Comparison of multianalyte proficiency test results by sum of ranking differences, principal component analysis, and hierarchical cluster analysis

Sum of ranking differences (SRD) was applied for comparing multianalyte results obtained by several analytical methods used in one or in different laboratories, i.e., for ranking the overall performances of the methods (or laboratories) in simultaneous determination of the same set of analytes. The data sets for testing of the SRD applicability contained the results reported during one of the proficiency tests (PTs) organized by EU Reference Laboratory for Polycyclic Aromatic Hydrocarbons (EU-RL-PAH). In this way, the SRD was also tested as a discriminant method alternative to existing average performance scores used to compare mutlianalyte PT results. SRD should be used along with the z scores-the most commonly used PT performance statistics. SRD was further developed to handle the same rankings (ties) among laboratories. Two benchmark concentration series were selected as reference: (a) the assigned PAH concentrations (determined precisely beforehand by the EU-RL-PAH) and (b) the averages of all individual PAH concentrations determined by each laboratory. Ranking relative to the assigned values and also to the average (or median) values pointed to the laboratories with the most extreme results, as well as revealed groups of laboratories with similar overall performances. SRD reveals differences between methods or laboratories even if classical test(s) cannot. The ranking was validated using comparison of ranks by random numbers (a randomization test) and using seven folds cross-validation, which highlighted the similarities among the (methods used in) laboratories. Principal component analysis and hierarchical cluster analysis justified the findings based on SRD ranking/grouping. If the PAH-concentrations are row-scaled, (i.e., z scores are analyzed as input for ranking) SRD can still be used for checking the normality of errors. Moreover, cross-validation of SRD on z scores groups the laboratories similarly. The SRD technique is general in nature, i.e., it can be applied to any experimental problem in which multianalyte results obtained either by several analytical procedures, analysts, instruments, or laboratories need to be compared. [Figure not available: see fulltext.] © 2013 Springer-Verlag Berlin Heidelberg

Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells

Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS) resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies

Soil mobility of surface applied polyaromatic hydrocarbons in response to simulated rainfall

Polyaromatic hydrocarbons (PAHs) are emitted from a variety of sources and can accumulate on and within surface soil layers. To investigate the level of potential risk posed by surface contaminated soils, vertical soil column experiments were conducted to assess the mobility, when leached with simulated rainwater, of six selected PAHs (naphthalene, phenanthrene, fluoranthene, pyrene, benzo(e)pyrene and benzo(ghi)perylene) with contrasting hydrophobic characteristics and molecular weights/sizes. The only PAH found in the leachate within the experimental period of 26 days was naphthalene. The lack of migration of the other applied PAHs were consistent with their low mobilities within the soil columns which generally parallelled their log Koc values. Thus only 2.3% of fluoranthene, 1.8% of pyrene, 0.2% of benzo(e)pyrene and 0.4% of benzo(ghi)perylene were translocated below the surface layer. The PAH distributions in the soil columns followed decreasing power relationships with 90% reductions in the starting levels being shown to occur within a maximum average depth of 0.94 cm compared to an average starting depth of 0.5 cm. A simple predictive model identifies the extensive time periods, in excess of 10 years, required to mobilise 50% of the benzo(e)pyrene and benzo(ghi)perylene from the surface soil layer. Although this reduces to between 2 and 7 years for fluoranthene and pyrene, it is concluded that the possibility of surface applied PAHs reaching and contaminating a groundwater aquifer is unlikely

Impact of Dreissena fouling on the physiological condition of native and invasive bivalves : interspecific and temporal variations

The impact of Dreissena fouling on unionids has hardly been studied in Europe, despite the fact that in some ecosystems (e.g. Lake Balaton, Hungary) infestations of several hundreds to a thousand individuals per unionid have been observed. At present, the zebra mussel Dreissena polymorpha is a dominant species in Lake Balaton and in the last decade three other invasive bivalves were introduced, potentially increasing the pressure on native unionid survival. We examined whether the fouling of dreissenids (zebra and quagga (D. rostriformis bugensis) mussels) has a negative impact on native (Anodonta anatina, Unio pictorum and U. tumidus) and invasive (Corbicula fluminea and Sinanodonta woodiana) bivalves and whether there are any interspecific and temporal variations in fouling intensity and physiological condition measured by standard condition index and glycogen content. A significant negative impact was detected on native unionids only in July and September (no impact was detected in May), when the fouling rate was high. For invasive species, a significant negative impact was detected on S. woodiana with a high level of dressenid infestation; whereas no significant impact was detected on C. fluminea. Overall, this study confirms that Dreissena may threaten unionid species including the invasive S. woodiana, although high interspecific and temporal variations were observed. This situation should be taken into account in future ecological and conservational assessments because species respond differently to Dreissena fouling and effects seem to be more pronounced in late summer/early autumn. In addition, this study provides the first evidence that the invasive C. fluminea appear to be less vulnerable to dressenid fouling.The study was supported by the Hungarian Scientific Fund (KTIA-OTKA) under the contract No. CNK80140

