Engineering Bacillus megaterium for production of functional intracellular materials

Background: Over the last 10-15 years, a technology has been developed to engineer bacterial polyhydroxybutyrate (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. So far, beads have mainly been produced in recombinant Escherichia coli which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals. Results: In this study, we have engineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding β-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled/effected PHB production by B. megaterium PHA05. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40-50 mg IgG/g beads. As B. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads. Conclusions: This study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.fals

Distribution and composition of macrobenthic communities along a Victoria-Land Transect (Ross Sea, Antarctica)

The Victoria-Land Transect project onboard the Italian research vessel ‘‘Italica’’ in February 2004, was a large-scale attempt to obtain benthic samples of smaller macrozoobenthic specimens systematically along a latitudinal and a depth transect along the Victoria- Land coast. Data presented from this survey are based on Rauschert dredge samples, which were taken at four areas at depth ranging from 84 to 515 m. A cluster analysis based on relative numbers of abundance was performed and demonstrated a change in community structure depending on the location along the latitudinal transect. A change in community structure with depth was not recorded. Dominant taxa of the Ross Sea fauna along the Victoria-Land coast were the Arthropoda (65.7%), followed by Annelida (20.7%), Mollusca (9.6%) and Echinodermata (2.5%). Total number of abundance decreased with depth with an exception at Cape Russell, whereas a trend in biomass was not documented. Abundance and biomass proportions of major taxa changed gradually along the latitudinal transect

Who receives treatment for alcohol use disorders in the European Union? A cross-sectional representative study in primary and specialized health care

Background Alcohol use disorders (AUDs) are highly prevalent in Europe, but only a minority of those affected receive treatment. It is therefore important to identify factors that predict treatment in order to reframe strategies aimed at improving treatment rates. Methods Representative cross-sectional study with patients aged 18\u201364 from primary health care (PC, six European countries, n = 8476, data collection 01/13\u201301/14) and from specialized health care (SC, eight European countries, n = 1762, data collection 01/13\u201303/14). For descriptive purposes, six groups were distinguished, based on type of DSM-IV AUD and treatment setting. Treatment status (yes/no) for any treatment (model 1), and for SC treatment (model 2) were main outcome measures in logistic regression models. Results AUDs were prevalent in PC (12-month prevalence: 11.8%, 95% confidence interval (CI): 11.2\u201312.5%), with 17.6% receiving current treatment (95%CI: 15.3\u201319.9%). There were clear differences between the six groups regarding key variables from all five predictor domains. Prediction of any treatment (model 1) or SC treatment (model 2) was successful with high overall accuracy (both models: 95%), sufficient sensitivity (model 1: 79%/model 2: 76%) and high specificity (both models: 98%). The most predictive single variables were daily drinking level, anxiety, severity of mental distress, and number of inpatient nights during the last 6 months. Conclusions Variables from four domains were highly predictive in identifying treatment for AUD, with SC treatment groups showing very high levels of social disintegration, drinking, comorbidity and functional losses. Earlier intervention and formal treatment for AUD in PC should be implemented to reduce these high levels of adverse outcomes

Linear approaches to intramolecular Förster Resonance Energy Transfer probe measurements for quantitative modeling

Numerous unimolecular, genetically-encoded Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R<sub>alt</sub>) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R<sub>alt</sub> are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purpose

The 8^8B Neutrino Spectrum

Knowledge of the energy spectrum of $^8$B neutrinos is an important ingredient for interpreting experiments that detect energetic neutrinos from the Sun. The neutrino spectrum deviates from the allowed approximation because of the broad alpha-unstable $^8$Be final state and recoil order corrections to the beta decay. We have measured the total energy of the alpha particles emitted following the beta decay of $^8$B. The measured spectrum is inconsistent with some previous measurements, in particular with a recent experiment of comparable precision. The beta decay strength function for the transition from $^8$B to the accessible excitation energies in $^8$Be is fit to the alpha energy spectrum using the R-matrix approach. Both the positron and neutrino energy spectra, corrected for recoil order effects, are constructed from the strength function. The positron spectrum is in good agreement with a previous direct measurement. The neutrino spectrum disagrees with previous experiments, particularly for neutrino energies above 12 MeV.Comment: 15 pages, 13 figures, 4 tables, submitted to Phys. Rev. C, typos correcte

Determining the best population-level alcohol consumption model and its impact on estimates of alcohol-attributable harms

BACKGROUND: The goals of our study are to determine the most appropriate model for alcohol consumption as an exposure for burden of disease, to analyze the effect of the chosen alcohol consumption distribution on the estimation of the alcohol Population- Attributable Fractions (PAFs), and to characterize the chosen alcohol consumption distribution by exploring if there is a global relationship within the distribution. METHODS: To identify the best model, the Log-Normal, Gamma, and Weibull prevalence distributions were examined using data from 41 surveys from Gender, Alcohol and Culture: An International Study (GENACIS) and from the European Comparative Alcohol Study. To assess the effect of these distributions on the estimated alcohol PAFs, we calculated the alcohol PAF for diabetes, breast cancer, and pancreatitis using the three above-named distributions and using the more traditional approach based on categories. The relationship between the mean and the standard deviation from the Gamma distribution was estimated using data from 851 datasets for 66 countries from GENACIS and from the STEPwise approach to Surveillance from the World Health Organization. RESULTS: The Log-Normal distribution provided a poor fit for the survey data, with Gamma and Weibull distributions providing better fits. Additionally, our analyses showed that there were no marked differences for the alcohol PAF estimates based on the Gamma or Weibull distributions compared to PAFs based on categorical alcohol consumption estimates. The standard deviation of the alcohol distribution was highly dependent on the mean, with a unit increase in alcohol consumption associated with a unit increase in the mean of 1.258 (95% CI: 1.223 to 1.293) (R2 = 0.9207) for women and 1.171 (95% CI: 1.144 to 1.197) (R2 = 0. 9474) for men. CONCLUSIONS: Although the Gamma distribution and the Weibull distribution provided similar results, the Gamma distribution is recommended to model alcohol consumption from population surveys due to its fit, flexibility, and the ease with which it can be modified. The results showed that a large degree of variance of the standard deviation of the alcohol consumption Gamma distribution was explained by the mean alcohol consumption, allowing for alcohol consumption to be modeled through a Gamma distribution using only average consumption

Beam heat load analysis with COLDDIAG: a cold vacuum chamber for diagnostics

The knowledge of the heat intake from the electron beam is essential to design the cryogenic layout of superconducting insertion devices. With the aim of measuring the beam heat load to a cold bore and understanding the responsible mechanisms, a cold vacuum chamber for diagnostics (COLDDIAG) has been built. The instrumentation comprises temperature sensors, pressure gauges, mass spectrometers and retarding field analyzers, which allow to study the beam heat load and the influence of the cryosorbed gas layer. COLDDIAG was installed in the storage ring of the Diamond Light Source from September 2012 to August 2013. During this time measurements were performed for a wide range of machine conditions, employing the various measuring capabilities of the device. Here we report on the analysis of the measured beam heat load, pressure and gas content, as well as the low energy charged particle flux and spectrum as a function of the electron beam parameters