Probing the internal micromechanical properties of Pseudomonas aeruginosa biofilms by Brillouin imaging

© 2017 The Author(s). Biofilms are organised aggregates of bacteria that adhere to each other or surfaces. The matrix of extracellular polymeric substances that holds the cells together provides the mechanical stability of the biofilm. In this study, we have applied Brillouin microscopy, a technique that is capable of measuring mechanical properties of specimens on a micrometre scale based on the shift in frequency of light incident upon a sample due to thermal fluctuations, to investigate the micromechanical properties of an active, live Pseudomonas aeruginosa biofilm. Using this non-contact and label-free technique, we have extracted information about the internal stiffness of biofilms under continuous flow. No correlation with colony size was found when comparing the averages of Brillouin shifts of two-dimensional cross-sections of randomly selected colonies. However, when focusing on single colonies, we observed two distinct spatial patterns: In smaller colonies, stiffness increased towards their interior, indicating a more compact structure of the centre of the colony, whereas, larger (over 45 μm) colonies were found to have less stiff interiors

Is late-life dependency increasing or not? A comparison of the Cognitive Function and Ageing Studies (CFAS)

Background: Little is known about how dependency levels have changed between generational cohorts of older people. We estimated years lived in different care states at age 65 in 1991 and 2011 and new projections of future demand for care. Methods: Two population-based studies of older people in defined geographical areas conducted two decades apart (the Cognitive Function and Ageing Studies) provided prevalence estimates of dependency in four states: high (24-hour care); medium (daily care); low (less than daily); independent. Years in each dependency state were calculated by Sullivan’s method. To project future demand, the proportions in each dependency state (by age group and sex) were applied to the 2014 England population projections. Findings: Between 1991 and 2011 there were significant increases in years lived from age 65 with low (men:1·7 years, 95%CI 1·0-2·4; women:2·4 years, 95%CI 1·8-3·1) and high dependency (men:0·9 years, 95%CI 0·2-1·7; women:1·3 years, 95%CI 0·5-2·1). The majority of men’s extra years of life were independent (36%) or with low dependency (36%) whilst for women the majority were spent with low dependency (58%), only 5% being independent. There were substantial reductions in the proportions with medium and high dependency who lived in care homes, although, if these dependency and care home proportions remain constant in the future, further population ageing will require an extra 71,000 care home places by 2025. Interpretation: On average older men now spend 2.4 years and women 3.0 years with substantial care needs (medium or high dependency), and most will live in the community. These findings have considerable implications for older people’s families who provide the majority of unpaid care, but the findings also supply valuable new information for governments and care providers planning the resources and funding required for the care of their future ageing populations

High-resolution large-scale onshore wind energy assessments: A review of potential definitions, methodologies and future research needs

The rapid uptake of renewable energy technologies in recent decades has increased the demand of energy researchers, policymakers and energy planners for reliable data on the spatial distribution of their costs and potentials. For onshore wind energy this has resulted in an active research field devoted to analysing these resources for regions, countries or globally. A particular thread of this research attempts to go beyond purely technical or spatial restrictions and determine the realistic, feasible or actual potential for wind energy. Motivated by these developments, this paper reviews methods and assumptions for analysing geographical, technical, economic and, finally, feasible onshore wind potentials. We address each of these potentials in turn, including aspects related to land eligibility criteria, energy meteorology, and technical developments of wind turbine characteristics such as power density, specific rotor power and spacing aspects. Economic aspects of potential assessments are central to future deployment and are discussed on a turbine and system level covering levelized costs depending on locations, and the system integration costs which are often overlooked in such analyses. Non-technical approaches include scenicness assessments of the landscape, constraints due to regulation or public opposition, expert and stakeholder workshops, willingness to pay/accept elicitations and socioeconomic cost-benefit studies. For each of these different potential estimations, the state of the art is critically discussed, with an attempt to derive best practice recommendations and highlight avenues for future research

Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa.

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection

Bayesian Model Selection Applied to the Analysis of Fluorescence Correlation Spectroscopy Data of Fluorescent Proteins in Vitro and in Vivo

Fluorescence correlation spectroscopy (FCS) is a powerful technique to investigate molecular dynamics with single molecule sensitivity. In particular, in the life sciences it has found widespread application using fluorescent proteins as molecularly specific labels. However, FCS data analysis and interpretation using fluorescent proteins remains challenging due to typically low signal-to-noise ratio of FCS data and correlated noise in autocorrelated data sets. As a result, naive fitting procedures that ignore these important issues typically provide similarly good fits for multiple competing models without clear distinction of which model is preferred given the signal-to-noise ratio present in the data. Recently, we introduced a Bayesian model selection procedure to overcome this issue with FCS data analysis. The method accounts for the highly correlated noise that is present in FCS data sets and additionally penalizes model complexity to prevent over interpretation of FCS data. Here, we apply this procedure to evaluate FCS data from fluorescent proteins assayed in vitro and in vivo. Consistent with previous work, we demonstrate that model selection is strongly dependent on the signal-to-noise ratio of the measurement, namely, excitation intensity and measurement time, and is sensitive to saturation artifacts. Under fixed, low intensity excitation conditions, physical transport models can unambiguously be identified. However, at excitation intensities that are considered moderate in many studies, unwanted artifacts are introduced that result in nonphysical models to be preferred. We also determined the appropriate fitting models of a GFP tagged secreted signaling protein, Wnt3, in live zebrafish embryos, which is necessary for the investigation of Wnt3 expression and secretion in development. Bayes model selection therefore provides a robust procedure to determine appropriate transport and photophysical models for fluorescent proteins when appropriate models are provided, to help detect and eliminate experimental artifacts in solution, cells, and in living organisms.National Science Foundation (U.S.). Physics of Living Systems ProgramNational Institute of Mental Health (U.S.) (Award U01MH106011

Perspectives of Patients with Insulin-Treated Type 1 and Type 2 Diabetes on Hypoglycemia: Results of the HAT Observational Study in Central and Eastern European Countries

INTRODUCTION: The aim of this study was to determine the level of awareness of hypoglycemia, the level of fear for hypoglycemia, and the response to hypoglycemic events among insulin-treated diabetes patients from Central and Eastern Europe (CEE). The impact of hypoglycemia on the use of healthcare resources and patient productivity was also assessed. METHODS: This was a multicenter, non-interventional, two-part, patient self-reported questionnaire study that comprised both a retrospective cross-sectional evaluation and a prospective observational evaluation. Study participants were insulin-treated adult patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) from CEE. RESULTS: Most patients (85.4% T1DM and 83.6% T2DM) reported normal hypoglycemia awareness. The median hypoglycemia fear score was 5 out of 10 for T1DM and 4 out of 10 for T2DM patients. Patients increased glucose monitoring, consulted a doctor/nurse, and/or reduced the insulin dose in response to hypoglycemia. As a consequence of hypoglycemia, patients took leave from work/studies or arrived late and/or left early. Hospitalization was required for 31 (1.2%) patients with T1DM and 66 (2.1%) patients with T2DM. CONCLUSION: Hypoglycemia impacts patients' personal and social functioning, reduces productivity, and results in additional costs, both direct (related to increased use of healthcare resources) and indirect (related to absenteeism. FUNDING: Novo Nordisk

Is late-life dependency increasing or not? A comparison of the Cognitive Function and Ageing Studies (CFAS)

Background: Little is known about how the proportions of dependency states have changed between generational cohorts of older people. We aimed to estimate years lived in different dependency states at age 65 years in 1991 and 2011, and new projections of future demand for care. Methods: In this population-based study, we compared two Cognitive Function and Ageing Studies (CFAS I and CFAS II) of older people (aged ≥65 years) who were permanently registered with a general practice in three defined geographical areas (Cambridgeshire, Newcastle, and Nottingham; UK). These studies were done two decades apart (1991 and 2011). General practices provided lists of individuals to be contacted and were asked to exclude those who had died or might die over the next month. Baseline interviews were done in the community and care homes. Participants were stratified by age, and interviews occurred only after written informed consent was obtained. Information collected included basic sociodemographics, cognitive status, urinary incontinence, and self-reported ability to do activities of daily living. CFAS I was assigned as the 1991 cohort and CFAS II as the 2011 cohort, and both studies provided prevalence estimates of dependency in four states: high dependency (24-h care), medium dependency (daily care), low dependency (less than daily), and independent. Years in each dependency state were calculated by Sullivan's method. To project future demands for social care, the proportions in each dependency state (by age group and sex) were applied to the 2014 England population projections. Findings: Between 1991 and 2011, there were significant increases in years lived from age 65 years with low dependency (1·7 years [95% CI 1·0-2·4] for men and 2·4 years [1·8-3·1] for women) and increases with high dependency (0·9 years [0·2-1·7] for men and 1·3 years [0·5-2·1] for women). The majority of men's extra years of life were spent independent (36·3%) or with low dependency (36·3%) whereas for women the majority were spent with low dependency (58·0%), and only 4·8% were independent. There were substantial reductions in the proportions with medium and high dependency who lived in care homes, although, if these dependency and care home proportions remain constant in the future, further population ageing will require an extra 71 215 care home places by 2025. Interpretation: On average older men now spend 2·4 years and women 3·0 years with substantial care needs, and most will live in the community. These findings have considerable implications for families of older people who provide the majority of unpaid care, but the findings also provide valuable new information for governments and care providers planning the resources and funding required for the care of their future ageing populations. Funding: Medical Research Council (G9901400) and (G06010220), with support from the National Institute for Health Research Comprehensive Local research networks in West Anglia and Trent, UK, and Neurodegenerative Disease Research Network in Newcastle, UK

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

10.1371/journal.pone.0051222PLoS ONE712

Resolving fluorescent species by their brightness and diffusion using correlated photon-counting histograms

Fluorescence fluctuation spectroscopy (FFS) refers to techniques that analyze fluctuations in the fluorescence emitted by fluorophores diffusing in a small volume and can be used to distinguish between populations of molecules that exhibit differences in brightness or diffusion. For example, fluorescence correlation spectroscopy (FCS) resolves species through their diffusion by analyzing correlations in the fluorescence over time; photon counting histograms (PCH) and related methods based on moment analysis resolve species through their brightness by analyzing fluctuations in the photon counts. Here we introduce correlated photon counting histograms (cPCH), which uses both types of information to simultaneously resolve fluorescent species by their brightness and diffusion. We define the cPCH distribution by the probability to detect both a particular number of photons at the current time and another number at a later time. FCS and moment analysis are special cases of the moments of the cPCH distribution, and PCH is obtained by summing over the photon counts in either channel. cPCH is inherently a dual channel technique, and the expressions we develop apply to the dual colour case. Using simulations, we demonstrate that two species differing in both their diffusion and brightness can be better resolved with cPCH than with either FCS or PCH. Further, we show that cPCH can be extended both to longer dwell times to improve the signal-to-noise and to the analysis of images. By better exploiting the information available in fluorescence fluctuation spectroscopy, cPCH will be an enabling methodology for quantitative biology