Potential local adaptation in populations of invasive reed canary grass (Phalaris arundinacea) across an urbanization gradient.

Urban stressors represent strong selective gradients that can elicit evolutionary change, especially in non-native species that may harbor substantial within-population variability. To test whether urban stressors drive phenotypic differentiation and influence local adaptation, we compared stress responses of populations of a ubiquitous invader, reed canary grass (Phalaris arundinacea). Specifically, we quantified responses to salt, copper, and zinc additions by reed canary grass collected from four populations spanning an urbanization gradient (natural, rural, moderate urban, and intense urban). We measured ten phenotypic traits and trait plasticities, because reed canary grass is known to be highly plastic and because plasticity may enhance invasion success. We tested the following hypotheses: (a) Source populations vary systematically in their stress response, with the intense urban population least sensitive and the natural population most sensitive, and (b) plastic responses are adaptive under stressful conditions. We found clear trait variation among populations, with the greatest divergence in traits and trait plasticities between the natural and intense urban populations. The intense urban population showed stress tolerator characteristics for resource acquisition traits including leaf dry matter content and specific root length. Trait plasticity varied among populations for over half the traits measured, highlighting that plasticity differences were as common as trait differences. Plasticity in root mass ratio and specific root length were adaptive in some contexts, suggesting that natural selection by anthropogenic stressors may have contributed to root trait differences. Reed canary grass populations in highly urbanized wetlands may therefore be evolving enhanced tolerance to urban stressors, suggesting a mechanism by which invasive species may proliferate across urban wetland systems generally.

Development of Microbiome Biobanks - Challenges and Opportunities.

The microbiome research field is rapidly evolving, but the required biobanking infrastructure is currently fragmented and not prepared for the biobanking of microbiomes. The rapid advancement of technologies requires an urgent assessment of how biobanks can underpin research by preserving microbiome samples and their functional potential.

Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.

Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the (13)C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential.

Prevalence, correlates, and risk perception of HIV infection among heroin users in Central Taiwan.

We investigated the prevalence and correlated factors of human immunodeficiency virus (HIV) among heroin users attending methadone maintenance treatment (MMT) programs in Central Taiwan, and explored the degree of risk perception of HIV infection among the participants. Our study participants were 781 heroin users seeking treatment at the MMT program at Tsaotun Psychiatric Center in Taiwan. The presence of HIV antibodies was identified by microparticle enzyme immunoassay and confirmed by western blot. Multivariate logistic regression was used to identify the independent correlates of HIV infection. The mean age of the sample was 36.1 years [standard deviation (SD) = 7.6]; of the patients, 710 (90.9%) were men. The prevalence of HIV infection among our study population was 20.7%. Multivariate logistic regression analysis revealed that HIV infection was independently associated with the age of the patients of initial heroin use, heroin injection use, nondrug-related criminal convictions, needle-sharing behaviors, and sharing injection paraphernalia. A strong agreement existed between self-reported HIV serostatus and the results of laboratory analyses, with 88.8% of patients reporting their condition correctly. We found a high rate of HIV infection among patients in the MMT program. Factors associated with HIV infection were mostly related to drug-use behaviors. These findings stress the importance of education regarding drug-risk behaviors.

Chronic obstructive pulmonary disease: a risk factor for type 2 diabetes: a nationwide population-based study.

