Search

Comparative studies of mortality in the wild are necessary to understand the evolution of aging; yet, ectothermic tetrapods are underrepresented in this comparative landscape, despite their suitability for testing evolutionary hypotheses. We present a study of aging rates and longevity across wild tetrapod ectotherms, using data from 107 populations (77 species) of nonavian reptiles and amphibians. We test hypotheses of how thermoregulatory mode, environmental temperature, protective phenotypes, and pace of life history contribute to demographic aging. Controlling for phylogeny and body size, ectotherms display a higher diversity of aging rates compared with endotherms and include phylogenetically widespread evidence of negligible aging. Protective phenotypes and life-history strategies further explain macroevolutionary patterns of aging. Analyzing ectothermic tetrapods in a comparative context enhances our understanding of the evolution of aging.

Cuticular hydrocarbons (CHCs) are organic compounds found on the cuticles of all insects which can act as close-contact pheromones, while also providing a hydrophobic barrier to water loss. Given their widespread importance in sexual behaviour and survival, CHCs have likely contributed heavily to the adaptation and speciation of insects. Despite this, the patterns and mechanisms of their diversification have been studied in very few taxa. Here, we perform the first study of CHC diversification in blowflies, focussing on wild populations of the ecologically diverse genus Chrysomya. We convert CHC profiles into qualitative and quantitative traits and assess their inter- and intra-specific variation across 10 species. We also construct a global phylogeny of Chrysomya, onto which CHCs were mapped to explore the patterns of their diversification. For the first time, we demonstrate that blowflies express an exceptional diversity of CHCs, which have diversified in a non-phylogenetic and punctuated manner, are species-specific, and sexually dimorphic. It is likely that both ecological and sexual selection have shaped these patterns of CHC diversification, and our study now provides a comprehensive framework for testing such hypotheses.

Wing interference patterns (WIPs) are stable structural colours displayed on insect wings which are only visible at specific viewing geometries and against certain backgrounds. These patterns are widespread among flies and wasps, and growing evidence suggests that they may function as species- and sex-specific mating cues in a range of taxa. As such, it is expected that WIPs should differ between species and show clear sexual dimorphisms. However, the true extent to which WIPs vary between species, sexes, and individuals is currently unclear, as previous studies have only taken a qualitative approach, without considering how WIPs might be perceived by the insect. Here, we perform the first quantitative analysis of inter- and intra-specific variation in WIPs across seven Australian species of the blowfly genus Chrysomya. Using multispectral digital imaging and a tentative model of blowfly colour vision, we provide quantitative evidence that WIPs are species-specific, highlight that the extent of divergence is greater in males than in females, and demonstrate sexual dimorphisms in several species. These data provide evidence that WIPs have diversified substantially in blowflies and suggests that sexual selection may have played a role in this process.

Workholding and fixturing is a critically important aspect of manufacturing that has direct implications for the quality of the manufactured component during processing as well as a direct impact on the cost of the component. The field of workholding is mature with numerous techniques employed, mostly using contact pressure, but also using magnetics and adhesives. Looking to nature for inspiration presents us with the use of ice as a mechanism for adhesion, referred to as cryo-adhesion. Cryo-adhesion offers some advantages over more traditional fixturing methods such as removing contact pressure and therefore reducing the dependence on the machining of complex, intricate bespoke fixtures. While the concept of ice adhesion is known, there is minimal research presented on the application of ice adhesion in manufacturing processes. This research reports on the development of a novel Peltier-based cryo-cooling fixture for workholding in manufacturing operations. The research provides insight into the main interactions that might be experienced in manufacturing type scenarios and presents findings on the cryo fixture's thermal and geometric characteristics, the use of the novel cryo fixture for holding various materials under tensile and shear loads, and a consideration of contact area and surface roughness on the cryo fixture performance. Keywords: Ice adhesion, Workholding, Grasping, Peltier cooling, Thermoelectric, Manufacturing technology

