The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA

SummaryThe amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA− mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals

Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Duplications of stretches of the genome are an important source of individual genetic variation, but their unrecognized presence in laboratory organisms would be a confounding variable for genetic analysis. RESULTS: We report here that duplications of 15 kb or more are common in the genome of the social amoeba Dictyostelium discoideum. Most stocks of the axenic 'workhorse' strains Ax2 and Ax3/4 obtained from different laboratories can be expected to carry different duplications. The auxotrophic strains DH1 and JH10 also bear previously unreported duplications. Strain Ax3/4 is known to carry a large duplication on chromosome 2 and this structure shows evidence of continuing instability; we find a further variable duplication on chromosome 5. These duplications are lacking in Ax2, which has instead a small duplication on chromosome 1. Stocks of the type isolate NC4 are similarly variable, though we have identified some approximating the assumed ancestral genotype. More recent wild-type isolates are almost without large duplications, but we can identify small deletions or regions of high divergence, possibly reflecting responses to local selective pressures. Duplications are scattered through most of the genome, and can be stable enough to reconstruct genealogies spanning decades of the history of the NC4 lineage. The expression level of many duplicated genes is increased with dosage, but for others it appears that some form of dosage compensation occurs. CONCLUSION: The genetic variation described here must underlie some of the phenotypic variation observed between strains from different laboratories. We suggest courses of action to alleviate the problem.Published versio

Targets downstream of Cdk8 in Dictyostelium development.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production. RESULTS: Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)). CONCLUSIONS: This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.Published versio

Cognitive ability does not predict objectively measured sedentary behaviour: evidence from three older cohorts

Higher cognitive ability is associated with being more physically active. Much less is known about the associations between cognitive ability and sedentary behavior. Ours is the first study to examine whether historic and contemporaneous cognitive ability predicts objectively measured sedentary behavior in older age. Participants were drawn from 3 cohorts (Lothian Birth Cohort, 1936 [LBC1936] [n = 271]; and 2 West of Scotland Twenty-07 cohorts: 1950s [n = 310] and 1930s [n = 119]). Regression models were used to assess the associations between a range of cognitive tests measured at different points in the life course, with sedentary behavior in older age recorded over 7 days. Prior simple reaction time (RT) was significantly related to later sedentary time in the youngest, Twenty-07 1950s cohort (p = .04). The relationship was nonsignificant after controlling for long-standing illness or employment status, or after correcting for multiple comparisons in the initial model. None of the cognitive measures were related to sedentary behavior in either of the 2 older cohorts (LBC1936, Twenty-07 1930s). There was no association between any of the cognitive tests and the number of sit-to-stand transitions in any of the 3 cohorts. The meta-analytic estimates for the measures of simple and choice RT that were identical in all cohorts (n = 700) were also not significant. In conclusion, we found no evidence that objectively measured sedentary time in older adults is associated with measures of cognitive ability at different time points in life, including cognitive change from childhood to older age

Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Pseudomonas aeruginosa is one of the most relevant human opportunistic bacterial pathogens. Two strains (PAO1 and PA14) have been mainly used as models for studying virulence of P. aeruginosa. The strain PA14 is more virulent than PAO1 in a wide range of hosts including insects, nematodes and plants. Whereas some of the differences might be attributable to concerted action of determinants encoded in pathogenicity islands present in the genome of PA14, a global analysis of the differential host responses to these P. aeruginosa strains has not been addressed. Little is known about the host response to infection with P. aeruginosa and whether or not the global host transcription is being affected as a defense mechanism or altered in the benefit of the pathogen. Since the social amoeba Dictyostelium discoideum is a suitable host to study virulence of P. aeruginosa and other pathogens, we used available genomic tools in this model system to study the transcriptional host response to P. aeruginosa infection. RESULTS: We have compared the virulence of the P. aeruginosa PAO1 and PA14 using D. discoideum and studied the transcriptional response of the amoeba upon infection. Our results showed that PA14 is more virulent in Dictyostelium than PA01using different plating assays. For studying the differential response of the host to infection by these model strains, D. discoideum cells were exposed to either P. aeruginosa PAO1 or P. aeruginosa PA14 (mixed with an excess of the non-pathogenic bacterium Klebsiella aerogenes as food supply) and after 4 hours, cellular RNA extracted. A three-way comparison was made using whole-genome D. discoideum microarrays between RNA samples from cells treated with the two different strains and control cells exposed only to K. aerogenes. The transcriptomic analyses have shown the existence of common and specific responses to infection. The expression of 364 genes changed in a similar way upon infection with one or another strain, whereas 169 genes were differentially regulated depending on whether the infecting strain was either P. aeruginosa PAO1 or PA14. Effects on metabolism, signalling, stress response and cell cycle can be inferred from the genes affected. CONCLUSION: Our results show that pathogenic Pseudomonas strains invoke both a common transcriptional response from Dictyostelium and a strain specific one, indicating that the infective process of bacterial pathogens can be strain-specific and is more complex than previously thought.Published versio

Reliability, minimal detectable change and responsiveness to change: indicators to select the best method to measure sedentary behaviour in older adults in different study designs

