Fluorescence characterization of clinically-important bacteria

Healthcare-associated infections (HCAI/HAI) represent a substantial threat to patient health during hospitalization and incur billions of dollars additional cost for subsequent treatment. One promising method for the detection of bacterial contamination in a clinical setting before an HAI outbreak occurs is to exploit native fluorescence of cellular molecules for a hand-held, rapid-sweep surveillance instrument. Previous studies have shown fluorescence-based detection to be sensitive and effective for food-borne and environmental microorganisms, and even to be able to distinguish between cell types, but this powerful technique has not yet been deployed on the macroscale for the primary surveillance of contamination in healthcare facilities to prevent HAI. Here we report experimental data for the specification and design of such a fluorescence-based detection instrument. We have characterized the complete fluorescence response of eleven clinically-relevant bacteria by generating excitation-emission matrices (EEMs) over broad wavelength ranges. Furthermore, a number of surfaces and items of equipment commonly present on a ward, and potentially responsible for pathogen transfer, have been analyzed for potential issues of background fluorescence masking the signal from contaminant bacteria. These include bedside handrails, nurse call button, blood pressure cuff and ward computer keyboard, as well as disinfectant cleaning products and microfiber cloth. All examined bacterial strains exhibited a distinctive double-peak fluorescence feature associated with tryptophan with no other cellular fluorophore detected. Thus, this fluorescence survey found that an emission peak of 340nm, from an excitation source at 280nm, was the cellular fluorescence signal to target for detection of bacterial contamination. The majority of materials analysed offer a spectral window through which bacterial contamination could indeed be detected. A few instances were found of potential problems of background fluorescence masking that of bacteria, but in the case of the microfiber cleaning cloth, imaging techniques could morphologically distinguish between stray strands and bacterial contamination

‘Sub-Prime’ Water, Low-Security Entitlements and Policy Challenges in Over-Allocated River Basins: the Case of the Murray–Darling Basin

Environmental policy is often implemented using market instruments. In some cases, including carbon taxing, the links between financial products and the environmental objectives, are transparent. In other cases, including water markets, the links are less transparent. In Australia’s Murray–Darling Basin (MDB), financial water products are known as ‘entitlements’, and are similar to traditional financial products, such as shares. The Australian water market includes ‘Low Security’ entitlements, which are similar to ‘sub-prime’ mortgage bonds because they are unlikely to yield an amount equal to their financial worth. Nearly half the water purchased under the Murray–Darling Basin Plan for environmental purposes is ‘Low Security’. We suggest that the current portfolio of water held by the Australian Government for environmental purposes reflects the mortgage market in the lead-up to the global financial crisis. Banks assumed that the future value of the mortgage market would reflect past trends. Similarly, it is assumed that the future value of water products will reflect past trends, without considering climate change. Historic records of allocations to ‘Low Security’ entitlements in the MDB suggest that, in the context of climate change, the Basin Plan water portfolio may fall short of the target annual average yield of 2075 GL by 511 GL. We recommend adopting finance sector methods including ‘hedging’ ‘Low Security’ entitlements by purchasing an additional 322–2755 GL of ‘Low Security’, or 160–511 GL of ‘High Security’ entitlements. Securing reliable environmental water is a global problem. Finance economics present opportunities for increasing the reliability of environmental flows

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Can we predict personality in fish? searching for consistency over time and across contexts

The interest in animal personality, broadly defined as consistency of individual behavioural traits over time and across contexts, has increased dramatically over the last years. Individual differences in behaviour are no longer recognised as noise around a mean but rather as adaptive variation and thus, essentially, raw material for evolution. Animal personality has been considered evolutionary conserved and has been shown to be present in all vertebrates including fish. Despite the importance of evolutionary and comparative aspects in this field, few studies have actually documented consistency across situations in fish. In addition, most studies are done with individually housed fish which may pose additional challenges when interpreting data from social species. Here, we investigate, for the first time in fish, whether individual differences in behavioural responses to a variety of challenges are consistent over time and across contexts using both individual and grouped-based tests. Twenty-four juveniles of Gilthead seabream Sparus aurata were subjected to three individual-based tests: feed intake recovery in a novel environment, novel object and restraining and to two group-based tests: risk-taking and hypoxia. Each test was repeated twice to assess consistency of behavioural responses over time. Risk taking and escape behaviours during restraining were shown to be significantly consistent over time. In addition, consistency across contexts was also observed: individuals that took longer to recover feed intake after transfer into a novel environment exhibited higher escape attempts during a restraining test and escaped faster from hypoxia conditions. These results highlight the possibility to predict behaviour in groups from individual personality traits.European Commission [265957 COPEWELL]; European Social Fund of Andalusia; Foundation for Science and Technology, Portugal [SFRH/BPD/77210/2011]info:eu-repo/semantics/publishedVersio

Changes in muscle contractile characteristics and jump height following 24 days of unilateral lower limb suspension

