than constitutive. Subcellular localization studies revealed that signal-induced nuclear translocation and euchromatic compartmentalization constitute an additional mechanism for regulating KLF16 function. Thus, this study lends insights on key biochemical mechanisms for regulating KLF sites involved in reproductive biology. These data also contribute to the new functional information that is applicable to understanding KLF16 and other highly related KLF proteins. PMID:22203677

  5. Human factors and pathways essential for mediating epigenetic gene silencing.

    PubMed

    Poleshko, Andrey; Kossenkov, Andrew V; Shalginskikh, Natalia; Pecherskaya, Anna; Einarson, Margret B; Marie Skalka, Anna; Katz, Richard A

    2014-09-01

    Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming.

  6. Loss of Smu1 function de-represses DNA replication and over-activates ATR-dependent replication checkpoint.

    PubMed

    Ren, Laifeng; Liu, Yao; Guo, Liandi; Wang, Haibin; Ma, Lei; Zeng, Ming; Shao, Xin; Yang, Chunlei; Tang, Yaxiong; Wang, Lei; Liu, Cong; Li, Mingyuan

    2013-06-28

    Smu1 is an evolutionarily conserved gene that encodes a member of the WD40-repeat protein family. Disruption of Smu1 function leads to multiple cellular defects including chromosomal instability, aberrant DNA replication and alternative RNA splicing events. In this paper, we show that Smu1 is a chromatin-bound protein that functions as a negative regulator of DNA replication. Knockdown of Smu1 gene expression promotes excessive incorporation of dNTP analogue, implicating the acceleration of DNA synthesis. Smu1-silenced cells show an excessive activation of replication checkpoint in response to ultraviolate (UV) or hydroxyurea treatment, indicating that abnormal stimulation of DNA replication leads to instability of genomic structure. Hence, we propose that Smu1 participates in the protection of genomic integrity by negatively regulating the process of DNA synthesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. 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.

  8. Breast cancer immunobiology driving immunotherapy: vaccines and immune checkpoint blockade

    PubMed Central

    Emens, Leisha A

    2013-01-01

    Breast cancer is immunogenic, and infiltrating immune cells in primary breast tumors convey important clinical prognostic and predictive information. Furthermore, the immune system is critically involved in clinical responses to some standard cancer therapies. Early breast cancer vaccine trials have established the safety and bioactivity of breast cancer immunotherapy, with hints of clinical activity. Novel strategies for modulating regulators of immunity, including regulatory T cells, myeloid-derived suppressor cells and immune checkpoint pathways (monoclonal antibodies specific for the cytotoxic T-lymphocyte antigen-4 or programmed death), are now available. In particular, immune checkpoint blockade has enormous therapeutic potential. Integrative breast cancer immunotherapies that strategically combine established breast cancer therapies with breast cancer vaccines, immune checkpoint blockade or both should result in durable clinical responses and increased cures. PMID:23253225

  9. 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.

  10. CDK5RAP2 is required for spindle checkpoint function

    PubMed Central

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

    2009-01-01

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

  11. 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

  12. 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

  13. Retrovirus silencer blocking by the cHS4 insulator is CTCF independent

    PubMed Central

    Yao, Shuyuan; Osborne, Cameron S.; Bharadwaj, Rikki R.; Pasceri, Peter; Sukonnik, Tanya; Pannell, Dylan; Recillas-Targa, Félix; West, Adam G.; Ellis, James

    2003-01-01

    Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRβ-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design. PMID:12954767

  14. Induction of a G1-S checkpoint in fission yeast.

    PubMed

    Bøe, Cathrine A; Krohn, Marit; Rødland, Gro Elise; Capiaghi, Christoph; Maillard, Olivier; Thoma, Fritz; Boye, Erik; Grallert, Beáta

    2012-06-19

    Entry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G(1)-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G(1)-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G(1)-S delay depends on at least two different inputs.

  15. 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

  16. The emerging world of small silencing RNAs in protozoan parasites

    PubMed Central

    Atayde, Vanessa D.; Tschudi, Christian; Ullu, Elisabetta

    2011-01-01

    A new RNA world has emerged in the past 10 years with the discovery of a plethora of 20- to 30-nucleotide long small RNAs that are involved in various gene silencing mechanisms. These small RNAs have considerably changed our view of the regulation of gene expression in eukaryotic organisms, with a major shift towards epigenetic and post-transcriptional mechanisms. Here we focus on the striking diversity of small silencing RNAs that have been identified in a number of protozoan parasites and their potential biological role. PMID:21497553

  17. RNA-guided genetic silencing systems in bacteria and archaea.

    PubMed

    Wiedenheft, Blake; Sternberg, Samuel H; Doudna, Jennifer A

    2012-02-15

    Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.

  18. Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

    PubMed

    Ocampo Ocampo, T; Gabriel Peralta, S M; Bacheller, N; Uiterwaal, S; Knapp, A; Hennen, A; Ochoa-Martinez, D L; Garcia-Ruiz, H

    2016-06-17

    In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.

  19. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence.

    PubMed

    Pumplin, Nathan; Voinnet, Olivier

    2013-11-01

    RNA silencing is a central regulator of gene expression in most eukaryotes and acts both at the transcriptional level through DNA methylation and at the post-transcriptional level through direct mRNA interference mediated by small RNAs. In plants and invertebrates, the same pathways also function directly in host defence against viruses by targeting viral RNA for degradation. Successful viruses have consequently evolved diverse mechanisms to avoid silencing, most notably through the expression of viral suppressors of RNA silencing. RNA silencing suppressors have also been recently identified in plant pathogenic bacteria and oomycetes, suggesting that disruption of host silencing is a general virulence strategy across several kingdoms of plant pathogens. There is also increasing evidence that plants have evolved specific defences against RNA-silencing suppression by pathogens, providing yet another illustration of the never-ending molecular arms race between plant pathogens and their hosts.

  20. Recent patents in RNA silencing in plants: constructs, methods and applications in plant biotechnology.

    PubMed

    López-Gomollón, Sara; Dalmay, Tamas

    2010-11-01

    RNA silencing is a recently discovered mechanism to regulate gene expression at transcriptional and posttranscriptional levels. It is based on the recognition and methylation of target genes or cleavage of target mRNAs by small RNA molecules, with length varying from 21 to 24 nucleotides. RNA silencing plays an important role modulating most of the important cell processes, such as growth, development or stress response. During the past few years, diverse strategies have been applied to exploit RNA silencing as a tool to create plants with enhanced economical properties or able to cope with pathogens or abiotic stress. This review describes the most important patents related to RNA silencing in plants, which disclose vectors designed to induce RNA silencing by hairpin RNAs, amplicons or virus-based plasmids, methods for detection and quantification of silencing as well as general uses in plant biotechnology.

  1. Kcnq1ot1/Lit1 Noncoding RNA Mediates Transcriptional Silencing by Targeting to the Perinucleolar Region ▿ †

    PubMed Central

    Mohammad, Faizaan; Pandey, Radha Raman; Nagano, Takashi; Chakalova, Lyubomira; Mondal, Tanmoy; Fraser, Peter; Kanduri, Chandrasekhar

    2008-01-01

    The Kcnq1ot1 antisense noncoding RNA has been implicated in long-range bidirectional silencing, but the underlying mechanisms remain enigmatic. Here we characterize a domain at the 5′ end of the Kcnq1ot1 RNA that carries out transcriptional silencing of linked genes using an episomal vector system. The bidirectional silencing property of Kcnq1ot1 maps to a highly conserved repeat motif within the silencing domain, which directs transcriptional silencing by interaction with chromatin, resulting in histone H3 lysine 9 trimethylation. Intriguingly, the silencing domain is also required to target the episomal vector to the perinucleolar compartment during mid-S phase. Collectively, our data unfold a novel mechanism by which an antisense RNA mediates transcriptional gene silencing of chromosomal domains by targeting them to distinct nuclear compartments known to be rich in heterochromatic machinery. PMID:18299392

  2. Mechanisms and Components of the DNA Damage Checkpoint

    DTIC Science & Technology

    2002-09-01

    S Phase in Saccharomyces cerevisiae. Genetics 157, 567-77. 35. Tercero , J.A. and Diffley, J.F. (2001) Regulation of DNA replication fork progression...The 16-mer Rad9 T390 peptides were combined with biotin-LC- the Weinert laboratory (Gardner et al., 1999; Lydall and Weinert, NHS (Pierce) at a molar

  3. MIDGET unravels functions of the Arabidopsis topoisomerase VI complex in DNA endoreduplication, chromatin condensation, and transcriptional silencing.

    PubMed

    Kirik, Viktor; Schrader, Andrea; Uhrig, Joachim F; Hulskamp, Martin

    2007-10-01

    The plant homologs of the archaeal DNA topoisomerase VI complex are required for the progression of endoreduplication cycles. Here, we describe the identification of MIDGET (MID) as a novel component of topoisomerase VI. We show that mid mutants show the same phenotype as rhl1, rhl2, and top6B mutants and that MID protein physically interacts with RHL1. The phenotypic analysis revealed new phenotypes, indicating that topoisomerase VI is involved in chromatin organization and transcriptional silencing. In addition, genetic evidence is provided suggesting that the ATR-dependent DNA damage repair checkpoint is activated in mid mutants, and CYCB1;1 is ectopically activated. Finally, we demonstrate that overexpression of CYCB1;2 can rescue the endoreduplication defects in mid mutants, suggesting that in mid mutants, a specific checkpoint is activated preventing further progression of endoreduplication cycles.

  4. Aurora B prevents premature removal of spindle assembly checkpoint proteins from the kinetochore: A key role for Aurora B in mitosis.

    PubMed

    Gurden, Mark D; Anderhub, Simon J; Faisal, Amir; Linardopoulos, Spiros

    2016-07-18

    Accurate chromosome segregation is dependent on the spindle assembly checkpoint (SAC). In current models, the key direct role of Aurora B in the SAC has been suggested to be to promote rapid kinetochore localisation of MPS1, allowing MPS1 to generate the checkpoint signal. However, Aurora B is also thought to play an indirect role in the SAC through the destabilisation of kinetochore-microtubule (KT-MT) attachments. Here, we demonstrate that Aurora B activity is not required for the kinetochore recruitment of the majority of SAC proteins. More importantly, we show that the primary role of Aurora B in the SAC is to prevent the premature removal of SAC proteins from the kinetochore, which is strictly dependent on KT-MT interactions. Moreover, in the presence of KT-MT interactions, Aurora B inhibition silences a persistent SAC induced by tethering MPS1 to the kinetochore. This explains the highly synergistic interaction between Aurora B and MPS1 inhibitors to override the SAC, which is lost when cells are pre-arrested in nocodazole. Furthermore, we show that Aurora B and MPS1 inhibitors synergistically kill a panel of breast and colon cancer cell lines, including cells that are otherwise insensitive to Aurora B inhibitors alone. These data demonstrate that the major role of Aurora B in SAC is to prevent the removal of SAC proteins from tensionless kinetochores, thus inhibiting premature SAC silencing, and highlights a therapeutic strategy through combination of Aurora B and MPS1 inhibitors.

  5. Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae

    PubMed Central

    Rossodivita, Alyssa A.; Boudoures, Anna L.; Mecoli, Jonathan P.; Steenkiste, Elizabeth M.; Karl, Andrea L.; Vines, Eudora M.; Cole, Arron M.; Ansbro, Megan R.; Thompson, Jeffrey S.

    2014-01-01

    Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways. PMID:24748660

  6. Riding in silence: a little snowboarding, a lot of small RNAs.

    PubMed

    Ameres, Stefan L; Fukunaga, Ryuya

    2010-03-15

    The recent symposium, RNA silencing: Mechanism, Biology and Applications, organized by Phillip D. Zamore (University of Massachusetts Medical School) and Beverly Davidson (University of Iowa), and held in Keystone, Colorado, brought together scientists working on diverse aspects of RNA silencing, a field that comprises a multitude of gene regulatory pathways guided by microRNAs, small interfering RNAs and PIWI-interacting RNAs.

  7. Functional Diversity of Silencers in Budding Yeasts

    PubMed Central

    Sjöstrand, Jimmy O. O.; Kegel, Andreas; Åström, Stefan U.

    2002-01-01

    We studied the silencing of the cryptic mating-type loci HMLα and HMRa in the budding yeast Kluyveromyces lactis. A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HMLα. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant K. lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the REB1 gene derepressed the transcription of the HMLα1 gene at the nonpermissive temperature. A functional silencer element from the K. lactis cryptic HMRa locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K. lactis silencing. Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces. PMID:12456003

  8. Immune Checkpoint Blockade Biology in Mouse Models of Glioblastoma.

    PubMed

    Yeo, Alan T; Charest, Alain

    2017-09-01

    Glioblastoma Multiforme (GBM) is a highly malignant primary brain cancer that is associated with abysmal prognosis. The median survival of GBM patients is ∼15 months and there have not been any significant advance in therapies in over a decade, leaving treatment options limited. There is clearly an unmet need for GBM treatment. Immunotherapies are treatments based on usurping the power of the host's immune system to recognize and eliminate cancer cells. They have recently proven to be a successful strategy for combating a variety of cancers. Of the various types of immunotherapies, checkpoint blockade approaches have thus far produced significant clinical responses in several cancers including melanoma, non small-cell lung cancer, renal cancer, and prostate cancer. This review focuses on the biological rationale for using checkpoint blockade immunotherapeutic approaches in primary brain cancer and an up-to-date summary of current and ongoing checkpoint inhibitors-based clinical trials for malignant glioma. In addition, we expand on new concepts for further improving checkpoint blockade treatments, with a particular focus on the advantages of using genetically engineered mouse models for studies of immunotherapies in GBM. J. Cell. Biochem. 118: 2516-2527, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  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. 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

  11. Alteration of the metaphase checkpoint by B chromosomes.

    PubMed

    Herron, W B; Lluesma, S M; Arana, P; Wise, D

    2004-01-01

    The B chromosome polymorphism in Spanish populations of the grasshopper, Eyprepocnemis plorans (Charpentier) is ancient and widespread. Meiocytes containing B chromosomes were analyzed in our laboratory using the 3F3/2 monoclonal antibody, which binds to a kinetochore phosphoepitope whose degree of phosphorylation is sensitive to tension applied to the kinetochore. Further, the tension created by the spindle at metaphase controls a checkpoint (the metaphase checkpoint) that allows the cell to begin anaphase when all chromosomes are aligned at the metaphase plate. Fluorescence patterns of the 3F3/2 phosphoepitope in cells containing B chromosomes were determined using confocal laser scanning microscopy. The phosphorylation pattern of kinetochores in these cells was shown to be different from that of cells without Bs. This suggests that the metaphase checkpoint has been modified in some way. We propose that B chromosomes in these grasshopper populations may have survived during evolution due to an alteration of the metaphase checkpoint, making it more permissive to the presence of misaligned chromosomes. Copyright 2004 S. Karger AG, Basel

  12. 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

  13. Final checkup of neoplastic DNA replication: evidence for failure in decision-making at the mitotic cell cycle checkpoint G(1)/S.

    PubMed

    Prindull, Gregor

    2008-11-01

    Processing of epigenomic transcriptional information by cell cycle phase G(1) and decision-making at checkpoint G(1)/S are the final organizational steps preceding gene replication in transcriptional reorientation programs (i.e., switches from proliferation to cycle arrest and neoplastic transformation). Further analyses of cycle progression will open up new approaches in antineoplastic therapy. The following bibliographic databases were consulted: Central Medical Library Cologne, PubMed (English), the last search was done on April 23,2008 and key words searched were: cell cycle, cell memory, DNA methylation, embryonal/neoplastic stem cells, enzyme-modulated chromatin, G(1)-G(1)/S checkpoint, genomic/epigenomics, genomic viral DNA, histones, telomere/telomerases, transcription factors, neoplastic transformation, senescence. Gene transcription and epigenomic surveillance form a functional entity. In proliferation programs, transcriptional information is mediated by chromatin and DNA methylation, analyzed and processed in G(1) phase, and converged on the parental checkpoint G(1)/S for final decision-making on DNA replication. Genomic reorientation appears to be associated with transcriptional instability, which normally is corrected, possibly during the G(2)/M phase, to new levels of epigenomic equilibria. We speculate that daughter stem cells inherit persistent neoplasm-specific transcriptional instabilities through failure of the parental G(1)/S checkpoint. Foreign, silenced, potentially oncogenic DNA sequences, i.e. regular components of the human genome such as endogenous retroviruses, could conceivably be activated for expression in neoplastic transformation by epigenomic histone deacetylase/acetyl transferase/histone methyltransferase-mixed lineage leukemia deregulations. Failure of cell cycle G(1)/S decision-making for DNA replication is the final and possibly a major cause in neoplastic transformation. Therefore, further analysis of the dynamics of G(1)-G(1

  14. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

    PubMed Central

    2013-01-01

    Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry

  15. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

    PubMed

    Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

    2013-01-24

    In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF

  16. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    NASA Astrophysics Data System (ADS)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  17. Role of the ATM-Checkpoint Kinase 2 Pathway in CDT-Mediated Apoptosis of Gingival Epithelial Cells

    PubMed Central

    Alaoui-El-Azher, Mounia; Mans, Jeffrey J.; Baker, Henry V.; Chen, Casey; Progulske-Fox, Ann; Lamont, Richard J.; Handfield, Martin

    2010-01-01

    The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT. PMID:20668524

  18. Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells.

    PubMed

    Alaoui-El-Azher, Mounia; Mans, Jeffrey J; Baker, Henry V; Chen, Casey; Progulske-Fox, Ann; Lamont, Richard J; Handfield, Martin

    2010-07-23

    The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

  19. Breaking the Code of Silence.

    ERIC Educational Resources Information Center

    Halbig, Wolfgang W.

    2000-01-01

    Schools and communities must break the adolescent code of silence concerning threats of violence. Schools need character education stressing courage, caring, and responsibility; regular discussions of the school discipline code; formal security discussions with parents; 24-hour hotlines; and protocols for handling reports of potential violence.…

  20. Herpes Simplex Virus Type 1 Suppresses RNA-Induced Gene Silencing in Mammalian Cells▿

    PubMed Central

    Wu, Zetang; Zhu, Yali; Bisaro, David M.; Parris, Deborah S.

