Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Estimating the abundance of burrow‐nesting species through the statistical analysis of combined playback and visual surveys

The conservation of elusive species relies on our ability to obtain unbiased estimates of their abundance trends. Many species live or breed in cavities, making it easy to define the search units (the cavity) yet hard to ascertain their occupancy. One such example is that of certain colonial seabirds like petrels and shearwaters, which occupy burrows to breed. In order to increase the chances of detection for these types of species, their sampling can be done using two independent methods to check for cavity occupancy: visual inspection, and acoustic response to a playback call. This double‐detection process allows us to estimate the probability of burrow occupancy by accounting for the probability of detection associated with each method. Here we provide a statistical framework to estimate the occupancy and population size of burrow‐dwelling species. We show how to implement the method using both maximum likelihood and Bayesian approaches, and test its precision and bias using simulated datasets. We subsequently illustrate how to extend the method to situations where two different species may occupy the burrows, and apply it to a dataset on wedge‐tailed shearwaters Puffinus pacificus and tropical shearwaters P. bailloni on Aride Island, Seychelles. The simulations showed that the single‐species model performed well in terms of error and bias except when detection probabilities and occupancies were very low. The two‐species model applied to shearwaters showed that detection probabilities were highly heterogeneous. The population sizes of wedge‐tailed and tropical shearwaters were estimated at 13 716 (95% CI: 12 909–15 874) and 25 550 (23 667–28 777) pairs respectively. The advantages of formulating the call‐playback sampling method statistically is that it provides a framework to calculate uncertainty in the estimates and model assumptions. This method is applicable to a variety of cavity‐dwelling species where two methods can be used to detect cavity occupancy.     

The glycomes of Caenorhabditis elegans and other model organisms

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fuc alpha 1-2 Gal 1-2 Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.     

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants.     

4-Acylamino-6-arylfuro[2,3-d]pyrimidines: potent and selective glycogen synthase kinase-3 inhibitors

Modeling studies of a furo[2,3-d]pyrimidine GSK-3 hit compound 1 superimposed onto the X-ray crystal structure of a legacy pyrazolo[3,4-c]pyridazine GSK-3 inhibitor 2 led to the identification of 4-acylamino-6-arylfuro[2,3-d]pyrimidine template 3. Synthesis of analogues based on template 3 has resulted in a number of potent and selective GSK-3beta inhibitors. The most potent and selective compound was the m-pyridyl analogue 24.     

Cost-Effectiveness of an Intervention to Reduce Emergency Re-Admissions to Hospital among Older Patients

Background

The objective is to estimate the cost-effectiveness of an intervention that reduces hospital re-admission among older people at high risk. A cost-effectiveness model to estimate the costs and health benefits of the intervention was implemented.

Methodology/Principal Findings

The model used data from a randomised controlled trial conducted in an Australian tertiary metropolitan hospital. Participants were acute medical admissions aged >65 years with at least one risk factor for re-admission: multiple comorbidities, impaired functionality, aged >75 years, recent multiple admissions, poor social support, history of depression. The intervention was a comprehensive nursing and physiotherapy assessment and an individually tailored program of exercise strategies and nurse home visits with telephone follow-up; commencing in hospital and continuing following discharge for 24 weeks. The change to cost outcomes, including the costs of implementing the intervention and all subsequent use of health care services, and, the change to health benefits, represented by quality adjusted life years, were estimated for the intervention as compared to existing practice. The mean change to total costs and quality adjusted life years for an average individual over 24 weeks participating in the intervention were: cost savings of $333 (95% Bayesian credible interval $ -1,932∶1,282) and 0.118 extra quality adjusted life years (95% Bayesian credible interval 0.1∶0.136). The mean net-monetary-benefit per individual for the intervention group compared to the usual care condition was $7,907 (95% Bayesian credible interval $5,959∶$9,995) for the 24 week period.

Conclusions/Significance

The estimation model that describes this intervention predicts cost savings and improved health outcomes. A decision to remain with existing practices causes unnecessary costs and reduced health. Decision makers should consider adopting this program for elderly hospitalised patients.     

Spontaneous autoimmunity in 129 and C57BL/6 mice-implications for autoimmunity described in gene-targeted mice

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 × C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.     

Role of the transcriptional corepressor Bcor in embryonic stem cell differentiation and early embryonic development

Bcor (BCL6 corepressor) is a widely expressed gene that is mutated in patients with X-linked Oculofaciocardiodental (OFCD) syndrome. BCOR regulates gene expression in association with a complex of proteins capable of epigenetic modification of chromatin. These include Polycomb group (PcG) proteins, Skp-Cullin-F-box (SCF) ubiquitin ligase components and a Jumonji C (Jmjc) domain containing histone demethylase. To model OFCD in mice and dissect the role of Bcor in development we have characterized two loss of function Bcor alleles. We find that Bcor loss of function results in a strong parent-of-origin effect, most likely indicating a requirement for Bcor in extraembryonic development. Using Bcor loss of function embryonic stem (ES) cells and in vitro differentiation assays, we demonstrate that Bcor plays a role in the regulation of gene expression very early in the differentiation of ES cells into ectoderm, mesoderm and downstream hematopoietic lineages. Normal expression of affected genes (Oct3/4, Nanog, Fgf5, Bmp4, Brachyury and Flk1) is restored upon re-expression of Bcor. Consistent with these ES cell results, chimeric animals generated with the same loss of function Bcor alleles show a low contribution to B and T cells and erythrocytes and have kinked and shortened tails, consistent with reduced Brachyury expression. Together these results suggest that Bcor plays a role in differentiation of multiple tissue lineages during early embryonic development.     

PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit

The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit.