Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.     

Diabetic Retinopathy and Estimated Cerebrospinal Fluid Pressure. The Beijing Eye Study 2011

Purpose

The cerebrospinal fluid pressure (CSFP) is a major determinant of central retinal vein pressure and thus of retinal capillary pressure. We tested the hypothesis whether prevalence and severity of diabetic retinopathy are associated with CSFP.

Methods

The population-based Beijing Eye Study 2011 included 3468 individuals with a mean age of 64.6±9.8 years. A detailed ophthalmic examination was performed including fundus photography for the assessment of diabetic retinopathy according. Based on a previous study with lumbar cerebrospinal fluid pressure (CSFP) measurements, CSFP was calculated as CSFP[mmHg] = 0.44xBody Mass Index[kg/m²]+0.16 Diastolic Blood Pressure[mmHg]–0.18xAge[Years]−1.91.

Results

In binary regression analysis, presence of diabetic retinopathy was significantly associated with higher levels of HbA1c (P<0.001; regression coefficient B:0.25; odds ratio (OR):1.28; 95% confidence interval (CI):1.15,1.43), higher blood concentration of glucose (P<0.001; B:0.40;OR:1.49;95%CI:1.36,1.63), longer known duration of diabetes mellitus (P<0.001; B:0.14;OR:1.15; 95%CI:1.11,1.19), higher systolic blood pressure (P<0.001; B:0.03;OR:1.03;95%CI:1.02,1.04), lower diastolic blood pressure (P<0.001; B:−0.06;OR:0.94;95%CI:0.91,0.97), and higher CSFP (P = 0.002; B:0.13;OR:1.14;95%CI:1.05,1.24). Severity of diabetic retinopathy was significantly associated with higher HbA1c value (P<0.001; standardized coefficient beta: 0.19; correlation coefficient B: 0.07;95%CI:0.05,0.08), higher blood concentration of glucose (P<0.001; beta:0.18;B:0.04;95%CI:0.04,0.05), longer known duration of diabetes mellitus (P<0.001; beta:0.20;B:0.03;95%CI:0.02,0.03), lower level of education (P = 0.001; beta:−0.05;B:−0.02;95%CI:−0.03,−0.01), lower diastolic blood pressure (P = 0.002; beta:−0.08;B:−0.001;95%CI:−0.004,−0.001), higher systolic blood pressure (P = 0.006; beta:0.06;B:0.001;95%CI:0.000,0.001), and higher CSFP (P = 0.006; beta:0.06;B:0.006;95%CI:0.002,0.010).

Conclusions

Higher prevalence and severity of diabetic retinopathy were associated with higher estimated CSFP after adjusting for systemic parameters. Higher CSFP through a higher retinal vein pressure may lead to more marked retinal venous congestion and vascular leakage in diabetic retinae.     

IN VIVO MEASUREMENT OF ACTIVE OXYGEN PRODUCTION IN THE BROWN ALGA FUCUS EVANESCENS USING 2′,7′-DICHLOROHYDROFLUORESCEIN DIACETATE1

Intracellular production of active oxygen in the brown alga Fucus evanescens C. Ag. was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA) to the fluorescent dye 2′,7′-dichlorofluorescein (DCF), both in emersed and immersed seaweeds. Algae were incubated in seawater containing DCFH-DA under a range of conditions, and it was also possible to load algae with DCFH-DA and then follow subsequent DCF production in emersed tissue. DCF formation was linear for at least 2 h in both darkness and light, with the rate of formation increasing with the light level. DCF formation was temperature dependent. It also increased when algae were treated with H₂O₂ or methyl viologen (paraquat), which disrupts photosystem 1 electron transport and increases O^?₂ production. Exogenous catalase reduced in vivo DCF production, presumably by lowering cellular concentrations of H₂O₂. Hydrogen peroxide was released into the seawater by illuminated algae resulting in external dye conversion to DCF. However, this does not interfere with in vivo measurement of DCF by loaded, washed algae because DCF leakage appeared to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untreated controls. Overall, our data suggest that DCFH-DA is a potentially very useful probe for studying active oxygen metabolism in seaweeds subjected to environmental stresses.     

Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).

The goals of this study were to determine whether the Fas-dependent apoptosis pathway is active in the lungs of patients with the acute respiratory distress syndrome (ARDS), and whether this pathway can contribute to lung epithelial injury. We found that soluble Fas ligand (sFasL) is present in bronchoalveolar lavage (BAL) fluid of patients before and after the onset of ARDS. The BAL concentration of sFasL at the onset of ARDS was significantly higher in patients who died. BAL from patients with ARDS induced apoptosis of distal lung epithelial cells, which express Fas, and this effect was inhibited by blocking the Fas/FasL system using three different strategies: anti-FasL mAb, anti-Fas mAb, and a Fas-Ig fusion protein. In contrast, BAL from patients at risk for ARDS had no effect on distal lung epithelial cell apoptosis. These data indicate that sFasL is released in the airspaces of patients with acute lung injury and suggest that activation of the Fas/FasL system contributes to the severe epithelial damage that occurs in ARDS. These data provide the first evidence that FasL can be released as a biologically active, death-inducing mediator capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury.     

Vegetation types and patterns in Senegal based on multivariate analysis of field and NOAA-AVHRR satellite data

Abstract. Woody vegetation was studied in 44 sites in Senegal. Species composition and density were analysed with TWINSPAN and Canonical Correspondence Analysis; 16 vegetation types were defined. A supervised, multispectral and multitemporal classification of day and night, dry season NOAA-AVHRR imagery was used to identify their distribution with a classification accuracy ranging from 60–100%.     

Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.