Overlapping pK a of the Multiprotic Hemostyptic Eltrombopag using UV–Vis Multiwavelength Spectroscopy and Potentiometry

pH-potentiometric and WApH-spectrophotometric titrations of the multiprotic hemostyptic Eltrombopag for dissociation constants determination were compared. Hemostyptic and hemostatic Eltrombopag treats low blood platelet counts in adults with chronic immune idiopathic thrombocytopenia ITP. Eltrombopag exhibits five protonatable sites in a pH range of 2 to 10, where only two pK are well separated (ΔpK > 3), while the other three are near dissociation constants of overlapping equilibria. According to MARVIN prediction, in the neutral medium Eltrombopag occurs in the slightly water soluble form LH3 that can be protonated to the soluble species LH4 and LH5 The molecule LH3 can be dissociated to still difficultly soluble species LH2, LH and L. Due to limited solubility of Eltrombopag above pH 9.5 the protonation was studied up to pH 10. Five dissociation constants can be reliably determined with REACTLAB and SQUAD84 leading to the same value. From a dependence on ionic strength the thermodynamic dissociation constants were estimated at 25°C: pKa1T = 2.69, pKa2T = 6.97, pKa3T = 7.13, pKa4T = 7.65, pKa5T = 8.30. Since pH above 10 and pH down 5 occurs in a titrated solution the very fine precipitate of Eltrombopag which is initially forming a slight opalescence, this part of the potentiometric titration curve pH over 9 and pH below 5 was not taken into regression analysis to estimate pKa2 = 6.53(01), pKa3 = 7.60(04), pKa4 = 9.62(59), pKa5 = 10.55(340) at 25°C with ESAB and HYPERQUAD.Byly porovnávány potenciometrické hodnoty pH a WApH spektrofotometrické titrace multiprotického hemostyptického Eltrombopagu pro stanovení disociačních konstant. Hemostyptický a hemostatický Eltrombopag léčí nízké počty krevních destiček u dospělých pacientů s chronickou imunitní idiopatickou trombocytopenií ITP. Eltrombopag vykazuje pět protonovatelných míst v rozmezí pH od 2 do 10, kde jsou jen dva pK dobře oddělené (ΔpK> 3), zatímco ostatní tři jsou téměř disociační konstanty překrývající se rovnováhy. Podle predikce společnosti MARVIN se v neutrálním prostředí Eltrombopag vyskytuje ve formě lehce rozpustné ve vodě LH3, která může být protonována na rozpustné druhy LH4 a LH5. Molekula LH3 může být disociována na stále obtížně rozpustné druhy LH2, LH a L. Vzhledem k omezenému rozpustnosti Eltrombopagu nad pH 9,5 byla zkoumána protonace až na pH 10. Pět disociačních konstant může být spolehlivě stanoveno pomocí REACTLAB a SQUAD84, což vede k stejné hodnotě. Ze závislosti na iontové síle byly termodynamické disociační konstanty odhadnuty na 25 ° C: pKa1T = 2,69, pKa2T = 6,97, pKa3T = 7,13, pKa4T = 7,65, pKa5T = 8,30. Vzhledem k tomu, že pH v hodnotě nad 10 a pH 5 se vyskytuje v titrovaném roztoku, velmi jemná sraženina Eltrombopagu, která zpočátku vytváří mírnou opalescenci, tato část potenciometrické titrační křivky pH nad 9 a pH pod 5 nebyla převzata do regresní analýzy k odhadu pKa2 = 6,53 (01), pKa3 = 7,60 (04), pKa4 = 9,62 (59), pKa5 = 10,55 (340) při 25 ° C pomocí ESAB a HYPERQUAD