During its life cycle, the environmental pathogenLegionella pneumophilaalternates between a replicative and a transmissive cell type when cultured in broth, macrophages, or amoebae. Within a protozoan host,L. pneumophilafurther differentiates into the hardy cell type known as the Mature Infectious Form (MIF). The second messenger cyclic-di-GMP coordinates lifestyle changes in many bacterial species, but its role in theL. pneumophilalife cycle is less understood. Using anin vitrobroth culture model that approximates the intracellular transition from the replicative to transmissive form, here we investigate the contribution toL. pneumophiladifferentiation of a two-component system (TCS) that regulates cyclic-di-GMP metabolism. The TCS is encoded bylpg0278-lpg0277and is co-transcribed withlpg0279, which encodes a protein upregulated in MIF cells. Using agfp-reporter, we demonstrate that the promoter for this operon is RpoS-dependent and induced in nutrient-limiting conditions that do not support replication. The response regulator of the TCS (Lpg0277) is a bifunctional enzyme that both synthesizes and degrades cyclic-di-GMP. Using a panel of site-directed point mutants, we show that cyclic-di-GMP synthesis mediated by a conserved GGDEF domain promotes growth arrest of replicativeL. pneumophila, production of pigment and poly-3-hydroxybutyrate storage granules, and viability in nutrient-limiting conditions. Genetic epistasis tests predict that the MIF protein Lpg0279 acts upstream of the TCS as a negative regulator. Thus,L. pneumophilais equipped with a regulatory network in which cyclic-di-GMP stimulates the switch from a replicative to a resilient state equipped to survive in low-nutrient environments.IMPORTANCEAlthough an intracellular pathogen,L. pneumophilahas developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication ofL. pneumophilafrom contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that supportL. pneumophilapersistence in low-nutrient environments can inform design of remediation methods. Here we characterize a genetic locus that encodes a two-component signaling system (lpg0278-lpg0277) and a putative regulator protein (lpg0279) that modulates production of the messenger molecule cyclic-di-GMP. We show that this locus promotes bothL. pneumophilacell differentiation and survival in nutrient-limiting conditions, thus advancing our understanding of the mechanisms that contribute toL. pneumophilaenvironmental resilience.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have higher incidence of psychosocial disorders including depression. As suicide is the most severe manifestation of depression, we sought to identify if patients with IBD have a higher incidence of suicide through a systematic review/meta-analysis. AIMS: To determine the incidence of suicide in IBD and to determine if patients with IBD were more likely to die from suicide compared to control. METHODS: Systematic literature search for articles using EMBASE and MEDLINE was conducted to identify studies investigating suicide in IBD. We included studies reporting expected number of death or standarized mortality ratio (SMR) for suicide in IBD. Meta-analysis for Crohns disease and ulcerative colitis was conducted separately, as well as combined. RESULTS: Seven cohort studies were identified through our search strategy and were included in our systematic review/meta-analysis. In our analysis, the SMR for suicide in patients with IBD for all studies included was 1.20 (95% CI 0.94–1.54). The overall pooled SMR for CD and UC were 1.36 (95% CI 0.98–1.88) and 1.16 (95% CI 0.8–1.69) respectively. CONCLUSIONS: Patients with Crohn’s disease and ulcerative colitis may have an increased risk of suicide. These results highlight the importance physicians must place on ensuring the mental health of patients with IBD is both assessed and treated appropriately. FUNDING AGENCIES: None

The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations predicted a lipid binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. We propose that structural lipids have a central role in the formation of functional, dimeric UapA. We describe the first in-depth analysis of membrane lipid interactions with a eukaryotic transporter using native mass spectrometry. We demonstrate that the binding of structural lipids is essential to maintain the stability of the functional UapA dimer in both the gas phase and in vivo. © 2018 The Authors

Background Therapeutic mammaplasty, which combines breast reduction and mastopexy techniques with tumour excision, may extend the boundaries of breast-conserving surgery and improve outcomes for patients, but current practice is unknown and high-quality outcome data are lacking. This prospective multicentre cohort study aimed to explore the practice and short-term outcomes of the technique. Methods Consecutive patients undergoing therapeutic mammaplasty at participating centres between 1 September 2016 and 30 June 2017 were recruited to the study. Demographic, preoperative, operative, oncological and complication data were collected. The primary outcome was unplanned reoperation for complications within 30 days of surgery. Secondary outcomes included re-excision rates and time to adjuvant therapy. Results Overall, 880 patients underwent 899 therapeutic mammaplasty procedures at 50 centres. The most common indications were avoidance of poor cosmetic outcomes associated with standard breast-conserving surgery (702 procedures, 78·1 per cent) or avoidance of mastectomy (379, 42·2 per cent). Wise-pattern skin incisions were the most common (429 of 899, 47·7 per cent), but a range of incisions and nipple–areola pedicles were used. Immediate contralateral symmetrization was performed in one-third of cases (284 of 880, 32·3 per cent). In total, 205 patients (23·3 per cent) developed a complication, but only 25 (2·8 per cent) required reoperation. Median postoperative lesion size was 24·5 (i.q.r. 16–38) mm. Incomplete excision was seen in 132 procedures (14·7 per cent), but completion mastectomy was required for only 51 lesions (5·7 per cent). Median time to adjuvant therapy was 54 (i.q.r. 42–66) days. Conclusion Therapeutic mammaplasty is a safe and effective alternative to mastectomy or standard breast-conserving surgery. Further work is required to explore the impact of the technique on quality of life, and to establish cost-effectiveness.

Male courtship behavior and displays influence female mating decisions, and therefore affect mating success in a diverse range of organisms. While there is substantial evidence confirming that females prefer males who invest more in courtship, less attention has been paid to the relative importance of individual behaviors, and the discrete sequences of courtship that result in mating success. The small hairy maggot blow fly, Chrysomya varipes, performs stereotyped courtship behaviors, involving orienting, tapping, waving, arching, wing vibration and mounting. This study aimed to quantify male investment in specific courtship behaviors, and compare courtship investment and behavioral transitions between males who gained mating success (successful males) and those that did not (unsuccessful males). Our results show that mating success was influenced by the behaviors orienting, tapping, arching and mounting. Behavioral transitions revealed a distinct pattern of behaviors leading to a mating attempt, and some differences were observed between successful and unsuccessful males. Overall, our findings suggest that female mating decisions were based on differences in specific male courtship behaviors. This detailed observational study has quantified multiple courtship behaviors for the first time in C. varipes, and highlights the importance of considering multiple behavioral traits when exploring the influence of male courtship on mating success.

Nanomanufacturing involves scaled-up, reliable, and cost-effective manufacturing of nanoscale materials, structures, devices, and systems. Nanomanufacturing methods can be classified into top-down and bottom-up approaches, including additive, subtractive, and replication/mass conservation processes. These include a cluster of various techniques such as nanomachining, nanofabrication, and nanometrology to produce nanotechnology components and conduct evaluation. This paper mainly focuses on the manufacturing methods for complex shapes or structures, such as textures on curves and hierarchical structures, and outlines the research perspectives and the current application status of nanomanufacturing fundamentals and key technologies.

Bacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulator csrA . Using bioinformatic analyses, we deduced that specific csrA paralogs are coinherited with particular lineages of the type IV secretion system that mediates horizontal spread of its ICE, suggesting a conserved regulatory interaction. As a first step to investigate the contribution of csrA regulators to this class of mobile genetic elements, we analyzed here the activity of the csrA paralog encoded on Legionella pneumophila ICE-βox. Deletion of this gene, which we name csrT , had no observed effect under laboratory conditions. However, ectopic expression of csrT abrogated the protection to hydrogen peroxide and macrophage degradation that ICE-βox confers to L. pneumophila . When ectopically expressed, csrT also repressed L. pneumophila flagellin production and motility, a function similar to the core genome's canonical csrA . Moreover, csrT restored the repression of motility to csrA mutants of Bacillus subtilis , a finding consistent with the predicted function of CsrT as an mRNA binding protein. Since all known ICEs of legionellae encode coinherited csrA -type IV secretion system pairs, we postulate that CsrA superfamily proteins regulate ICE activity to increase their horizontal spread, thereby expanding L. pneumophila versatility. IMPORTANCE ICEs are mobile DNA elements whose type IV secretion machineries mediate spread among bacterial populations. All surveyed ICEs within the Legionella genus also carry paralogs of the essential life cycle regulator csrA . It is striking that the csrA loci could be classified into distinct families based on either their sequence or the subtype of the adjacent type IV secretion system locus. To investigate whether ICE-encoded csrA paralogs are bona fide regulators, we analyzed ICE-βox as a model system. When expressed ectopically, its csrA paralog inhibited multiple ICE-βox phenotypes, as well as the motility of not only Legionella but also Bacillus subtilis . Accordingly, we predict that CsrA regulators equip legionellae ICEs to promote their spread via dedicated type IV secretion systems.

