Caffeine as a tool for investigating the integration of Cdc25 phosphorylation, activity and ubiquitin-dependent degradation in Schizosaccharomyces pombe

The evolutionarily conserved Cdc25 phosphatase is an essential protein that removes inhibitory phosphorylation moieties on the mitotic regulator Cdc2. Together with the Wee1 kinase, a negative regulator of Cdc2 activity, Cdc25 is thus a central regulator of cell cycle progression in Schizosaccharomyces pombe. The expression and activity of Cdc25 is dependent on the activity of the Target of Rapamycin Complex 1 (TORC1). TORC1 inhibition leads to the activation of Cdc25 and repression of Wee1, leading to advanced entry into mitosis. Withdrawal of nitrogen leads to rapid Cdc25 degradation via the ubiquitin- dependent degradation pathway by the Pub1 E3- ligase. Caffeine is believed to mediate the override of DNA damage checkpoint signalling, by inhibiting the activity of the ataxia telangiectasia mutated (ATM)/Rad3 homologues. This model remains controversial, as TORC1 appears to be the preferred target of caffeine in vivo. Recent studies suggest that caffeine induces DNA damage checkpoint override by inducing the nuclear accumulation of Cdc25 in S. pombe. Caffeine may thus modulate Cdc25 activity and stability via inhibition of TORC1. A clearer understanding of the mechanisms by which caffeine stabilises Cdc25, may provide novel insights into how TORC1 and DNA damage signalling is integrated

Redundant Mechanisms Prevent Mitotic Entry Following Replication Arrest in the Absence of Cdc25 Hyper-Phosphorylation in Fission Yeast

Following replication arrest the Cdc25 phosphatase is phosphorylated and inhibited by Cds1. It has previously been reported that expressing Cdc25 where 9 putative amino-terminal Cds1 phosphorylation sites have been substituted to alanine results in bypass of the DNA replication checkpoint. However, these results were acquired by expression of the phosphorylation mutant using a multicopy expression vector in a genetic background where the DNA replication checkpoint is intact. In order to clarify these results we constructed a Cdc25(9A)-GFP native promoter integrant and examined its effect on the replication checkpoint at endogenous expression levels. In this strain the replication checkpoint operates normally, conditional on the presence of the Mik1 kinase. In response to replication arrest the Cdc25(9A)-GFP protein is degraded, suggesting the presence of a backup mechanism to eliminate the phosphatase when it cannot be inhibited through phosphorylation

Comparative analysis of rosaceous genomes and the reconstruction of a putative ancestral genome for the family

Abstract Background Comparative genome mapping studies in Rosaceae have been conducted until now by aligning genetic maps within the same genus, or closely related genera and using a limited number of common markers. The growing body of genomics resources and sequence data for both Prunus and Fragaria permits detailed comparisons between these genera and the recently released Malus × domestica genome sequence. Results We generated a comparative analysis using 806 molecular markers that are anchored genetically to the Prunus and/or Fragaria reference maps, and physically to the Malus genome sequence. Markers in common for Malus and Prunus, and Malus and Fragaria, respectively were 784 and 148. The correspondence between marker positions was high and conserved syntenic blocks were identified among the three genera in the Rosaceae. We reconstructed a proposed ancestral genome for the Rosaceae. Conclusions A genome containing nine chromosomes is the most likely candidate for the ancestral Rosaceae progenitor. The number of chromosomal translocations observed between the three genera investigated was low. However, the number of inversions identified among Malus and Prunus was much higher than any reported genome comparisons in plants, suggesting that small inversions have played an important role in the evolution of these two genera or of the Rosaceae.Apple genome research at FEM is supported by the research office of the Provincia autonoma di Trento. DJS and ELG acknowledge a grant from the East Malling Trust. Fragaria genomics at EMR is funded by the BBSRC. JMB is supported by a grant by Plant & Food Research's Excellence Programme. Apple genomics at Plant & Food Research is partially supported by the New Zealand Foundation for Research Science and Technology project C06X0812 "Exploiting Opportunities from Horticultural Genomics". Research conducted at IRTA was partly funded by the CONSOLIDER-INGENIO 2010 Program (CSD2007-00036) and project INIA-RTA2007-00063-00-00, both from the Spanish Ministry of Science and Innovation. RosCOS development at OSU/MSU was funded by the National Research Initiative Competitive Grant 2005-35300-15454 of USDA's National Institute of Food and Agriculture.Peer Reviewe