    2009-01-01

    RNA-induced silencing is a potent innate antiviral defense strategy in plants, and suppression of silencing is a hallmark of pathogenic plant viruses. However, the impact of silencing as a mammalian antiviral defense mechanism and the ability of mammalian viruses to suppress silencing in natural host cells have remained controversial. The ability of herpes simplex virus type 1 (HSV-1) to suppress silencing was examined in a transient expression system that employed an imperfect hairpin to target degradation of transcripts encoding enhanced green fluorescent protein (EGFP). HSV-1 infection suppressed EGFP-specific silencing as demonstrated by increased EGFP mRNA levels and an increase in the EGFP mRNA half-life. The increase in EGFP mRNA stability occurred despite the well-characterized host macromolecular shutoff functions of HSV-1 that globally destabilize mRNAs. Moreover, mutant viruses defective in these functions increased the stability of EGFP mRNA even more than did the wild-type virus in silenced cells compared to results in control cells. The importance of RNA silencing to HSV-1 replication was confirmed by a significantly enhanced virus burst size in cells in which silencing was knocked down with small inhibitory RNAs directed to Argonaute 2, an integral component of the silencing complex. Given that HSV-1 encodes several microRNAs, it is possible that a dynamic equilibrium exists between silencing and silencing suppression that is capable of modulating viral gene expression to promote replication, to evade host defenses, and/or to promote latency. PMID:19369325

  1. Chemoimmunotherapy by combining oxaliplatin with immune checkpoint blockades reduced tumor burden in colorectal cancer animal model.

    PubMed

    Wang, Weiwei; Wu, Ling; Zhang, Jiansheng; Wu, Huiguo; Han, Enkun; Guo, Qiang

    2017-05-20

    Colorectal cancer (CRC) is among one of the top common cancers worldwide. Developing novel comprehensive treatment strategies is critical for improving survival of late stage CRC patients. Recent advances in immune checkpoint blockades provided a novel strategy for treating cancers via stimulating the antitumor immune response. However, the effects of immune checkpoint blockades were limited in CRC due to intrinsic resistance. Oxaliplatin (OXA) based chemotherapy was the foundation of CRC adjuvant chemotherapy. Here, we investigated the potential roles of OXA in inducing immunogenicity and synergizing with immune checkpoints in CRC. Immunogenicity of OXA was tested in CRC cell lines. Immune checkpoint blockades sensitive and resistant CRC models were used to study the potential synergistic roles of OXA with immune checkpoint blockades. We found CT26 mouse model was sensitive to immune checkpoint blockades, while MC38 mouse model was resistant. OXA could induce immunogenic cell death in several human and mouse CRC cell lines. Short term OXA treatment increased immune cell infiltration in MC38 mouse model and therefore enhanced the efficacy of immune checkpoint in MC38 mouse model. As a response to the OXA and immune checkpoint blockades combination, inhibitory immune checkpoints were down-regulated in MC38 tumors, while immune enhancing cytokines were up-regulated. Short term OXA treatment induced antitumor immune response in an immune checkpoint blockades resistant mouse model, therefore synergized with immune checkpoint blockades. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Successive silencing of tandem reporter genes in potato (Solanum tuberosum) over 5 years of vegetative propagation.

    PubMed

    Nocarova, Eva; Opatrny, Zdenek; Fischer, Lukas

    2010-10-01

    Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene.

  3. Silence or the Sound of Limpid Water: Disability, Power, and the Educationalisation of Silence

    ERIC Educational Resources Information Center

    Verstraete, Pieter

    2017-01-01

    In this article the history of silence is looked at from an educational perspective. By closely examining the way three nineteenth-century authors--who all based their educational theories on concrete experiences with persons with disabilities--have related themselves to silence, it will be argued that silence has been educationalised. Silence has…

  4. Virus-induced gene silencing (VIGS) in barley seedling leaves

    USDA-ARS?s Scientific Manuscript database

    Virus-induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterization. This method exploits a dsRNA-mediated antiviral defense mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid ...

  5. Silence as the Foundation of Learning

    ERIC Educational Resources Information Center

    Caranfa, Angelo

    2004-01-01

    Past and present discussions on education all too frequently neglect the role that silence plays in learning. In this article I set out to demonstrate that silence is the very foundation of learning. My claim is that we must find ways of freeing silence in our pedagogical practices so that our discourse does not denigrate into mere empty words,…

  6. RNA editing regulates transposon-mediated heterochromatic gene silencing.

    PubMed

    Savva, Yiannis A; Jepson, James E C; Chang, Yao-Jen; Whitaker, Rachel; Jones, Brian C; St Laurent, Georges; Tackett, Michael R; Kapranov, Philipp; Jiang, Nan; Du, Guyu; Helfand, Stephen L; Reenan, Robert A

    2013-01-01

    Heterochromatin formation drives epigenetic mechanisms associated with silenced gene expression. Repressive heterochromatin is established through the RNA interference pathway, triggered by double-stranded RNAs (dsRNAs) that can be modified via RNA editing. However, the biological consequences of such modifications remain enigmatic. Here we show that RNA editing regulates heterochromatic gene silencing in Drosophila. We utilize the binding activity of an RNA-editing enzyme to visualize the in vivo production of a long dsRNA trigger mediated by Hoppel transposable elements. Using homologous recombination, we delete this trigger, dramatically altering heterochromatic gene silencing and chromatin architecture. Furthermore, we show that the trigger RNA is edited and that dADAR serves as a key regulator of chromatin state. Additionally, dADAR auto-editing generates a natural suppressor of gene silencing. Lastly, systemic differences in RNA editing activity generates interindividual variation in silencing state within a population. Our data reveal a global role for RNA editing in regulating gene expression.

  7. Is the Efficiency of RNA Silencing Evolutionarily Regulated?

    PubMed

    Ui-Tei, Kumiko

    2016-05-12

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate gene expression in a sequence-specific manner. Genes with partial complementarity to siRNA/miRNA sequences in their 3'-untranslated regions (UTRs) are suppressed by a mechanism referred to as the siRNA off-target effect or miRNA-mediated RNA silencing. However, the determinants of such RNA silencing efficiency are poorly understood. Previously, I and co-workers reported that the efficiency of RNA silencing is strongly correlated with the thermodynamic stability of base pairing in the duplex formed within an siRNA/miRNA and between the seed region and its target mRNA. In this review, I first summarize our previous studies that identified the thermodynamic parameter to estimate the silencing efficiency using the calculated base pairing stability: siRNAs downregulate the expression of off-target genes depending on the stability of binding between the siRNA seed region (nucleotides 2-8) and off-target mRNAs, and miRNAs downregulate target mRNA expression depending on the stability of the duplex formed between the 5' terminus of the miRNA and its target mRNA. I further discuss the possibility that such thermodynamic features of silencing efficiency may have arisen during evolution with increasing body temperature in various organisms.

  8. The meanings of silence in Brazilian women with urinary incontinence.

    PubMed

    Higa, Rosângela; Chvatal, Vera Lucia Soares; de Moraes Lopes, Maria Helena Baena; Turato, Egberto Ribeiro

    2011-01-01

    We explored the meanings of silence for Brazilian women with urinary incontinence (UI). The sample consisted of 8 women, aged 30 to 45 years. Respondents worked as housekeepers or cleaning staff and were from lower social, economic, and educational strata. Their years of formal education varied from 0 to 8 years and they earned up to 4.5 times the Brazilian minimum wage, which is equivalent to US$900. A qualitative method using semistructured interviews was employed to gather data. Individual semistructured interviews were recorded and subsequently transcribed, including researchers' observations of subjects' nonverbal behaviors. The interviews began with the question: "Can you tell me about your experience with urinary incontinence?" Data were analyzed using a content analysis technique. Respondents avoided discussing UI and initially resisted labeling themselves as incontinent, but their nonverbal behaviors provided clues to the psychosocial distress caused by urinary leakage. Results suggest that respondents' underprivileged social, economic, and cultural situation may aggravate their limitations when expressing their feelings. We found that the women employed silence as a means to contain the psychosocial distress created by their UI, and that the silence itself should be interpreted as an expression of distress associated with UI. The silence of Brazilian women with UI is an essential element of communication about incontinence. We believe that the silence used by these women expresses the pain and anxiety they experienced, and it acts as an adaptive psychosocial mechanism.

  9. Intergenic transcription through a polycomb group response element counteracts silencing.

    PubMed

    Schmitt, Sabine; Prestel, Matthias; Paro, Renato

    2005-03-15

    Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, we show that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex we tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system.

  10. Is silence killing your company?

    PubMed

    Perlow, Leslie; Williams, Stephanie

    2003-05-01

    Many times, often with the best of intentions, people at work decide it's more productive to remain silent about their differences than to air them. There's no time, they think, or no point in going against what the boss says. But as new research by the authors shows, silencing doesn't smooth things over or make people more productive. It merely pushes differences beneath the surface and can set in motion powerfully destructive forces. When people stay silent about important disagreements, they can begin to fill with anxiety, anger, and resentment. As long as the conflict is unresolved, their repressed feelings remain potent, making them increasingly distrustful, self-protective, and all the more fearful that if they speak up they will be embarrassed or rejected. Their sense of insecurity grows, leading to further acts of silence, more defensiveness, and more distrust, thereby setting into motion a destructive "spiral of silence." Sooner or later, they mentally opt out--sometimes merely doing what they're told but contributing nothing of their own, sometimes spreading discontent and frustration throughout the workplace that can lead them, and others, to leave without thinking it through. These vicious spirals of silence can be replaced with virtuous spirals of communication, but that requires individuals to find the courage to act differently and executives to create the conditions in which people will value the expression of differences. All too often, behind failed products, broken processes, and mistaken career decisions are people who chose to hold their tongues. Breaking the silence can bring an outpouring of fresh ideas from all levels of an organization--ideas that might just raise the organization's performance to a whole new level.

  11. In vivo chromatin accessibility correlates with gene silencing in Drosophila.

    PubMed Central

    Boivin, A; Dura, J M

    1998-01-01

    Gene silencing by heterochromatin is a well-known phenomenon that, in Drosophila, is called position effect variegation (PEV). The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct support only recently. Another gene-silencing phenomenon in Drosophila, although similar in its phenotype of variegation, has been shown to be associated with euchromatic sequences and is dependent on developmental regulators of the Polycomb group (Pc-G) of gene products. One model proposes that the Pc-G products may cause a local heterochromatinization that maintains a repressed state of transcription of their target genes. Here, we test these models by measuring the accessibility of white or miniwhite sequences, in different contexts, to the Escherichia coli dam DNA methyltransferase in vivo. We present evidence that PEV and Pc-G-mediated repression mechanisms, although based on different protein factors, may indeed involve similar higher-order chromatin structure. PMID:9832530

  12. Histone deacetylase inhibitors reverse gene silencing in Friedreich's ataxia.

    PubMed

    Herman, David; Jenssen, Kai; Burnett, Ryan; Soragni, Elisabetta; Perlman, Susan L; Gottesfeld, Joel M

    2006-10-01

    Expansion of GAA x TTC triplets within an intron in FXN (the gene encoding frataxin) leads to transcription silencing, forming the molecular basis for the neurodegenerative disease Friedreich's ataxia. Gene silencing at expanded FXN alleles is accompanied by hypoacetylation of histones H3 and H4 and trimethylation of histone H3 at Lys9, observations that are consistent with a heterochromatin-mediated repression mechanism. We describe the synthesis and characterization of a class of histone deacetylase (HDAC) inhibitors that reverse FXN silencing in primary lymphocytes from individuals with Friedreich's ataxia. We show that these molecules directly affect the histones associated with FXN, increasing acetylation at particular lysine residues on histones H3 and H4 (H3K14, H4K5 and H4K12). This class of HDAC inhibitors may yield therapeutics for Friedreich's ataxia.

  13. Gene Silencing in Crustaceans: From Basic Research to Biotechnologies

    PubMed Central

    Sagi, Amir; Manor, Rivka; Ventura, Tomer

    2013-01-01

    Gene silencing through RNA interference (RNAi) is gaining momentum for crustaceans, both in basic research and for commercial development. RNAi has proven instrumental in a growing number of crustacean species, revealing the functionality of novel crustacean genes essential among others to development, growth, metabolism and reproduction. Extensive studies have also been done on silencing of viral transcripts in crustaceans, contributing to the understanding of the defense mechanisms of crustaceans and strategies employed by viruses to overcome these. The first practical use of gene silencing in aquaculture industry has been recently achieved, through manipulation of a crustacean insulin-like androgenic gland hormone. This review summarizes the advancements in the use of RNAi in crustaceans, and assesses the advantages of this method, as well as the current hurdles that hinder its large-scale practice. PMID:24705266

  14. Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes

    USDA-ARS?s Scientific Manuscript database

    Gene silencing is a useful technique for elucidating biological function of genes by knocking down their expression. Recently developed artificial microRNAs (amiRNAs) exploit an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs that target transcript...

  15. Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

    PubMed Central

    Mulvey, Claire M.; Tudzarova, Slavica; Crawford, Mark; Williams, Gareth H.; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2014-01-01

    Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations. PMID:23320540

  16. Immunosuppressive networks and checkpoints controlling antitumor immunity and their blockade in the development of cancer immunotherapeutics and vaccines.

    PubMed

    Butt, A Q; Mills, K H G

    2014-09-18

    Vaccines that promote protective adaptive immune responses have been successfully developed against a range of infectious diseases, and these are normally administered prior to exposure with the relevant virus or bacteria. Adaptive immunity also plays a critical role in the control of tumors. Immunotherapeutics and vaccines that promote effector T cell responses have the potential to eliminate tumors when used in a therapeutic setting. However, the induction of protective antitumor immunity is compromised by innate immunosuppressive mechanisms and regulatory cells that often dominate the tumor microenvironment. Recent studies have shown that blocking these suppressor cells and immune checkpoints to allow induction of antitumor immunity is a successful immunotherapeutic modality for the treatment of cancer. Furthermore, stimulation of innate and consequently adaptive immune responses with concomitant inhibition of immune suppression, especially that mediated by regulatory T (Treg) cells, is emerging as a promising approach to enhance the efficacy of therapeutic vaccines against cancer. This review describes the immunosuppressive mechanisms controlling antitumor immunity and the novel strategies being employed to design effective immunotherapeutics against tumors based on inhibition of suppressor cells or blockade of immune checkpoints to allow induction of more potent effector T cell responses. This review also discusses the potential of using a combination of adjuvants with inhibition of immune checkpoint or suppressor cells for therapeutic vaccines and the translation of pre-clinical studies to the next-generation vaccines against cancer in humans.

  17. A Cell Biological Perspective on Past, Present and Future Investigations of the Spindle Assembly Checkpoint

    PubMed Central

    Joglekar, Ajit P.

    2016-01-01

    The spindle assembly checkpoint (SAC) is a quality control mechanism that ensures accurate chromosome segregation during cell division. It consists of a mechanochemical signal transduction mechanism that senses the attachment of chromosomes to the spindle, and a signaling cascade that inhibits cell division if one or more chromosomes are not attached. Extensive investigations of both these component systems of the SAC have synthesized a comprehensive understanding of the underlying molecular mechanisms. This review recounts the milestone results that elucidated the SAC, compiles a simple model of the complex molecular machinery underlying the SAC, and highlights poorly understood facets of the biochemical design and cell biological operation of the SAC that will drive research forward in the near future. PMID:27869759

  18. 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

  19. A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

    PubMed Central

    Dagdas, Yavuz S.; Chen, Janice S.; Sternberg, Samuel H.; Doudna, Jennifer A.; Yildiz, Ahmet

    2017-01-01

    The Cas9 endonuclease is widely used for genome engineering applications by programming its single-guide RNA, and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer, we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites. PMID:28808686

  20. 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.

  1. 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

  2. A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores

    PubMed Central

    Moudgil, Devinderjit K.; Westcott, Nathan; Famulski, Jakub K.; Patel, Kinjal; Macdonald, Dawn; Hang, Howard

    2015-01-01

    Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein–dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein–protein interaction. PMID:25825516

  3. Dermatologic complications of anti-PD-1/PD-L1 immune checkpoint antibodies.

    PubMed

    Sibaud, Vincent; Meyer, Nicolas; Lamant, Laurence; Vigarios, Emmanuelle; Mazieres, Julien; Delord, Jean Pierre

    2016-07-01

    The therapeutic use of anti-PD-1/PD-L1 antibodies (nivolumab, pembrolizumab) is rapidly increasing. Given their mechanism of action that triggers T-cell activation, these immune checkpoint inhibitors induce specific adverse events that are mostly of immunologic origin. In this way, cutaneous toxicities represent the most frequent immune-related adverse events (irAEs). The purpose of this review is to summarize the most prevalent dermatologic complications induced by PD-1/PD-L1 immune checkpoint-blocking antibodies and to compare their dermatologic safety profile with anti-CTLA-4 ipilimumab. More than 40% of melanoma patients treated with anti-PD-1 therapy are faced with dermatologic irAEs. However, these cutaneous complications usually remain self-limiting and readily manageable. Nonspecific macular papular rash and pruritus represent the most common manifestations. More characteristic lichenoid dermatitis or psoriasis may also develop. Vitiligo is also frequent in patients with melanoma but has not been reported in other types of solid cancers. Mucosal involvement may also occur, including xerostomia and lichenoid reactions. Although available data remain scarce, anti-PD-L1 antibodies present a similar dermatologic safety profile. Dermatologic irAEs induced by PD-1 or PD-L1 blockade therapy rarely result in significant morbidity or permanent discontinuation of treatment. However, early recognition and appropriate management are crucial for restricting dose-limiting toxicities.

  4. 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

  5. Spindly/CCDC99 is required for efficient chromosome congression and mitotic checkpoint regulation.

    PubMed

    Barisic, Marin; Sohm, Bénédicte; Mikolcevic, Petra; Wandke, Cornelia; Rauch, Veronika; Ringer, Thomas; Hess, Michael; Bonn, Günther; Geley, Stephan

    2010-06-15

    Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle-regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NDelta253), or the conserved Spindly box (DeltaSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.

  6. Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

    PubMed Central

    Barisic, Marin; Sohm, Bénédicte; Mikolcevic, Petra; Wandke, Cornelia; Rauch, Veronika; Ringer, Thomas; Hess, Michael; Bonn, Günther

    2010-01-01

    Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle–regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NΔ253), or the conserved Spindly box (ΔSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules. PMID:20427577

  7. Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint.