Transporters are integral membrane proteins with central roles in the efficient movement of molecules across biological membranes. Many transporters exist as oligomers in the membrane. Depending on the individual transport protein, oligomerization can have roles in membrane trafficking, function, regulation and turnover. For example, our recent studies on UapA, a nucleobase ascorbate transporter, from Aspergillus nidulans, have revealed both that dimerization of this protein is essential for correct trafficking to the membrane and the structural basis of how one UapA protomer can affect the function of the closely associated adjacent protomer. Here, we review the roles of oligomerization in many particularly well-studied transporters and transporter families. © 2016 The Author(s).

Secondary salinization has been identified as a major stressor to amphibians. Exposure to elevated salinity necessitates physiological adjustments and biochemical changes that may be energetically demanding. As such, exposure to non-lethal levels of salinity during development could potentially alter anuran metabolic rates and individual performance in both pre- and post-metamorphic life stages. We investigated the effects of non-lethal levels of salinity on metamorphic traits (time to reach metamorphosis and metamorphic mass), tadpole oxygen consumption, escape response behaviour (pre- and post-metamorphosis) and foraging ability post-metamorphosis in two native Australian frog species, the southern brown tree frog ( Litoria ewingii ) and the striped marsh frog ( Limnodynastes peronii ). We found that both Lit. ewingii and Lim. peronii exhibited differences in metamorphic traits in response to elevated salinity. Neither species showed significant change in oxygen consumption during development in response to salinity, relative to freshwater controls. Both species displayed impaired escape response behaviours in response to salinity during larval development, but flow-on effects to adult escape response behaviours and foraging performance were species-specific. Our results show that the influence of stressors during development can have consequences for anuran physiology and behaviour at multiple life stages, and emphasize the need for studies that examine the energetics of anuran responses in order to better understand the responses of biota to stressful environments.

Abnormalities in atrial myocardium increase the likelihood of arrhythmias, including atrial fibrillation (AF). The deposition of misfolded protein, or amyloidosis, plays an important role in the pathophysiology of many diseases, including human cardiomyopathies. We have shown that genes implicated in amyloidosis are activated in a cellular model of AF, with the development of preamyloid oligomers (PAOs). PAOs are intermediates in the formation of amyloid fibrils, and they are now recognized to be the cytotoxic species during amyloidosis. To investigate the presence of PAOs in human atrium, we developed a microscopic imaging-based protocol to enable robust and reproducible quantitative analysis of PAO burden in atrial samples harvested at the time of elective cardiac surgery. Using PAO- and myocardial-specific antibodies, we found that PAO distribution was typically heterogeneous within a myocardial sample. Rigorous imaging and analysis protocols were developed to quantify the relative area of myocardium containing PAOs, termed the Green/Red ratio (G/R), for a given sample. Using these methods, reproducible G/R values were obtained when different sections of a sample were independently processed, imaged, and analyzed by different investigators. This robust technique will enable studies to investigate the role of this novel structural abnormality in the pathophysiology of and arrhythmia generation in human atrial tissue.