BACKGROUND: Type 2 diabetes mellitus (DM) is a common medical disorder and a leading cause of morbidity and mortality worldwide. We investigated whether chronic obstructive pulmonary disease (COPD) was the risk factor for type 2 diabetes in an Asian population. MATERIALS AND METHODS: From Taiwan's National Health Insurance Research Database, we collected data from 16,088 patients, including 8044 COPD patients and 8044 age- and gender- matched control subjects. Cox proportional hazard regression was performed to evaluate independent risk factors for type 2 diabetes in all patients and identify risk factors in patients with COPD. RESULTS: During the 5.5-year follow-up, patients with COPD were found to have a significantly higher rate of incident type 2 diabetes than the control group (P < 0.001). COPD was significantly associated with type 2 diabetes hazard ratio (HR : 1.41, 1.23-1.63, P < 0.001) after adjusting sex, age, residential area, insurance premium, steroid use, hypertriglycemia, hypertension, coronary artery disease (CAD) and cerebrovascular disease. Cox regression analysis showed that hypertension (HR : 1.55, 1.33-1.80, P < 0.001) and hypertriglycemia (HR : 1.48, 1.15-1.90, P = 0.002) were important risk factors for type 2 diabetes in patients with COPD. CONCLUSIONS: Patients with COPD have a higher risk of type 2 diabetes compared with control subjects after adjusting for confounding factors such as sex, age, residential area, insurance premium, steroid use, hypertriglycemia, hypertension, CAD and cerebrovascular disease. Continuous surveillance of signals of dysglycemia may be incorporated into care programmes for patients with COPD.

Suicide and other-cause mortality among heroin users in Taiwan: a prospective study.

AIMS: The present study investigates one-year incidence of mortality from suicide and other causes among heroin users in Taiwan. DESIGN: A prospective national register-based cohort study. SETTING: All heroin users who attended the methadone maintenance treatment (MMT) programs in all treatment centers in Taiwan. PARTICIPANTS: The sample comprised 10,842 heroin users attending MMT. Between Jan 2006 and Dec 2007, cases were identified through the multiple-center register system and followed until Dec 2008 for date and cause of death on the Taiwan national mortality database. MEASUREMENTS: Standardized mortality ratios within one year of starting MMT were calculated as a ratio of actual versus expected numbers of deaths in the general population in Taiwan. Cox regression models were fitted to estimate the effects of gender, age, education and marital status as well as heroin related behaviors. FINDINGS: In total, 256 cases died, 67 through suicide. The mortality rate (per 100 person-years) in the first year of all-cause and suicide was 1.71 and 0.45 respectively, representing 7.5- and 18.4-fold age- and gender-standardized mortality ratio (SMR) compared to the general population. Besides, the mortality rate in the first year of overdoses, murder, HIV, somatic was 0.19, 0.02, 0.07, and 0.75 respectively, representing 68.4-, 27.7-, 76.8-, and 4.3-fold SMR increases to the general population. Older age and unemployment were independent risk factors for mortality. Females had higher standardized mortality ratio than males for suicide and all-cause mortality. CONCLUSIONS: Results showed higher risk of suicide and other-cause mortality among heroin users in MMT than general population. Suicide is an important contributor to overall excess mortality among heroin users in MMT, and especially among women. Suicide prevention and physical health monitoring are important components of MMT programs.

Risk and predictors of suicide and non-suicide mortality following non-fatal self-harm in Northern Taiwan.

OBJECTIVE: To investigate the risk and predictors of suicide and non-suicide mortality after self-harm in a Taiwanese population. METHOD: Between July 2006 and June 2008, 3,299 individuals who harmed themselves were recruited to a population-based self-harm register in Taoyuan County, Taiwan. They were followed until December 2008, with record linkage for date and cause of death in a national mortality database. RESULTS: In total, 115 individuals died, 52 through suicide. The risks of suicide and non-suicide mortality in the first year were 1.5 and 2.2%, respectively, representing an approximately 75-fold and 5-fold age- and gender-standardized increase compared with the general population in Taiwan. Male gender, rural residence, more lethal methods of self-harm and self-cited stressors for the index self-harm episode (unemployment, and chronic somatic illness) were independent risk factors for suicide mortality. Male gender, older age, rural residence and more lethal methods of self-harm were also independent risk factors for non-suicide mortality. The association between of unemployment as a cited reason for self-harm and later suicide was strongest in men and in those aged more than 45 years. CONCLUSIONS: Relatively high rates of suicide and non-suicide mortality were found following self-harm. Suicide prevention needs to take into account of risk factors for fatal repetition of self-harm.

Delay of flower senescence by bacterial endophytes expressing 1-aminocyclopropane-1-carboxylate deaminase.

AIMS: The ability of 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant growth-promoting bacterial (PGPB) endophytes Pseudomonas fluorescens YsS6 and Pseudomonas migulae 8R6, their ACC deaminase minus mutants and the rhizospheric plant growth-promoting bacterium Pseudomonas putida UW4 to delay the senescence of mini carnation cut flowers was assessed. METHODS AND RESULTS: Fresh cut flowers were incubated with either a bacterial cell suspension, the ethylene precursor ACC, the ethylene inhibitor l-α-(aminoethoxyvinyl)-glycine or 0·85% NaCl at room temperature for 11 days. Levels of flower senescence were recorded every other day. To verify the presence of endophytes inside the plant tissues, scanning electron microscopy was performed. Among all treatments, flowers treated with wild-type ACC deaminase-containing endophytic strains exhibited the most significant delay in flower senescence, while flowers treated with the ACC deaminase minus mutants senesced at a rate similar to the control. Flowers treated with Ps. putida UW4 senesced more rapidly than untreated control flowers. CONCLUSION: The only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity so that it may be concluded that this enzyme is directly responsible for the significant delay in flower senescence. Despite containing ACC deaminase activity, Ps. putida UW4 is not taken up by the cut flowers and therefore has no effect on prolonging their shelf life. SIGNIFICANCE AND IMPACT OF THE STUDY: The world-wide cut flower industry currently uses expensive and potentially environmentally dangerous chemical inhibitors of ethylene to prolong the shelf life of cut flowers. The use of PGPB endophytes with ACC deaminase activity has the potential to replace the chemicals that are currently used by the cut flower industry.

Open resource metagenomics: a model for sharing metagenomic libraries.

Both sequence-based and activity-based exploitation of environmental DNA have provided unprecedented access to the genomic content of cultivated and uncultivated microorganisms. Although researchers deposit microbial strains in culture collections and DNA sequences in databases, activity-based metagenomic studies typically only publish sequences from the hits retrieved from specific screens. Physical metagenomic libraries, conceptually similar to entire sequence datasets, are usually not straightforward to obtain by interested parties subsequent to publication. In order to facilitate unrestricted distribution of metagenomic libraries, we propose the adoption of open resource metagenomics, in line with the trend towards open access publishing, and similar to culture- and mutant-strain collections that have been the backbone of traditional microbiology and microbial genetics. The concept of open resource metagenomics includes preparation of physical DNA libraries, preferably in versatile vectors that facilitate screening in a diversity of host organisms, and pooling of clones so that single aliquots containing complete libraries can be easily distributed upon request. Database deposition of associated metadata and sequence data for each library provides researchers with information to select the most appropriate libraries for further research projects. As a starting point, we have established the Canadian MetaMicroBiome Library (CM(2)BL [1]). The CM(2)BL is a publicly accessible collection of cosmid libraries containing environmental DNA from soils collected from across Canada, spanning multiple biomes. The libraries were constructed such that the cloned DNA can be easily transferred to Gateway® compliant vectors, facilitating functional screening in virtually any surrogate microbial host for which there are available plasmid vectors. The libraries, which we are placing in the public domain, will be distributed upon request without restriction to members of both the academic research community and industry. This article invites the scientific community to adopt this philosophy of open resource metagenomics to extend the utility of functional metagenomics beyond initial publication, circumventing the need to start from scratch with each new research project.

Mortality and suicide after self-harm: community cohort study in Taiwan.