    PubMed

    Roy, Anita; Lordier, Larissa; Pioche-Durieu, Catherine; Souquere, Sylvie; Roy, Lydia; Rameau, Philippe; Lapierre, Valérie; Le Cam, Eric; Plo, Isabelle; Debili, Najet; Raslova, Hana; Vainchenker, William

    2016-12-01

    Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes. Copyright© Ferrata Storti Foundation.

  8. ILC2s regulate adaptive Th2 cell functions via PD-L1 checkpoint control.

    PubMed

    Schwartz, Christian; Khan, Adnan R; Floudas, Achilleas; Saunders, Sean P; Hams, Emily; Rodewald, Hans-Reimer; McKenzie, Andrew N J; Fallon, Padraic G

    2017-09-04

    Group 2 innate lymphoid cells (ILC2s) are important effector cells driving the initiation of type 2 immune responses leading to adaptive T helper 2 (Th2) immunity. Here we show that ILC2s dynamically express the checkpoint inhibitor molecule PD-L1 during type 2 pulmonary responses. Surprisingly, PD-L1:PD-1 interaction between ILC2s and CD4(+) T cells did not inhibit the T cell response, but PD-L1-expressing ILC2s stimulated increased expression of GATA3 and production of IL-13 by Th2 cells both in vitro and in vivo. Conditional deletion of PD-L1 on ILC2s impaired early Th2 polarization and cytokine production, leading to delayed worm expulsion during infection with the gastrointestinal helminth Nippostrongylus brasiliensis Our results identify a novel PD-L1-controlled mechanism for type 2 polarization, with ILC2s mediating an innate checkpoint to control adaptive T helper responses, which has important implications for the treatment of type 2 inflammation. © 2017 Schwartz et al.

  9. 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.

  10. Lessons from CTLA-4 deficiency and checkpoint inhibition.

    PubMed

    Lo, Bernice; Abdel-Motal, Ussama M

    2017-08-11

    CTLA-4 is a crucial negative regulator of immune responses. Absence of CTLA-4 in mice causes autoimmunity and lethal multiorgan lymphocytic infiltration and tissue destruction. Recently, heterozygous CTLA4 or biallelic LRBA mutations leading to functional CTLA-4 deficiency and autoimmunity have been discovered. LRBA was identified as a novel regulator of steady-state CTLA-4 protein levels in Tregs and activated T cells. CTLA-4 deficiency due to checkpoint blockade cancer immunotherapy has also been found to lead to autoimmune reactions. Studies investigating the variable efficacy and adverse autoimmune responses to checkpoint therapy elucidated a role of the microbiota in promoting antitumor and autoreactive immune responses that are regulated by CTLA-4. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. 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.

  12. Covering Resilience: A Recent Development for Binomial Checkpointing

    SciTech Connect

    Walther, Andrea; Narayanan, Sri Hari Krishna

    2016-09-12

    In terms of computing time, adjoint methods offer a very attractive alternative to compute gradient information, required, e.g., for optimization purposes. However, together with this very favorable temporal complexity result comes a memory requirement that is in essence proportional with the operation count of the underlying function, e.g., if algorithmic differentiation is used to provide the adjoints. For this reason, checkpointing approaches in many variants have become popular. This paper analyzes an extension of the so-called binomial approach to cover also possible failures of the computing systems. Such a measure of precaution is of special interest for massive parallel simulations and adjoint calculations where the mean time between failure of the large scale computing system is smaller than the time needed to complete the calculation of the adjoint information. We describe the extensions of standard checkpointing approaches required for such resilience, provide a corresponding implementation and discuss first numerical results.

  13. Genetic Control of the Trigger for the G2/M Checkpoint

    SciTech Connect

    Hall, Eric J.; Smilenov, Lubomir B.; Young, Erik F.

    2013-10-01

    The work undertaken in this project addressed two seminal areas of low dose radiation biology that are poorly understood and controversial. These areas are the challenge to the linear-no-threshold (LNT) paradigm at low doses of radiation and, the fundamental elements of radiation bystander effect biology Genetic contributions to low dose checkpoint engagement: The LNT paradigm is an extrapolation of known, measured cancer induction endpoints. Importantly, data for lower doses is often not available. Debatably, radiation protection standards have been introduced which are prudently contingent on the adherence of cancer risk to the established trend seen at higher doses. Intriguing findings from other labs have hinted at separate DNA damage response programs that engage at low or high levels of radiation. Individual radiation sensitivity commensurate with hemizygosity for a radiation sensitivity gene has been estimated at 1-2% in the U.S.. Careful interrogation of the DNA damage response at low doses of radiation became important and served as the basis for this grant. Several genes were tested in combinations to determine if combined haploinsufficiency for multiple radiosensitizing genes could render a cell more sensitive to lower levels of acute radiation exposure. We measured a classical radiation response endpoint, cell cycle arrest prior to mitosis. Mouse embryo fibroblasts were used and provided a uniform, rapidly dividing and genetically manipulable population of study. Our system did not report checkpoint engagement at acute doses of gamma rays below 100 mGy. The system did report checkpoint engagement reproducibly at 500 mGy establishing a threshold for activation between 100 and 500 mGy. Engagement of the checkpoint was ablated in cells nullizygous for ATM but was otherwise unperturbed in cells combinatorially haploinsufficient for ATM and Rad9, ATM and PTEN or PTEN and Rad9. Taken together, these experiments tell us that, in a sensitive fibroblast culture

  14. Tethering DNA damage checkpoint mediator proteins topoisomerase IIbeta-binding protein 1 (TopBP1) and Claspin to DNA activates ataxia-telangiectasia mutated and RAD3-related (ATR) phosphorylation of checkpoint kinase 1 (Chk1).

    PubMed

    Lindsey-Boltz, Laura A; Sancar, Aziz

    2011-06-03

    The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

  15. Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix

    PubMed Central

    Rieke, Damian T.; Ochsenreither, Sebastian; Klinghammer, Konrad; Seiwert, Tanguy Y.; Klauschen, Frederick; Tinhofer, Inge; Keilholz, Ulrich

    2016-01-01

    Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression. A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma. A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC. Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition. PMID:27683114

  16. 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.

  17. Recovery in Distributed Systems Using Optimistic Message Logging and Checkpointing

    DTIC Science & Technology

    1990-09-01

    approach used by these message logging and checkpointing methods has been called the state machine approach [18], which assumes that program execution for...each input is determin- istic and is based only on the program state at the time of the input and on the input itself. This approach is also used by...the Time Warp system [61, through its Virtual Time method [7], using message logging and che.kpointing. However, Virtual Time is designed to support the

  18. Enhancing Immune Checkpoint Inhibitor Therapy In Kidney Cancer

    DTIC Science & Technology

    2016-10-01

    that the funds were made available to Johns Hopkins. Hence the PI did not hire respective personnel as described in the SOW and animal studies were not... animal model of kidney cancer. Our plan is to combine focally ablative techniques such as radiation or cryotherapy with PD1 immune checkpoint inhibition...lab) b) obtain IACUC/ACURO approval for the animal protocol c) test the effect of radiation of radiation and cryotherapy +/- PD1 inhibition on the

  19. Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein▿

    PubMed Central

    Vogler, Hannes; Akbergenov, Rashid; Shivaprasad, Padubidri V.; Dang, Vy; Fasler, Monika; Kwon, Myoung-Ok; Zhanybekova, Saule; Hohn, Thomas; Heinlein, Manfred

    2007-01-01

    Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA. PMID:17634237

  20. Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein.

    PubMed

    Vogler, Hannes; Akbergenov, Rashid; Shivaprasad, Padubidri V; Dang, Vy; Fasler, Monika; Kwon, Myoung-Ok; Zhanybekova, Saule; Hohn, Thomas; Heinlein, Manfred

    2007-10-01

    Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

  1. Silencing of Repetitive DNA Is Controlled by a Member of an Unusual Caenorhabditis elegans Gene Family.

    PubMed

    Leyva-Díaz, Eduardo; Stefanakis, Nikolaos; Carrera, Inés; Glenwinkel, Lori; Wang, Guoqiang; Driscoll, Monica; Hobert, Oliver

    2017-08-11

    Repetitive DNA sequences are subject to gene silencing in various animal species. Under specific circumstances repetitive DNA sequences can escape such silencing. For example, when exogenously added, extrachromosomal DNA sequences that are stably inherited in multicopy repetitive arrays in the nematode Caenorhabditis elegans are frequently silenced in the germline, whereas such silencing often does not occur in the soma. This indicates that somatic cells might utilize factors that prevent repetitive DNA silencing. Indeed, such "anti-silencing" factors have been revealed through genetic screens that identified mutant loci in which repetitive transgenic arrays are aberrantly silenced in the soma. We describe here a novel locus, pals-22 (for protein containing ALS2CR12 signature), required to prevent silencing of repetitive transgenes in neurons and other somatic tissue types. pals-22 deficiency also severely impacts animal vigor and confers phenotypes reminiscent of accelerated aging. We find that pals-22 is a member of a large family of divergent genes (39 members), defined by homology to the ALS2CR12 protein family. While gene family members are highly divergent, they show striking patterns of chromosomal clustering. The family expansion appears C. elegans-specific and has not occurred to the same extent in other nematode species for which genome sequences are available. The transgene silencing phenotype observed upon loss of PALS-22 protein depends on the biogenesis of small RNAs, since silencing is abolished in the RNAi defective mutant rde-4, suggesting that pals-22 might regulate RNAi-dependent silencing in different somatic cell types. We speculate that the pals gene family may be part of a species-specific cellular defense mechanism. Copyright © 2017, Genetics.

  2. Posttranscriptional gene silencing in nuclei

    PubMed Central

    Hoffer, Paul; Ivashuta, Sergey; Pontes, Olga; Vitins, Alexa; Pikaard, Craig; Mroczka, Andrew; Wagner, Nicholas; Voelker, Toni

    2011-01-01

    In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybean FAD2-1A desaturase intron is sufficient to silence FAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. Silencing FAD2-1 using intronic or 3′-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA–specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants. PMID:21173264

  3. 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

  4. Checkpoint kinase 2 is required for efficient immunoglobulin diversification.

    PubMed

    Davari, Kathrin; Frankenberger, Samantha; Schmidt, Angelika; Tomi, Nils-Sebastian; Jungnickel, Berit

    2014-01-01

    Maintenance of genome integrity relies on multiple DNA repair pathways as well as on checkpoint regulation. Activation of the checkpoint kinases Chk1 and Chk2 by DNA damage triggers cell cycle arrest and improved DNA repair, or apoptosis in case of excessive damage. Chk1 and Chk2 have been reported to act in a complementary or redundant fashion, depending on the physiological context. During secondary immunoglobulin (Ig) diversification in B lymphocytes, DNA damage is abundantly introduced by activation-induced cytidine deaminase (AID) and processed to mutations in a locus-specific manner by several error-prone DNA repair pathways. We have previously shown that Chk1 negatively regulates Ig somatic hypermutation by promoting error-free homologous recombination and Ig gene conversion. We now report that Chk2 shows opposite effects to Chk1 in the regulation of these processes. Chk2 inactivation in B cells leads to decreased Ig hypermutation and Ig class switching, and increased Ig gene conversion activity. This is linked to defects in non-homologous end joining and increased Chk1 activation upon interference with Chk2 function. Intriguingly, in the context of physiological introduction of substantial DNA damage into the genome during Ig diversification, the 2 checkpoint kinases thus function in an opposing manner, rather than redundantly or cooperatively.

  5. 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.

  6. 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.

  7. A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs

    PubMed Central

    An, Ping; Burge, Christopher B

    2005-01-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes. PMID:15828859

  8. A combinatorial code for splicing silencing: UAGG and GGGG motifs.

    PubMed

    Han, Kyoungha; Yeo, Gene; An, Ping; Burge, Christopher B; Grabowski, Paula J

    2005-05-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

  9. Inactivation of ATM/ATR DNA Damage Checkpoint Promotes Androgen Induced Chromosomal Instability in Prostate Epithelial Cells

    PubMed Central

    Chiu, Yung-Tuen; Liu, Ji; Tang, Kaidun; Wong, Yong-Chuan; Khanna, Kum Kum; Ling, Ming-Tat

    2012-01-01

    The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene have been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR) to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen-induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced autophosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis. PMID:23272087

  10. RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion.

    PubMed

    Malkova, Anna; Naylor, Maria L; Yamaguchi, Miyuki; Ira, Grzegorz; Haber, James E

    2005-02-01

    Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time. However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms. Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome. Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G(2)/M DNA damage checkpoint. RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process. RAD51-dependent BIR occurs efficiently in G(2)-arrested cells. Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.

  11. Interferon-γ is a master checkpoint regulator of cytokine-induced differentiation.

    PubMed

    Zha, Zhao; Bucher, Felicitas; Nejatfard, Anahita; Zheng, Tianqing; Zhang, Hongkai; Yea, Kyungmoo; Lerner, Richard A

    2017-08-15

    Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved.

  12. Interferon-γ is a master checkpoint regulator of cytokine-induced differentiation

    PubMed Central

    Zha, Zhao; Bucher, Felicitas; Nejatfard, Anahita; Zheng, Tianqing; Zhang, Hongkai; Yea, Kyungmoo; Lerner, Richard A.

    2017-01-01

    Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved. PMID:28760993

  13. A novel duct silencer using dielectric elastomer absorbers

    NASA Astrophysics Data System (ADS)

    Lu, Zhenbo; Cui, Yongdong; Zhu, Jian; Debiasi, Marco

    2014-03-01

    A novel duct silencer was developed using dielectric elastomer absorbers (DEAs). Dielectric elastomer, a lightweight, high elastic energy density and large deformation under high DC/AC voltages smart material, was used to fabricate this new generation actuator. The acoustic performances of this duct silencer were experimentally investigated in a transmission loss (TL) measurement system using two-load method. It was found that the resonance peaks of this new duct silencer could be controlled by applying various DC voltages, a maximum resonance shift of 59.5Hz for the resonance peaks was achieved which indicated that this duct silencer could be adjusted to absorb broadband range noise without any addition mechanical part. Furthermore, the resonance shift and multiple resonances mechanisms using DEAs were proposed and discussed in the present paper which was aiming to achieve broadband noise reduction. The present results also provide insight into the appropriateness of the absorber for possible use as new acoustic treatment to replace the traditional acoustic treatment.

  14. RNA silencing in the life cycle of soybean: multiple restriction systems and spatiotemporal variation associated with plant architecture.

    PubMed

    Mori, Ayumi; Sato, Hiroshi; Kasai, Megumi; Yamada, Tetsuya; Kanazawa, Akira

    2017-06-01

    The expression of transgenes introduced into a plant genome is sometimes suppressed by RNA silencing. Although local and systemic spread of RNA silencing have been studied, little is known about the mechanisms underlying spatial and temporal variation in transgene silencing between individual plants or between plants of different generations, which occurs seemingly stochastically. Here, we analyzed the occurrence, spread, and transmission of RNA silencing of the green fluorescent protein (GFP) gene over multiple generations of the progeny of a single soybean transformant. Observation of GFP fluorescence in entire plants of the T3-T5 generations indicated that the initiation and subsequent spread of GFP silencing varied between individuals, although this GFP silencing most frequently began in the primary leaves. In addition, GFP silencing could spread into the outer layer of seed coat tissues but was hardly detectable in the embryos. These results are consistent with the notion that transgene silencing involves its reset during reproductive phase, initiation after germination, and systemic spread in each generation. GFP silencing was absent in the pulvinus, suggesting that its cortical cells inhibit cell-to-cell spread or induction of RNA silencing. The extent of GFP silencing could differ between the stem and a petiole or between petiolules, which have limited vascular bundles connecting them and thus deter long-distant movement of silencing. Taken together, these observations indicate that the initiation and/or spread of RNA silencing depend on specific features of the architecture of the plant in addition to the mechanisms that can be conserved in higher plants.

  15. Limitations of silencing at native yeast telomeres.

    PubMed Central

    Pryde, F E; Louis, E J

    1999-01-01

    Silencing at native yeast telomeres, in which the subtelomeric elements are intact, is different from silencing at terminal truncations. The repression of URA3 inserted in different subtelomeric positions at several chromosome ends was investigated. Many ends exhibit very little silencing close to the telomere, while others exhibit substantial repression in limited domains. Silencing at native ends is discontinuous, with maximal repression found adjacent to the ARS consensus sequence in the subtelomeric core X element. The level of repression declines precipitously towards the centromere. Mutation of the ARS sequence or an adjacent Abf1p-binding site significantly reduces silencing. The subtelomeric Y' elements are resistant to silencing along their whole length, yet silencing can be re-established at the proximal X element. Deletion of PPR1, the transactivator of URA3, and SIR3 overexpression do not increase repression or extend spreading of silencing to the same extent as with terminally truncated ends. sir1Delta causes partial derepression at X-ACS, in contrast to the lack of effect seen at terminal truncations. orc2-1 and orc5-1 have no effect on natural silencing yet cause derepression at truncated ends. X-ACS silencing requires the proximity of the telomere and is dependent on SIR2, SIR3, SIR4 and HDF1. The structures found at native yeast telomeres appear to limit the potential of repressive chromatin. PMID:10228167

  16. Silencing polygalacturonase expression inhibits tomato petiole abscission.

    PubMed

    Jiang, Cai-Zhong; Lu, Feng; Imsabai, Wachiraya; Meir, Shimon; Reid, Michael S

    2008-01-01

    Virus-induced gene silencing (VIGS) was used as a tool for functional analysis of cell wall-associated genes that have been suggested to be involved in leaf abscission. Tobacco rattle virus is an effective vector for VIGS in tomato (Lycopersicon esculentum). Silencing was more efficient when the plants were grown at 22 degrees C than when they were grown at 20 degrees C or 25 degrees C. The photobleaching phenotype resulting from silencing phytoene desaturase varied, depending on cultivar, from barely visible to photobleaching of entire leaves. To study the function of abscission-related genes, a purple transgenic tomato line constitutively expressing the maize anthocyanin regulatory gene, Leaf colour (Lc) was used. Silencing Lc expression in this line resulted in reduced anthocyanin production (reversing the colour from purple to green), thus providing a convenient silencing 'reporter'. Silencing tomato abscission-related polygalacturonases (TAPGs), using a TAPG1 fragment, delayed abscission and increased break strength of the abscission zone in explants treated with 1 mul l(-1) ethylene. The abundance of TAPG1 transcripts in the green (silenced) abscission zone tissues was <1% of that in the purple (non-silenced) controls. As a highly homologous region was used for all five polygalacturonases it is assumed that the effect of delayed abscission is the result of silencing all the genes in this family. By contrast, silencing abscission-related expansins (LeEXP11 and LeEXP12) and endoglucanases (LeCEL1 and LeCEL2) had no discernible effect on break strength, even when the two endoglucanase genes were silenced concurrently. Simultaneous silencing of TAPG and LeCEL1 was no more effective than silencing TAPG alone. The data demonstrate the importance of TAPGs in the abscission of leaf petioles.