Background: Intraoperative hemolysis and inflammation are associated with acute kidney injury (AKI) following cardiac surgery. Plasma-free hemoglobin induces heme oxygenase-1 (HO-1) expression. HO-1 degrades heme but increases in experimental models of AKI. This study tested the hypothesis that plasma HO-1 concentrations are associated with intraoperative hemolysis and are increased in patients that develop AKI following cardiac surgery. Methods: We measured plasma HO-1, free hemoglobin, and inflammatory markers in 74 patients undergoing cardiopulmonary bypass (CPB). AKI was defined as an increase in serum creatinine concentration of 50% or 0.3 mg/dl within 72 h of surgery. Results: Twenty-eight percent of patients developed AKI. HO-1 concentrations increased from 4.2 ± 0.2 ng/ml at baseline to 6.6 ± 0.5 ng/ml on postoperative day (POD) 1 (p < 0.001). POD1 HO-1 concentrations were 3.1 ng/ml higher (95% CI 1.1-5.1) in AKI patients, as was the change in HO-1 from baseline to POD1 (4.4 ± 1.3 ng/ml in AKI patients vs. 1.5 ± 0.3 ng/ml in no-AKI patients, p = 0.006). HO-1 concentrations remained elevated in AKI patients even after controlling for AKI risk factors and preoperative drug therapy. Peak-free hemoglobin concentrations correlated with peak HO-1 concentrations on POD1 in patients that developed AKI (p = 0.02). Duration of CPB and post-CPB IL-6 and IL-10 concentrations were also associated with increased HO-1 on POD1. Conclusion: Plasma HO-1 is increased in patients that develop AKI, and CPB duration, hemolysis, and inflammation are associated with increased HO-1 concentrations following cardiac surgery. Strategies that alter hemolysis and HO-1 expression during cardiac surgery may affect risk for AKI.

Significant advances have been made to understand the mechanisms involved in cardiac cell-based therapies. The early translational application of basic science knowledge has led to several animal and human clinical trials. The initial promising beneficial effect of stem cells on cardiac function restoration has been eclipsed by the inability of animal studies to translate into sustained clinical improvements in human clinical trials.In this review, the authors cover an updated overview of various stem cell populations used in chronic heart failure. A critical review of clinical trials conducted in advanced heart failure patients is proposed, and finally promising avenues for developments in the field of cardiac cell-based therapies are presented.Several questions remain unanswered, and this limits our ability to understand basic mechanisms involved in stem cell therapeutics. Human studies have revealed critical unresolved issues. Further elucidation of the proper timing, mode delivery and prosurvival factors is imperative, if the field is to advance. The limited benefits seen to date are simply not enough if the potential for substantial recovery of nonfunctioning myocardium is to be realized.

Azospira suillum strain PS (formally Dechlorosoma suillum strain PS) is a metabolically versatile betaproteobacterium first identified for its ability to grow by dissimilatory reduction of perchlorate and chlorate [denoted (per)chlorate]. Together with Dechloromonas species, these two genera represent the dominant (per)chlorate-reducing bacteria in mesophilic freshwater environments. In addition to (per)chlorate reduction, A. suillum is capable of the anaerobic oxidation of humic substances and is the first anaerobic nitrate-dependent Fe(II) oxidizer outside the Diaphorobacter and Acidovorax genera for which there is a completed genome sequence.

The levels of integron abundance and diversity in soil amended with pig slurry were studied. Real-time PCR illustrated a significant increase in class 1 integron prevalence after slurry application, with increased prevalence still evident at 10 months after application. Culture-dependent data revealed 10 genera, including putative human pathogens, carrying class 1 and 2 integrons.

The Nucleobase-Ascorbate Transporters (NATs) family includes carriers with fundamental functions in uptake of key cellular metabolites, such as uric acid or vitamin C. The best studied example of a NAT transporter is the uric acid-xanthine permease (UapA) from the model ascomycete Aspergillus nidulans. Detailed genetic and biochemical analyses have revealed much about the mechanism of action of this protein; however, the difficulties associated with handling eukaryotic membrane proteins have limited efforts to elucidate the precise structure-function relationships of UapA by structural analysis. In this manuscript, we describe the heterologous overexpression of functional UapA as a fusion with GFP in different strains of Saccharomyces cerevisiae. The UapA-GFP construct expressed to 2.3 mg/L in a pep4Δ deletion strain lacking a key vacuolar endopeptidase and 3.8 mg/L in an npi1-1 mutant strain with defective Rsp5 ubiquitin ligase activity. Epifluorescence microscopy revealed that the UapA-GFP was predominately localized to the plasma membrane in both strains, although a higher intensity of fluorescence was observed for the npi1-1 mutant strain plasma membrane. In agreement with these observations, the npi1-1 mutant strain demonstrated a ∼5-fold increase in uptake of [3H]-xanthine compared to the pep4Δ deletion strain. Despite yielding the best results for functional expression, in-gel fluorescence of the UapA-GFP expressed in the npi1-1 mutant strain revealed that the protein was subject to significant proteolytic degradation. Large scale expression of the protein using the pep4Δ deletion strain followed by purification produced mg quantities of pure, monodispersed protein suitable for further structural and functional studies. In addition, this work has generated a yeast cell based system for performing reverse genetics and other targeted approaches, in order to further understand the mechanism of action of this important model protein. © 2010 Elsevier Inc. All rights reserved.