We measured changes in maximal voluntary and electrically evoked torque and rate of torque development because of limb unloading. We investigated whether these changes during single joint isometric muscle contractions were related to changes in jump performance involving dynamic muscle contractions and several joints. Six healthy male subjects (21 ± 1 years) underwent 3 weeks of unilateral lower limb suspension (ULLS) of the right limb. Plantar flexor and knee extensor maximal voluntary contraction (MVC) torque and maximal rate of torque development (MRTD), voluntary activation, and maximal triplet torque (thigh; 3 pulses at 300 Hz) were measured next to squat jump height before and after ULLS. MVC of plantar flexors and knee extensors (MVCke) and triplet torque decreased by 12% (P = 0.012), 21% (P = 0.001) and 11% (P = 0.016), respectively. Voluntary activation did not change (P = 0.192). Absolute MRTD during voluntary contractions decreased for plantar flexors (by 17%, P = 0.027) but not for knee extensors (P = 0.154). Absolute triplet MRTD decreased by 17% (P = 0.048). The reduction in MRTD disappeared following normalization to MVC. Jump height with the previously unloaded leg decreased significantly by 28%. No significant relationships were found between any muscle variable and jump height (r < 0.48), but decreases in torque were (triplet, r = 0.83, P = 0.04) or tended to be (MVCke r = 0.71, P = 0.11) related to decreases in jump height. Thus, reductions in isometric muscle torque following 3 weeks of limb unloading were significantly related to decreases in the more complex jump task, although torque in itself (without intervention) was not related to jump performance

The kinome of Phytophthora infestans reveals oomycete-specific innovations and links to other taxonomic groups

<p>Abstract</p> <p>Background</p> <p>Oomycetes are a large group of economically and ecologically important species. Its most notorious member is <it>Phytophthora infestans</it>, the cause of the devastating potato late blight disease. The life cycle of <it>P. infestans </it>involves hyphae which differentiate into spores used for dispersal and host infection. Protein phosphorylation likely plays crucial roles in these stages, and to help understand this we present here a genome-wide analysis of the protein kinases of <it>P. infestans </it>and several relatives. The study also provides new insight into kinase evolution since oomycetes are taxonomically distant from organisms with well-characterized kinomes.</p> <p>Results</p> <p>Bioinformatic searches of the genomes of <it>P. infestans</it>, <it>P. ramorum</it>, and <it>P. sojae </it>reveal they have similar kinomes, which for <it>P. infestans </it>contains 354 eukaryotic protein kinases (ePKs) and 18 atypical kinases (aPKs), equaling 2% of total genes. After refining gene models, most were classifiable into families seen in other eukaryotes. Some ePK families are nevertheless unusual, especially the tyrosine kinase-like (TKL) group which includes large oomycete-specific subfamilies. Also identified were two tyrosine kinases, which are rare in non-metazoans. Several ePKs bear accessory domains not identified previously on kinases, such as cyclin-dependent kinases with integral cyclin domains. Most ePKs lack accessory domains, implying that many are regulated transcriptionally. This was confirmed by mRNA expression-profiling studies that showed that two-thirds vary significantly between hyphae, sporangia, and zoospores. Comparisons to neighboring taxa (apicomplexans, ciliates, diatoms) revealed both clade-specific and conserved features, and multiple connections to plant kinases were observed. The kinome of <it>Hyaloperonospora arabidopsidis</it>, an oomycete with a simpler life cycle than <it>P. infestans</it>, was found to be one-third smaller. Some differences may be attributable to gene clustering, which facilitates subfamily expansion (or loss) through unequal crossing-over.</p> <p>Conclusion</p> <p>The large sizes of the <it>Phytophthora </it>kinomes imply that phosphorylation plays major roles in their life cycles. Their kinomes also include many novel ePKs, some specific to oomycetes or shared with neighboring groups. Little experimentation to date has addressed the biological functions of oomycete kinases, but this should be stimulated by the structural, evolutionary, and expression data presented here. This may lead to targets for disease control.</p

Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL

Introduction It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal. Methods Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation. Results Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10%-30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment. Conclusions We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes. [PROVISIONAL

A correction for sample overlap in genome-wide association studies in a polygenic pleiotropy-informed framework.

BACKGROUND: There is considerable evidence that many complex traits have a partially shared genetic basis, termed pleiotropy. It is therefore useful to consider integrating genome-wide association study (GWAS) data across several traits, usually at the summary statistic level. A major practical challenge arises when these GWAS have overlapping subjects. This is particularly an issue when estimating pleiotropy using methods that condition the significance of one trait on the signficance of a second, such as the covariate-modulated false discovery rate (cmfdr). RESULTS: We propose a method for correcting for sample overlap at the summary statistic level. We quantify the expected amount of spurious correlation between the summary statistics from two GWAS due to sample overlap, and use this estimated correlation in a simple linear correction that adjusts the joint distribution of test statistics from the two GWAS. The correction is appropriate for GWAS with case-control or quantitative outcomes. Our simulations and data example show that without correcting for sample overlap, the cmfdr is not properly controlled, leading to an excessive number of false discoveries and an excessive false discovery proportion. Our correction for sample overlap is effective in that it restores proper control of the false discovery rate, at very little loss in power. CONCLUSIONS: With our proposed correction, it is possible to integrate GWAS summary statistics with overlapping samples in a statistical framework that is dependent on the joint distribution of the two GWAS

Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera

BACKGROUND: Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. CONCLUSIONS/SIGNIFICANCE: These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey

Systematic review of methods used in meta-analyses where a primary outcome is an adverse or unintended event

addresses: Peninsula College of Medicine and Dentistry, St Luke's Campus, University of Exeter, Exeter, UK. [email protected]: PMCID: PMC3528446types: Journal Article; Research Support, Non-U.S. Gov't© 2012 Warren et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Adverse consequences of medical interventions are a source of concern, but clinical trials may lack power to detect elevated rates of such events, while observational studies have inherent limitations. Meta-analysis allows the combination of individual studies, which can increase power and provide stronger evidence relating to adverse events. However, meta-analysis of adverse events has associated methodological challenges. The aim of this study was to systematically identify and review the methodology used in meta-analyses where a primary outcome is an adverse or unintended event, following a therapeutic intervention