  17. Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

    PubMed Central

    Kontra, Levente; Tavazza, Mario; Lucioli, Alessandra; Tavazza, Raffaela; Moxon, Simon; Medzihradszky, Anna; Burgyán, József

    2016-01-01

    RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination. PMID:27711201

  18. Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica

    PubMed Central

    Morf, Laura; Pearson, Richard J.; Wang, Angelia S.; Singh, Upinder

    2013-01-01

    RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5′-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a ‘trigger’ and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5′-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects. PMID:23935116

  19. 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. © 2011 APJPH.

  20. 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

  1. Sequences throughout the basic beta-1,3-glucanase mRNA coding region are targets for homology dependent post-transcriptional gene silencing.

    PubMed

    Jacobs; Sanders; Bots; Andriessen; Van Eldik GJ; Litière; Van Montagu M; Cornelissen

    1999-10-01

    In the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post-transcriptional silencing of basic beta-1,3-glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing-target glucanase mRNAs. Using a two-component viral reporter system facilitated a comparison, in a quantitat- ive manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 mRNA. Similar-sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5' and 3' terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn1 transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA-internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism.

  2. 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

  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. Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation.

    PubMed

    Wang, Guoliang; Tong, Xiangyan; Weng, Stephanie; Zhou, Huilin

    2012-10-15

    The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.

  5. [E. M. Jellinek's silenced and silencing transgenerational story].

    PubMed

    Kelemen, Gábor; Márk, Mónika

    2013-01-01

    Jellinek is a kind of archetypal character for future generations in the field of addiction studies. His implosion in the arena of alcoholism around the age of 50 was an unexpected challenge to medical science. We know very little about his own role models giving an intellectual and moral compass to his pragmatic creativity. More than 30 years has passed since Jellinek's death when an American sociologist Ron Roizen started unearthing his silent story. Roizen discerned that there are a lot of unsaid and muted issues in his personal Hungarian past. Our paper, based on the authors' research in Hungarian archives and other sources reveals that not just Jellinek's personal but his transgenerational narrative has been not-yet-said. This silenced and silencing history appears an unfinished business of acculturation of the family, which started prior to four generations. Authors have been concluding that the issue of religious conversion is a critical point in the process of acculturation. They examine the counter move of loyalty to family values and driving force of assimilation making their story unspeakable.

  6. Silence Is Consent, or Curse Ye Meroz!

    ERIC Educational Resources Information Center

    Levin, Richard

    1997-01-01

    Examines assumptions of "oppositional" literary criticism, namely the assumption that older-style "objective" literary criticism must, in its political silence, be supportive of dominant ideologies. (TB)

  7. G2 Checkpoint Responses in Arabidopsis

    SciTech Connect

    Britt, Anne

    2013-03-18

    This project focused on the mechanism and biological significance of the G2 arrest response to replication stress in plants. We employed both forward and reverse genetic approaches to identify genes required for this response. A total of 3 different postdocs, 5 undergraduates, and 2 graduate students participated in the project. We identified several genes required for damage response in plants, including homologs of genes previously identified in animals (ATM and ATR), novel, a plant-specific genes (SOG1) and a gene known in animals but previously thought to be missing from the Arabidopsis genome (ATRIP). We characterized the transcriptome of gamma-irradiated plants, and found that plants, unlike animals, express a robust transcriptional response to damage, involving genes that regulate the cell cycle and DNA metabolism. This response requires both ATM and the transcription factor SOG1. We found that both ATM and ATR play a role in meiosis in plants. We also found that plants have a cell-type-specific programmed cell death response to ionizing radiation and UV light, and that this response requires ATR, ATM, and SOG1. These results were published in a series of 5 papers.

  8. Silence

    NASA Astrophysics Data System (ADS)

    Cogswell, J.

    2011-06-01

    On the occasion of the International Year of Astronomy, I was commissioned to create a mural for the University of Michigan Department of Astronomy, responding to an array of scientific images based on astronomical research, with special focus on the work of University of Michigan astronomers carried out within the building. My paper illustrates the development of this and several subsequent projects, explaining the implications for my artistic practice of entering into this conversation with astronomers and their work.

  9. Tissue homogeneity requires inhibition of unequal gene silencing during development

    PubMed Central

    Le, Hai H.; Looney, Monika; Strauss, Benjamin; Bloodgood, Michael

    2016-01-01

    Multicellular organisms can generate and maintain homogenous populations of cells that make up individual tissues. However, cellular processes that can disrupt homogeneity and how organisms overcome such disruption are unknown. We found that ∼100-fold differences in expression from a repetitive DNA transgene can occur between intestinal cells in Caenorhabditis elegans. These differences are caused by gene silencing in some cells and are actively suppressed by parental and zygotic factors such as the conserved exonuclease ERI-1. If unsuppressed, silencing can spread between some cells in embryos but can be repeat specific and independent of other homologous loci within each cell. Silencing can persist through DNA replication and nuclear divisions, disrupting uniform gene expression in developed animals. Analysis at single-cell resolution suggests that differences between cells arise during early cell divisions upon unequal segregation of an initiator of silencing. Our results suggest that organisms with high repetitive DNA content, which include humans, could use similar developmental mechanisms to achieve and maintain tissue homogeneity. PMID:27458132

  10. Gene silencing triggered by non-LTR retrotransposons in the female germline of Drosophila melanogaster.

    PubMed Central

    Robin, Stéphanie; Chambeyron, Séverine; Bucheton, Alain; Busseau, Isabelle

    2003-01-01

    Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing. PMID:12807773

  11. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA.

    PubMed

    Decker, Logan M; Xiao, Hua; Boone, Erin C; Vierling, Michael M; Shanker, Benjamin S; Kingston, Shanika L; Boone, Shannon F; Haynes, Jackson B; Shiu, Patrick K T

    2017-02-07

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus to combat these repetitive elements. One of these defense mechanisms is known as meiotic silencing by unpaired DNA (MSUD), which is an RNA silencing system that identifies and silences unpaired genes during meiosis. Utilizing common RNAi proteins such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified another silencing component known as the cap-binding complex (CBC). Made up of CBP20 and CBP80 (cap-binding proteins 20 and 80), CBC associates with the 5' cap of nascent mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. Similar to animals but unlike plants, CBP20's robust nuclear re-entry is shown to be dependent on CBP80. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors.

  12. Short-Lived Antigen Recognition but Not Viral Infection at a Defined Checkpoint Programs Effector CD4 T Cells To Become Protective Memory.

    PubMed

    Bautista, Bianca L; Devarajan, Priyadharshini; McKinstry, K Kai; Strutt, Tara M; Vong, Allen M; Jones, Michael C; Kuang, Yi; Mott, Daniel; Swain, Susan L

    2016-11-15

    Although memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are still poorly defined. We find that following murine influenza infection, most effector CD4 T cells undergo apoptosis unless they encounter cognate Ag at a defined stage near the peak of effector generation. Ag recognition at this memory checkpoint blocks default apoptosis and programs their transition to long-lived memory. Strikingly, we find that viral infection is not required, because memory formation can be restored by the addition of short-lived, Ag-pulsed APC at this checkpoint. The resulting memory CD4 T cells express an enhanced memory phenotype, have increased cytokine production, and provide protection against lethal influenza infection. Finally, we find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered during this checkpoint. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. We also suggest that administering Ag at this checkpoint may improve vaccine efficacy. Copyright © 2016 by The American Association of Immunologists, Inc.

  13. Obtusilactone A and (-)-sesamin induce apoptosis in human lung cancer cells by inhibiting mitochondrial Lon protease and activating DNA damage checkpoints.

    PubMed

    Wang, Hui-Min; Cheng, Kuo-Chen; Lin, Cheng-Jung; Hsu, Shu-Wei; Fang, Wei-Cheng; Hsu, Tai-Feng; Chiu, Chien-Chih; Chang, Hsueh-Wei; Hsu, Chun-Hua; Lee, Alan Yueh-Luen

    2010-12-01

    Several compounds from Cinnamomum kotoense show anticancer activities. However, the detailed mechanisms of most compounds from C. kotoense remain unknown. In this study, we investigated the anticancer activity of obtusilactone A (OA) and (-)-sesamin in lung cancer. Our results show that human Lon is upregulated in non-small-cell lung cancer (NSCLC) cell lines, and downregulation of Lon triggers caspase-3 mediated apoptosis. Through enzyme-based screening, we identified two small-molecule compounds, obtusilactone A (OA) and (-)-sesamin from C. kotoense, as potent Lon protease inhibitors. Obtusilactone A and (-)-sesamin interact with Ser855 and Lys898 residues in the active site of the Lon protease according to molecular docking analysis. Thus, we suggest that cancer cytotoxicity of the compounds is partly due to the inhibitory effects on Lon protease. In addition, the compounds are able to cause DNA double-strand breaks and activate checkpoints. Treatment with OA and (-)-sesamin induced p53-independent DNA damage responses in NSCLC cells, including G(1) /S checkpoint activation and apoptosis, as evidenced by phosphorylation of checkpoint proteins (H2AX, Nbs1, and Chk2), caspase-3 cleavage, and sub-G(1) accumulation. In conclusion, OA and (-)-sesamin act as both inhibitors of human mitochondrial Lon protease and DNA damage agents to activate the DNA damage checkpoints as well induce apoptosis in NSCLC cells. These dual functions open a bright avenue to develop more selective chemotherapy agents to overcome chemoresistance and sensitize cancer cells to other chemotherapeutics.