Acidovorax ebreus strain TPSY is the first anaerobic nitrate-dependent Fe(II) oxidizer for which there is a completed genome sequence. Preliminary protein annotation revealed an organism optimized for survival in a complex environmental system. Here, we briefly report the completed and annotated genome sequence of strain TPSY.

Legionella pneumophila, a motile opportunistic pathogen of humans, is restricted from replicating in the lungs of C57BL/6 mice. Resistance of mouse macrophages toL. pneumophiladepends on recognition of cytosolic flagellin. Once detected by the NOD-like receptors Naip5 and Ipaf (Nlrc4), flagellin triggers pyroptosis, a proinflammatory cell death. In contrast, motile strains ofL. parisiensisandL. tucsonensisreplicate profusely within C57BL/6 macrophages, similar to flagellin-deficientL. pneumophila. To gain insight into how motile species escape innate defense mechanisms of mice, we compared their impacts on macrophages.L. parisiensisandL. tucsonensisdo not induce proinflammatory cell death, as measured by lactate dehydrogenase (LDH) release and interleukin-1β (IL-1β) secretion. However, flagellin isolated fromL. parisiensisandL. tucsonensistriggers cell death and IL-1β secretion when transfected into the cytosol of macrophages. Neither strain displays three characteristics of the canonicalL. pneumophilaDot/Icm type IV secretion system: sodium sensitivity, LAMP-1 evasion, and pore formation. Therefore, we postulate that whenL. parisiensisandL. tucsonensisinvade a mouse macrophage, flagellin is confined to the phagosome, protecting the bacteria from recognition by the cytosolic surveillance system and allowingLegionellato replicate. Despite their superior capacity to multiply in mouse macrophages,L. parisiensisandL. tucsonensishave been associated with only two cases of disease, both in renal transplant patients. These results point to the complexity of disease, a product of the pathogenic potential of the microbe, as defined in the laboratory, and the capacity of the host to mount a measured defense.

The prevalences of three sulfonamide resistance genes, sul1 , sul2 , and sul3 and sulfachloropyridazine (SCP) resistance were determined in bacteria isolated from manured agricultural clay soils and slurry samples in the United Kingdom over a 2-year period. Slurry from tylosin-fed pigs amended with SCP and oxytetracycline was used for manuring. Isolates positive for sul genes were further screened for the presence of class 1 and 2 integrons. Phenotypic resistance to SCP was significantly higher in isolates from pig slurry and postapplication soil than in those from preapplication soil. Of 531 isolates, 23% carried sul1 , 18% sul2 , and 9% sul3 only. Two percent of isolates contained all three sul genes. Class 1 and class 2 integrons were identified in 5% and 11.7%, respectively, of sul -positive isolates. In previous reports, sul1 was linked to class 1 integrons, but in this study only 8% of sul1 -positive isolates carried the intI1 gene. Sulfonamide-resistant pathogens, including Shigella flexneri , Aerococcus spp., and Acinetobacter baumannii , were identified in slurry-amended soil and soil leachate, suggesting a potential environmental reservoir. Sulfonamide resistance in Psychrobacter , Enterococcus , and Bacillus spp. is reported for the first time, and this study also provides the first description of the genotypes sul1 , sul2 , and sul3 outside the Enterobacteriaceae and in the soil environment.

Legionella pneumophila , a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella -containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB , encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB , encoding components of the Type IV secretion system. Quantitative similarity of the katA / B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H 2 O 2 compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii , indicating that certain dotA / B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages.