BACKGROUND: Little is known about outcomes after self-harm in East Asia. AIMS: To investigate mortality after self-harm in a Taiwanese population. METHOD: Between 2000 and 2003, 1083 individuals who self-harmed were identified through a population self-harm register in Nantou County, Taiwan, and followed until 2007 for date and cause of death on a national mortality database. RESULTS: In total, 145 individuals died, 48 through suicide. The risks of all-cause and suicide mortality in the first year were 4.7% and 2.1% respectively, representing 8- and 131-fold age- and gender-standardised increases. Male gender and older age were independent risk factors for both suicide and non-suicide mortality. Use of more lethal methods in the index episode was associated with higher mortality but this was accounted for by gender. CONCLUSIONS: Results in this sample support the recommendation that people with a history of recent self-harm should be a major target for suicide prevention programmes.

Development of a Chinese version of the Suicide Intent Scale.

This study established the psychometric properties of the Chinese version of the Suicide Intent Scale (SIS) in a clinic- and community-based sample of 36 patients and 592 respondents, respectively. Results showed that the Chinese SIS demonstrated good inter-rater and test-retest reliability. Factor analysis generated three factors (Precautions, Planning, and Seriousness) explaining 92.9% of the total variance with high internal consistency. It was moderately correlated with depressive symptoms. Results suggest that the Chinese SIS is a reliable and valid instrument for use in assessing the extent of suicidal intention among subjects with deliberate self-harm in ethnic Chinese populations.

The influence of media reporting of the suicide of a celebrity on suicide rates: a population-based study.

BACKGROUND: The impact of media reporting of suicides of entertainment celebrities may affect suicide rates due to an imitation effect. We investigated the impact on suicides of the media reporting of the suicide of a male television celebrity. METHODS: All suicides during 2003-2005 in Taiwan (n = 10,945) were included in this study. A Poisson time series autoregression analysis was conducted to examine whether there was an increase in suicides during the 4-week period after extensive media reporting of the celebrity suicide. RESULTS: After controlling for seasonal variation, calendar year, temperature, humidity and unemployment rate, there was a marked increase in the number of suicides during the 4-week period after media reporting (relative risk = 1.17, 95% CI 1.04-1.31). The increase was in men (relative risk = 1.30, 95% CI 1.14-1.50) and for the individuals using the same highly lethal method (hanging) as the TV actor did (relative risk = 1.51, 95% CI 1.25-1.83). However, the age groups in which the increase occurred were younger than the age of the celebrity. CONCLUSIONS: The extensive media reporting of the celebrity suicide was followed by an increase in suicides with a strong implication of a modelling effect. The results provide further support for the need for more restrained reporting of suicides as part of suicide prevention strategies to decrease the imitation effect.

A novel bacteriocin, thuricin 17, produced by plant growth promoting rhizobacteria strain Bacillus thuringiensis NEB17: isolation and classification.

AIMS: The aim of this study was to identify and characterize a compound produced by the plant growth promoting bacterium, Bacillus thuringiensis non-Bradyrhizobium Endophytic Bacterium 17. METHODS AND RESULTS: The bacterial peptide was analysed and purified via HPLC. Using the disk diffusion assay this peptide inhibited the growth of 16/19 B. thuringiensis strains, 4/4 Bacillus cereus strains, among others, as well as a Gram-negative strain Escherichia coli MM294 (pBS42). Both bactericidal and bacteristatic effects were observed on B. cereus ATCC 14579 and bactericidal effects were observed on B. thuringiensis ssp. thuringiensis Bt1267. The molecular weight of the peptide was estimated via SDS-PAGE and confirmed with Matrix Assisted Laser Desorption Ionization Quadrapole Time of Flight mass spectrometry; its weight is 3162 Da. The peptide is biologically active after exposure to 100 degrees C for 15 min, and within the pH range 1.00-9.25. Its activity disappeared when treated with proteinase K and protease, but not with alpha-amylase or catalase. CONCLUSIONS: We conclude that this is the first report of a bacteriocin produced by a plant growth promoting rhizobacteria (B. thuringiensis) species and have named the bacteriocin thuricin 17. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work has characterized a bacteriocin produced by a plant growth promoting bacterium. This strain is previously reported to increase soya bean nodulation.

Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants.

The competitive abilities of Sinorhizobium meliloti mutant strains containing lesions in the PHB synthesis (phbC) and degradation (bdhA) pathways were compared. While the bdhA mutant showed no noticeable symbiotic defects on alfalfa host plants when inoculated alone, in mixed inoculation experiments it was found to be less competitive than the wild type for nodule occupancy. Long-term survival of the bdhA mutant on a carbon-limiting medium was not affected. However, when subjected to competition with the wild-type strain in periodic subculturing through alternating carbon-limiting and carbon-excess conditions, the bdhA mutant performed poorly. A more severe defect in competition for growth and nodule occupancy was observed with a mutant unable to synthesize PHB (phbC). These results indicate that the ability to efficiently deposit cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability, whereas the ability to use these stores is less important.

Effect of experimental contamination with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine on soil bacterial communities.

The effect of contamination with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on an indigenous soil bacterial community was examined in two uncontaminated loam soil columns possessing native grasses. One column was spiked twice with RDX crystals for a total RDX load of 1000 mg (kg soil)(-1). The reduced metabolite of RDX degradation, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine, was observed in the column leachate, suggesting anaerobic degradation of RDX. Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA from both contaminated and uncontaminated columns produced identical banding patterns which were stable over the course of the experimental period. The bacterial diversity remained high in the contaminated column, as determined by restriction fragment length polymorphism and rarefaction analyses of random 16S rDNA clones. These combined results suggested that long-term exposure to 1000 mg RDX (kg soil)(-1) did not produce an observable effect on bacterial diversity or the numerically dominant members of the indigenous soil bacterial community.

Bradyrhizobium japonicum mutants with enhanced sensitivity to genistein resulting in altered nod gene regulation.

Bradyrhizobium japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. Plasmid pZB32, carrying a nodY::lacZ transcriptional gene fusion, was introduced into B. japonicum cells that had been subjected to UV mutagenesis. Ten independent transformants producing a blue color on plates containing 5bromo-4chloro-3indolyl-beta-D-galactopyranoside but lacking genistein, indicative of constitutive expression of the nodY::lacZ reporter gene, were isolated. Beta-galactosidase activity assays revealed that while all of the 10 strains were sensitive to low concentrations of genistein, none exhibited truly constitutive nodY::lacZ expression in liquid culture. Soybean plants inoculated with three of the mutants were chlorotic and stunted, with shoot dry weights close to those of the uninoculated plants, indicating the absence of nitrogen fixation. Differences in the kinetics of nodY::lacZ expression and lipochitin oligosaccharide Nod signal production suggested that the strains carried different mutations. Some of these strains may be useful in mitigating the low root zone temperature-associated delay in soybean nodulation at the northern extent of soybean cultivation.

The chvH locus of Agrobacterium encodes a homologue of an elongation factor involved in protein synthesis.

The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in Escherichia coli. The analysis of vir-lacZ and vir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH and efp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed a chvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.

An ACC deaminase minus mutant of Enterobacter cloacae UW4 no longer promotes root elongation.

The ACC deaminase gene (acdS) from Enterobacter cloacae UW4 was replaced by homologous recombination with the acdS gene with a tetracycline resistance gene inserted within the coding region. Upon characterization of this AcdS minus mutant, it was determined that both ACC deaminase activity and the ability to promote the elongation of canola roots under gnotobiotic conditions were greatly diminished. This result is consistent with a previously postulated model that suggests that a major mechanism utilized by plant growth-promoting bacteria involves the lowering of plant ethylene levels, and hence ethylene inhibition of root elongation, by bacterial ACC deaminase.

Requirement for the enzymes acetoacetyl coenzyme A synthetase and poly-3-hydroxybutyrate (PHB) synthase for growth of Sinorhizobium meliloti on PHB cycle intermediates.

We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.