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

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

Gene expression patterns associated with blood-feeding in the malaria mosquito Anopheles gambiae

BACKGROUND: Blood feeding, or hematophagy, is a behavior exhibited by female mosquitoes required both for reproduction and for transmission of pathogens. We determined the expression patterns of 3,068 ESTs, representing ~2,000 unique gene transcripts using cDNA microarrays in adult female Anopheles gambiae at selected times during the first two days following blood ingestion, at 5 and 30 min during a 40 minute blood meal and at 0, 1, 3, 5, 12, 16, 24 and 48 hours after completion of the blood meal and compared their expression to transcript levels in mosquitoes with access only to a sugar solution. RESULTS: In blood-fed mosquitoes, 413 unique transcripts, approximately 25% of the total, were expressed at least two-fold above or below their levels in the sugar-fed mosquitoes, at one or more time points. These differentially expressed gene products were clustered using k-means clustering into Early Genes, Middle Genes, and Late Genes, containing 144, 130, and 139 unique transcripts, respectively. Several genes from each group were analyzed by quantitative real-time PCR in order to validate the microarray results. CONCLUSION: The expression patterns and annotation of the genes in these three groups (Early, Middle, and Late genes) are discussed in the context of female mosquitoes' physiological responses to blood feeding, including blood digestion, peritrophic matrix formation, egg development, and immunity

Gaia Data Release 2 Mapping the Milky Way disc kinematics

Context. The second Gaia data release (Gaia DR2) contains high-precision positions, parallaxes, and proper motions for 1.3 billion sources as well as line-of-sight velocities for 7.2 million stars brighter than G(RVS) = 12 mag. Both samples provide a full sky coverage. Aims. To illustrate the potential of Gaia DR2, we provide a first look at the kinematics of the Milky Way disc, within a radius of several kiloparsecs around the Sun. Methods. We benefit for the first time from a sample of 6.4 million F-G-K stars with full 6D phase-space coordinates, precise parallaxes (sigma((omega) over bar)/(omega) over bar Results. Gaia DR2 allows us to draw 3D maps of the Galactocentric median velocities and velocity dispersions with unprecedented accuracy, precision, and spatial resolution. The maps show the complexity and richness of the velocity field of the galactic disc. We observe streaming motions in all the components of the velocities as well as patterns in the velocity dispersions. For example, we confirm the previously reported negative and positive galactocentric radial velocity gradients in the inner and outer disc, respectively. Here, we see them as part of a non-axisymmetric kinematic oscillation, and we map its azimuthal and vertical behaviour. We also witness a new global arrangement of stars in the velocity plane of the solar neighbourhood and in distant regions in which stars are organised in thin substructures with the shape of circular arches that are oriented approximately along the horizontal direction in the U - V plane. Moreover, in distant regions, we see variations in the velocity substructures more clearly than ever before, in particular, variations in the velocity of the Hercules stream. Conclusions. Gaia DR2 provides the largest existing full 6D phase-space coordinates catalogue. It also vastly increases the number of available distances and transverse velocities with respect to Gaia DR1. Gaia DR2 offers a great wealth of information on the Milky Way and reveals clear non-axisymmetric kinematic signatures within the Galactic disc, for instance. It is now up to the astronomical community to explore its full potential.Peer reviewe

Lenalidomide maintenance versus observation for patients with newly diagnosed multiple myeloma (Myeloma XI): a multicentre, open-label, randomised, phase 3 trial.