We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores of Mycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of M. brassicicola correlation coefficients ( r 2 ) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of M. brassicicola per liter of air sampled and the amount of disease expressed on exposed plants of Brassica oleracea (Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of B. cinerea , a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of M. brassicicola ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments.

Background Conservation Breeding Programs (CBP's) are playing an important role in the protection of critically endangered anuran amphibians, but for many species recruitment is not successful enough to maintain captive populations, or provide individuals for release. In response, there has been an increasing focus on the use of Assisted Reproductive Technologies (ART), including the administration of reproductive hormones to induce gamete release followed by in vitro fertilisation. The objective of this study was to test the efficacy of two exogenous hormones to induce gamete release, for the purpose of conducting in vitro fertilisation (IVF), in one of Australia's most critically endangered frog species, Pseudophryne corroboree. Methods Male frogs were administered a single dose of either human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRHa), while female frogs received both a priming and ovulatory dose of LHRHa. Spermiation responses were evaluated at 3, 7, 12, 24, 36, 48, 60 and 72 h post hormone administration (PA), and sperm number and viability were quantified using fluorescent microscopy. Ovulation responses were evaluated by stripping females every 12 h PA for 5 days. Once gametes were obtained, IVF was attempted by combining spermic urine with oocytes in a dilute solution of simplified amphibian ringer (SAR). Results Administration of both hCG and LHRHa induced approximately 80% of males to release sperm over 72 h. Peak sperm release occurred at 12 h PA for hCG treated males and 36 h PA for LHRHa treated males. On average, LHRHa treated males released a significantly higher total number of live sperm, and a higher concentration of sperm, over a longer period. In female frogs, administration of LHRHa induced approximately 30% of individuals to release eggs. On average, eggs were released between 24 and 48 h PA, with a peak in egg release at 36 h PA. IVF resulted in a moderate percentage (54.72%) of eggs being fertilised, however all resultant embryos failed prior to gastrulation. Conclusions Hormone treatment successfully induced spermiation and ovulation in P. corroboree, but refinement of gamete induction and IVF techniques will be required before ART protocols can be used to routinely propagate this species.

1. The action of noradrenaline on cells isolated from the rabbit ear artery was studied with the whole-cell configuration of the patch clamp technique. In normal potassium-containing solutions at a holding potential of -50 mV noradrenaline elicited either outward, inward or mixed outward and inward currents. These responses were blocked by the alpha-adrenoceptor antagonist, phentolamine (10(-6) M). 2. The outward current occurred as a consequence of an increase in membrane conductance and the reversal potential was close to the potassium equilibrium potential (EK). It was possible to record outward currents without external calcium but not when the concentration of EGTA in the pipette was increased to 10 mM or when potassium was absent from the pipette solution. It is concluded that the outward current evoked by alpha-adrenoceptor stimulation is produced by a calcium mediated increase in potassium conductance. 3. The ionic basis of the inward current was investigated in potassium-free external and pipette solutions. When sodium chloride was the major constituent of the external and pipette solutions the reversal potential (Er) of the noradrenaline-induced current was 0.63 mV, close to ENa and ECl. 4. When most of the external sodium chloride was replaced by sucrose Er was intermediate between ENa and ECl but had shifted significantly towards ENa. Further ion substitution experiments suggest that noradrenaline increased the membrane conductance to both anions and cations. 5. When the current was carried predominantly by anions, depolarizing steps (from -50 mV) produced outward current relaxations with a time constant of about 40 ms. Bath-applied caffeine also produced a membrane current which rectified in the outward direction. 6. When the response to noradrenaline was mediated mainly by cations, the relationship between the membrane current and clamp potential was non-linear and the amplitude of the currents at potentials positive to -50 mV became disproportionately smaller. In addition on repolarization to -50 mV the instantaneous current was followed by an inward relaxation. 7. In high external barium solution noradrenaline evoked a membrane current with a reversal potential much more positive than ENa or ECl. This current was recorded in the presence of 10(-5) M-nifedipine and diltiazem and in 10(-4) M-cadmium which suggests that the voltage-dependent calcium channel is not implicated in the generation of the non-selective cation current to noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)