Two Distinct Cdc2 Pools Regulate Cell Cycle Progression and the DNA Damage Response in the Fission Yeast S.pombe

The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage

Feasibility of time-lapse seismic methodology for monitoring the injection of small quantities of CO2 into a saline formation, CO2CRC Otway Project

A key objective of Stage 2 of the CO2CRC Otway Project is to explore the ability of geophysical methods to detect and monitor injection of greenhouse gas into a saline formation. For this purpose, injection of some 10,000 30,000 tonnes of CO2-rich mixture into the Paaratte formation, a saline aquifer located at a depth of about 1,400 m, is planned. Before such an injection experiment is undertaken, we assess the feasibility of geophysical monitoring using computer modelling. To examine the detectability of the plume we need to estimate the time-lapse signal and time- lapse noise. The time lapse signal is modelled using flow simulations, fluid substitution and seismic forward modelling. In order to assess the applicability of time-lapse seismic to monitor the injection, the predicted signal is compared to the time-lapse noise level from the recent 4D seismic survey acquired at the Otway site in 2009-2010. The methodology is applied to two alternative reservoir intervals located at a depth of 1392-1399 m and 1445-1465 m below the sea level, respectively. These intervals are considered to be the two possible options for the injection. The results show that injection into the lower interval will produce a plume of a larger thickness and smaller lateral extent, and a seismic response that is more likely to be detectable. The developed feasibility assessment workflow, and the results of its application to the Otway site, can be used to assess the ability of seismic methods to detect and monitor greenhouse gas leakage in other CCS projects

Icing mitigation by mems-fabricated surface dielectric barrier discharge

Avoiding ice accumulation on aerodynamic components is of enormous importance to flight safety. Novel approaches utilizing surface dielectric barrier discharges (SDBDs) are expected to be more efficient and effective than conventional solutions for preventing ice accretion on aerodynamic components. In this work, the realization of SDBDs based on thin-film substrates by means of micro-electro-mechanical-systems (MEMS) technology is presented. The anti-icing performance of the MEMS SDBDs is presented and compared to SDBDs manufactured by printed circuit board (PCB) technology. It was observed that the 35 µm thick electrodes of the PCB SDBDs favor surface icing with an initial accumulation of supercooled water droplets at the electrode impact edges. This effect was not observed for 0.3 µm thick MEMS-fabricated electrodes indicating a clear advantage for MEMS-technology SDBDs for anti-icing applications. Titanium was identified as the most suitable material for MEMS electrodes. In addition, an optimization of the MEMS-SDBDs with respect to the dielectric materials as well as SDBD design is discussed

Influence of dielectric thickness and electrode structure on the ion wind generation by micro fabricated plasma actuators

Abstract Surface dielectric barrier discharges are investigated in order to explore the combined effects of barrier thickness and microstructure of the exposed electrode on the ion wind generation. Actuators with straight and structured high voltage electrodes with characteristic sizes of 200 and 250 µm and dielectric thicknesses of 0.5, 1 and 2 mm are compared. It is observed that: i) actuator efficiency of ion wind generation strongly depends on the applied voltage amplitude; ii) operation voltage depends on the dielectric thickness logarithmically; iii) electrode microstructure slightly increases the dynamic pressure (few percent in maximum), however the effect decreases with thicker dielectrics and smaller electrode structures; iv) the pattern of the most intensive discharge parts as well as the dielectric erosion repeats the regular structure of the electrodes down to 200 µm. Several identical samples are tested during different days to estimate the impact of the air humidity and the degradation of the dielectric. The microscale precision of the sample manufacture was accomplished by a commercial facility for printed circuit boards

Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle

SUMO chain formation is required for response to replication arrest in S. pombe

SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved betabetaalphabetabetaalphabeta fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pomb

Consensus over Random Graph Processes: Network Borel-Cantelli Lemmas for Almost Sure Convergence

Distributed consensus computation over random graph processes is considered. The random graph process is defined as a sequence of random variables which take values from the set of all possible digraphs over the node set. At each time step, every node updates its state based on a Bernoulli trial, independent in time and among different nodes: either averaging among the neighbor set generated by the random graph, or sticking with its current state. Connectivity-independence and arc-independence are introduced to capture the fundamental influence of the random graphs on the consensus convergence. Necessary and/or sufficient conditions are presented on the success probabilities of the Bernoulli trials for the network to reach a global almost sure consensus, with some sharp threshold established revealing a consensus zero-one law. Convergence rates are established by lower and upper bounds of the $\epsilon$-computation time. We also generalize the concepts of connectivity/arc independence to their analogues from the $*$-mixing point of view, so that our results apply to a very wide class of graphical models, including the majority of random graph models in the literature, e.g., Erd\H{o}s-R\'{e}nyi, gossiping, and Markovian random graphs. We show that under $*$-mixing, our convergence analysis continues to hold and the corresponding almost sure consensus conditions are established. Finally, we further investigate almost sure finite-time convergence of random gossiping algorithms, and prove that the Bernoulli trials play a key role in ensuring finite-time convergence. These results add to the understanding of the interplay between random graphs, random computations, and convergence probability for distributed information processing.Comment: IEEE Transactions on Information Theory, In Pres

Sixty Years of Modern Human Origins in the American Anthropological Association

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65197/1/aa.2003.105.1.89.pd