BACKGROUND: Patients with multiple myeloma treated with lenalidomide maintenance therapy have improved progression-free survival, primarily following autologous stem-cell transplantation. A beneficial effect of lenalidomide maintenance therapy on overall survival in this setting has been inconsistent between individual studies. Minimal data are available on the effect of maintenance lenalidomide in more aggressive disease states, such as patients with cytogenetic high-risk disease or patients ineligible for transplantation. We aimed to assess lenalidomide maintenance versus observation in patients with newly diagnosed multiple myeloma, including cytogenetic risk and transplantation status subgroup analyses. METHODS: The Myeloma XI trial was an open-label, randomised, phase 3, adaptive design trial with three randomisation stages done at 110 National Health Service hospitals in England, Wales, and Scotland. There were three potential randomisations in the study: induction treatment (allocation by transplantation eligibility status); intensification treatment (allocation by response to induction therapy); and maintenance treatment. Here, we report the results of the randomisation to maintenance treatment. Eligible patients for maintenance randomisation were aged 18 years or older and had symptomatic or non-secretory multiple myeloma, had completed their assigned induction therapy as per protocol and had achieved at least a minimal response to protocol treatment, including lenalidomide. Patients were randomly assigned (1:1 from Jan 13, 2011, to Jun 27, 2013, and 2:1 from Jun 28, 2013, to Aug 11, 2017) to lenalidomide maintenance (10 mg orally on days 1-21 of a 28-day cycle) or observation, and stratified by allocated induction and intensification treatment, and centre. The co-primary endpoints were progression-free survival and overall survival, analysed by intention to treat. Safety analysis was per protocol. This study is registered with the ISRCTN registry, number ISRCTN49407852, and clinicaltrialsregister.eu, number 2009-010956-93, and has completed recruitment. FINDINGS: Between Jan 13, 2011, and Aug 11, 2017, 1917 patients were accrued to the maintenance treatment randomisation of the trial. 1137 patients were assigned to lenalidomide maintenance and 834 patients to observation. After a median follow-up of 31 months (IQR 18-50), median progression-free survival was 39 months (95% CI 36-42) with lenalidomide and 20 months (18-22) with observation (hazard ratio [HR] 0·46 [95% CI 0·41-0·53]; p<0·0001), and 3-year overall survival was 78·6% (95% Cl 75·6-81·6) in the lenalidomide group and 75·8% (72·4-79·2) in the observation group (HR 0·87 [95% CI 0·73-1·05]; p=0·15). Progression-free survival was improved with lenalidomide compared with observation across all prespecified subgroups. On prespecified subgroup analyses by transplantation status, 3-year overall survival in transplantation-eligible patients was 87·5% (95% Cl 84·3-90·7) in the lenalidomide group and 80·2% (76·0-84·4) in the observation group (HR 0·69 [95% CI 0·52-0·93]; p=0·014), and in transplantation-ineligible patients it was 66·8% (61·6-72·1) in the lenalidomide group and 69·8% (64·4-75·2) in the observation group (1·02 [0·80-1·29]; p=0·88). By cytogenetic risk group, in standard-risk patients, 3-year overall survival was 86·4% (95% CI 80·0-90·9) in the lenalidomide group compared with 81·3% (74·2-86·7) in the observation group, and in high-risk patients, it was 74.9% (65·8-81·9) in the lenalidomide group compared with 63·7% (52·8-72·7) in the observation group; and in ultra-high-risk patients it was 62·9% (46·0-75·8) compared with 43·5% (22·2-63·1). Since these subgroup analyses results were not powered they should be interpreted with caution. The most common grade 3 or 4 adverse events for patients taking lenalidomide were haematological, including neutropenia (362 [33%] patients), thrombocytopenia (72 [7%] patients), and anaemia (42 [4%] patients). Serious adverse events were reported in 494 (45%) of 1097 patients receiving lenalidomide compared with 150 (17%) of 874 patients on observation. The most common serious adverse events were infections in both the lenalidomide group and the observation group. 460 deaths occurred during maintenance treatment, 234 (21%) in the lenalidomide group and 226 (27%) in the observation group, and no deaths in the lenalidomide group were deemed treatment related. INTERPRETATION: Maintenance therapy with lenalidomide significantly improved progression-free survival in patients with newly diagnosed multiple myeloma compared with observation, but did not improve overall survival in the intention-to-treat analysis of the whole trial population. The manageable safety profile of this drug and the encouraging results in subgroup analyses of patients across all cytogenetic risk groups support further investigation of maintenance lenalidomide in this setting. FUNDING:Cancer Research UK, Celgene, Amgen, Merck, and Myeloma UK