Introduction : Prolonged sedentary behaviour (SB) is associated with poor health. It is unclear which SB measure is most appropriate for interventions and population surveillance to measure and interpret change in behaviour in older adults. The aims of this study: to examine the relative and absolute reliability, Minimal Detectable Change (MDC) and responsiveness to change of subjective and objective methods of measuring SB in older adults and give recommendations of use for different study designs. Methods : SB of 18 older adults (aged 71 (IQR 7) years) was assessed using a systematic set of six subjective tools, derived from the TAxonomy of Self report Sedentary behaviour Tools (TASST), and one objective tool (activPAL3c), over 14 days. Relative reliability (Intra Class Correlation coefficients-ICC), absolute reliability (SEM), MDC, and the relative responsiveness (Cohen's d effect size (ES) and Guyatt's Responsiveness coefficient (GR)) were calculated for each of the different tools and ranked for different study designs. Results : ICC ranged from 0.414 to 0.946, SEM from 36.03 to 137.01 min, MDC from 1.66 to 8.42 hours, ES from 0.017 to 0.259 and GR from 0.024 to 0.485. Objective average day per week measurement ranked as most responsive in a clinical practice setting, whereas a one day measurement ranked highest in quasi-experimental, longitudinal and controlled trial study designs. TV viewing Previous Week Recall (PWR) ranked as most responsive subjective measure in all study designs. Conclusions : The reliability, Minimal Detectable Change and responsiveness to change of subjective and objective methods of measuring SB is context dependent. Although TV viewing-PWR is the more reliable and responsive subjective method in most situations, it may have limitations as a reliable measure of total SB. Results of this study can be used to guide choice of tools for detecting change in sedentary behaviour in older adults in the contexts of population surveillance, intervention evaluation and individual care

Sex Determination in the Social Amoeba <em>Dictyostelium discoideum</em>

The genetics of sex determination remains mysterious in many organisms including some that are otherwise well-studied. Here we report the discovery and analysis of the mating-type locus of the model organism Dictyostelium discoideum. Three forms of a single genetic locus specifies this species’ three mating-types: two versions of the locus are entirely different in sequence, and the third resembles a composite of the other two. Single, unrelated genes are sufficient to determine two of the mating-types, while homologues of both these genes are required in the composite type. The key genes encode polypeptides that possess no recognisable similarity to established protein families. Sex determination in the social amoebae thus appears to use regulators unrelated to any currently known

White-tailed Deer Browsing and Rubbing Preferences for Trees and Shrubs That Produce Nontimber Forest Products

Nontimber forest products (food, herbal medicinals, and woody floral and handicraft products) produced in forest, agroforestry, and horticultural systems can be important sources of income to landowners. White-tailed deer (Odocoileus virginianus) can reduce the quality, quantity, and profitability of forest products by browsing twigs and rubbing stems, resulting in direct and indirect losses to production enterprises. We evaluated deer damage (frequency and intensity of browsing and rubbing) sustained by 26 species of trees and shrubs, the relationships among morphological features of trees and shrubs to damage levels, and the economic impacts of deer damage on the production of nontimber forest products. Levels of browsing were high (frequency \u3e93% and intensity \u3e50%) in most species of trees and shrubs, with the highest intensity (\u3e60%) occurring in chinese chestnut (Castanea mollisima) and dogwood (Cornus spp.), and the lowest (Ginkgo biloba), curly willow (Salix matsudana), ‘Scarlet Curls’ curly willow, smooth sumac (Rhus glabra), and pussy willow (Salix caprea). Species of trees or shrubs with one or a few stout stems unprotected by dense branching [e.g., american elderberry (Sambucus canadensis), smooth sumac, and curly willow] sustained the most damage by rubbing. Trees and shrubs with many small diameter stems or with dense tangled branching [e.g. redozier dogwood (Cornus sericea), forsythia (Forsythia suspensa), ‘Flame’ willow (Salix alba), and ‘Streamco’ basket willow (Salix purpurea)] were damaged the least by rubbing. Annual economic costs of deer damage to producers of nontimber forest products can range from $26/acre for pussy willow to$1595/acre for curly willow

SCH 48973: a Potent, Broad-Spectrum, Antienterovirus Compound.

SCH 48973 is a novel molecule with potent, selective, antienterovirus activity. In assays of the cytopathic effect against five picornaviruses, SCH 48973 had antiviral activity (50% inhibitory concentrations [IC50s]) of 0.02 to 0.11 microg/ml, with no detectable cytotoxicity at 50 microg/ml. SCH 48973 inhibited 80% of 154 recent human enterovirus isolates at an IC50 of 0.9 microg/ml. The antiviral activity of SCH 48973 is derived from its specific interaction with viral capsid, as confirmed by competition binding studies. The affinity constant (Ki) for SCH 48973 binding to poliovirus was 8.85 x 10(-8) M. In kinetic studies, a maximum of approximately 44 molecules of SCH 48973 were bound to poliovirus capsid. SCH 48973 demonstrated efficacy in a murine poliovirus model of enterovirus disease. SCH 48973 increased the survival of infected mice when it was administered orally at dosages of 3 to 20 mg/kg of body weight/day. Oral administration of SCH 48973 also reduced viral titers in the brains of infected mice. On the basis of its in vitro and in vivo profiles, SCH 48973 represents a potential candidate for therapeutic intervention against enterovirus infections