  14. Elm1 kinase activates the spindle position checkpoint kinase Kin4.

    PubMed

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I L; Hofmann, Astrid; Ibrahim, Bashar; Pereira, Gislene

    2010-09-20

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

  15. Elm1 kinase activates the spindle position checkpoint kinase Kin4

    PubMed Central

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I.L.; Hofmann, Astrid; Ibrahim, Bashar

    2010-01-01

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4. PMID:20855503

  16. The Aurora B Kinase in Chromosome Bi-Orientation and Spindle Checkpoint Signaling

    PubMed Central

    Krenn, Veronica; Musacchio, Andrea

    2015-01-01

    Aurora B, a member of the Aurora family of serine/threonine protein kinases, is a key player in chromosome segregation. As part of a macromolecular complex known as the chromosome passenger complex, Aurora B concentrates early during mitosis in the proximity of centromeres and kinetochores, the sites of attachment of chromosomes to spindle microtubules. There, it contributes to a number of processes that impart fidelity to cell division, including kinetochore stabilization, kinetochore–microtubule attachment, and the regulation of a surveillance mechanism named the spindle assembly checkpoint. In the regulation of these processes, Aurora B is the fulcrum of a remarkably complex network of interactions that feed back on its localization and activation state. In this review, we discuss the multiple roles of Aurora B during mitosis, focusing in particular on its role at centromeres and kinetochores. Many details of the network of interactions at these locations remain poorly understood, and we focus here on several crucial outstanding questions. PMID:26528436

  17. "The Silence Itself Is Enough of a Statement": The Day of Silence and LGBTQ Awareness Raising

    ERIC Educational Resources Information Center

    Woolley, Susan W.

    2012-01-01

    This ethnographic study of a high school gay-straight alliance club examines unintended consequences of silence during the Day of Silence, a day of action aimed at addressing anti-LGBTQ bias in schools. While this strategy calls for students to engage in intentional silences to raise awareness of anti-LGBTQ bias, it does not necessarily lead…

  18. "The Silence Itself Is Enough of a Statement": The Day of Silence and LGBTQ Awareness Raising

    ERIC Educational Resources Information Center

    Woolley, Susan W.

    2012-01-01

    This ethnographic study of a high school gay-straight alliance club examines unintended consequences of silence during the Day of Silence, a day of action aimed at addressing anti-LGBTQ bias in schools. While this strategy calls for students to engage in intentional silences to raise awareness of anti-LGBTQ bias, it does not necessarily lead…

  19. Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis

    PubMed Central

    Chandrasekhara, Chinmayi; Mohannath, Gireesha; Blevins, Todd; Pontvianne, Frederic; Pikaard, Craig S.

    2016-01-01

    In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2. PMID:26744421

  20. Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis.

    PubMed

    Chandrasekhara, Chinmayi; Mohannath, Gireesha; Blevins, Todd; Pontvianne, Frederic; Pikaard, Craig S

    2016-01-15

    In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2. © 2016 Chandrasekhara et al.; Published by Cold Spring Harbor Laboratory Press.

  1. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  2. 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.

  3. Has medical education killed "silence"?

    PubMed

    Lee, Shuh Shing

    2017-04-01

    There is an ignorance of "silence" observed from student selection methods to teaching and learning approaches. While selecting the candidates with suitable values to medical schools is crucial, most methods are unable to address fairness issue toward students from some disadvantaged background or certain personality specifically introversion. Similarly, teaching and learning approaches have shifted away from didactic to a more discursive methods such as brainstorming, team-based learning and case-based learning. These methods emphasize active participation and communication with team members, but having more discussion does not indicate that deep learning has taken place. Majority of these approaches require students to complete a task within an allocated time frame. Therefore, most of the time is utilized to complete a task instead of learning how to acquire a skill or learning how to learn. Important "silent" skills such as observation, reasoning process, and listening skills, are given less time or almost none due to time constraint within these discursive approaches, although these skills are extremely important as a doctor. Hence, it is time to think about on how best to balance the use of silence and externalize thought processes in medical education.

  4. 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.

  5. 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.

  6. 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.

  7. Cancer-associated TERT promoter mutations abrogate telomerase silencing.

    PubMed

    Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk

    2015-07-21

    Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.

  8. PhOBF1, a petunia ocs element binding factor, plays an important role in antiviral RNA silencing

    USDA-ARS?s Scientific Manuscript database

    Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, ...

  9. Polycomb Group-Dependent, Heterochromatin Protein 1-Independent, Chromatin Structures Silence Retrotransposons in Somatic Tissues Outside Ovaries

    PubMed Central

    Dufourt, J.; Brasset, E.; Desset, S.; Pouchin, P.; Vaury, C.

    2011-01-01

    Somatic cells are equipped with different silencing mechanisms that protect the genome against retrotransposons. In Drosophila melanogaster, a silencing pathway implicating the argonaute protein PIWI represses retrotransposons in cells surrounding the oocyte, whereas a PIWI-independent pathway is involved in other somatic tissues. Here, we show that these two silencing mechanisms result in distinct chromatin structures. Using sensor transgenes, we found that, in somatic tissues outside of the ovaries, these transgenes adopt a heterochromatic configuration implicating hypermethylation of H3K9 and K27. We identified the Polycomb repressive complexes (PRC1 and 2), but not heterochromatin protein 1 to be necessary factors for silencing. Once established, the compact structure is stably maintained through cell divisions. By contrast, in cells where the silencing is PIWI-dependent, the transgenes display an open and labile chromatin structure. Our data suggest that a post-transcriptional gene silencing (PTGS) mechanism is responsible for the repression in the ovarian somatic cells, whereas a mechanism that couples PTGS to transcriptional gene silencing operates to silence retrotransposons in the other somatic tissues. PMID:21908513

  10. Triticum mosaic poacevirus enlists P1 rather than HC-Pro to suppress RNA silencing-mediated host defense

    USDA-ARS?s Scientific Manuscript database

    RNA silencing, or posttranscriptional gene silencing (PTGS) is one of the most important defense mechanisms employed by higher plants and animals to defend against viral infections. Plant viruses evolved by adopting divergent proteins, even within single virus families, to counter this host defense ...

  11. viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence.

    PubMed

    Csorba, Tibor; Kontra, Levente; Burgyán, József

    2015-05-01

    RNA silencing is a homology-dependent gene inactivation mechanism that regulates a wide range of biological processes including antiviral defense. To deal with host antiviral responses viruses evolved mechanisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Besides working as silencing suppressors, these proteins may also fulfill other functions during infection. In many cases the interplay between the suppressor function and other "unrelated" functions remains elusive. We will present host factors implicated in antiviral pathways and summarize the current status of knowledge about the diverse viral suppressors' strategies acting at various steps of antiviral silencing in plants. Besides, we will consider the multi-functionality of these versatile proteins and related biochemical processes in which they may be involved in fine-tuning the plant-virus interaction. Finally, we will present the current applications and discuss perspectives of the use of these proteins in molecular biology and biotechnology.

  12. Communication to Enhance Silence: The Trappist Experience

    ERIC Educational Resources Information Center

    Jaksa, James A.; Stech, Ernest L.

    1978-01-01

    Investigates perceptions of the amount of interpersonal communication and attitudes towards communication frequency after the Trappist monk's rule of enforced silence and solitude was lifted in 1969. Concludes that increased interpersonal communication resulted in increased self-awareness and therefore more meaningful and effective silence. (MH)

  13. Silence amenity engineering: Past and present

    NASA Astrophysics Data System (ADS)

    Fujita, Hajime; Yokono, Yasuyuki

    1993-09-01

    Recent historical development of the noise control engineering, from mere noise reduction to silence amenity engineering, is reviewed, with social and psychological backgrounds behind it. Philosophical view points for fundamental approach to the silence amenity engineering and examples of noise source control in vibration and aerodynamic noises are described.

  14. Silence as the voice of trauma.

    PubMed

    Ritter, Maria

    2014-06-01

    Silence is a key to the unspoken world of the patient. Rather than interpreting silence as a defensive maneuver, the analyst may understand this disruption as a royal road to the patient's traumatic experiences. The author proposes to recognize traumatic silences in the analytic process and the transference as a re-experiencing of past, unpredictable traumatic affective states and memories. Silences in this context are both a repeat of a disconnecting experience as well as a manifestation of a silencing identification with the original silencer. The clinical material illustrates effects of a German mother's World War II (WWII) personal traumata and collective shame-based silence on her daughter's self and good object development. In the daughter's analysis, the patient and the analyst, who herself experienced similar WWII traumata, face the pain of trauma recovery and un-silencing. The author suggests that the deadening effect of past traumata may be reversed by an analytic process of re-membering and re-speaking for both the patient and analyst. This allows for a more transparent, subjective experience in the transference and a verbal integration of ego functions.