Quantification of gold nanoparticle cell uptake under controlled biological conditions and adequate resolution

Aim: We examined cellular uptake mechanisms of fluorescently labeled polymer-coated gold nanoparticles (NPs) under different biological conditions by two quantitative, microscopic approaches. Materials & methods: Uptake mechanisms were evaluated using endocytotic inhibitors that were tested for specificity and cytotoxicity. Cellular uptake of gold NPs was analyzed either by laser scanning microscopy or transmission electron microscopy, and quantified by means of stereology using cells from the same experiment. Results: Optimal inhibitor conditions were only achieved with chlorpromazine (clathrin-mediated endocytosis) and methyl-β-cyclodextrin (caveolin-mediated endocytosis). A significant methyl-β-cyclodextrin-mediated inhibition (63-69%) and chlorpromazine-mediated increase (43-98%) of intracellular NPs was demonstrated with both imaging techniques, suggesting a predominant uptake via caveolin-medicated endocytois. Transmission electron microscopy imaging revealed more than 95% of NPs localized in intracellular vesicles and approximately 150-times more NP events/cell were detected than by laser scanning microscopy. Conclusion: We emphasize the importance of studying NP-cell interactions under controlled experimental conditions and at adequate microscopic resolution in combination with stereology. Original submitted 10 July 2012; Revised submitted 23 January 2013.

Quantification of the spatiotemporal microstructural organization of the human brain association, projection and commissural pathways across the lifespan using diffusion tensor tractography.

Using diffusion tensor tractography, we quantified the microstructural changes in the association, projection, and commissural compact white matter pathways of the human brain over the lifespan in a cohort of healthy right-handed children and adults aged 6-68 years. In both males and females, the diffusion tensor radial diffusivity of the bilateral arcuate fasciculus, inferior longitudinal fasciculus, inferior fronto-occipital fasciculus, uncinate fasciculus, corticospinal, somatosensory tracts, and the corpus callosum followed a U-curve with advancing age; fractional anisotropy in the same pathways followed an inverted U-curve. Our study provides useful baseline data for the interpretation of data collected from patients.

Global ocean storage of anthropogenic carbon

The global ocean is a significant sink for anthropogenic carbon (Cant), absorbing roughly a third of human CO2 emitted over the industrial period. Robust estimates of the magnitude and variability of the storage and distribution of Cant in the ocean are therefore important for understanding the human impact on climate. In this synthesis we review observational and model-based estimates of the storage and transport of Cant in the ocean. We pay particular attention to the uncertainties and potential biases inherent in different inference schemes. On a global scale, three data-based estimates of the distribution and inventory of Cant are now available. While the inventories are found to agree within their uncertainty, there are considerable differences in the spatial distribution. We also present a review of the progress made in the application of inverse and data assimilation techniques which combine ocean interior estimates of Cant with numerical ocean circulation models. Such methods are especially useful for estimating the air–sea flux and interior transport of Cant, quantities that are otherwise difficult to observe directly. However, the results are found to be highly dependent on modeled circulation, with the spread due to different ocean models at least as large as that from the different observational methods used to estimate Cant. Our review also highlights the importance of repeat measurements of hydrographic and biogeochemical parameters to estimate the storage of Cant on decadal timescales in the presence of the variability in circulation that is neglected by other approaches. Data-based Cant estimates provide important constraints on forward ocean models, which exhibit both broad similarities and regional errors relative to the observational fields. A compilation of inventories of Cant gives us a "best" estimate of the global ocean inventory of anthropogenic carbon in 2010 of 155 ± 31 PgC (±20% uncertainty). This estimate includes a broad range of values, suggesting that a combination of approaches is necessary in order to achieve a robust quantification of the ocean sink of anthropogenic CO2.

Quantifying uncertainty in cost effectiveness analyses

This study compared four alternative approaches (Taylor, Fieller, percentile bootstrap, and bias-corrected bootstrap methods) to estimating confidence intervals (CIs) around cost-effectiveness (CE) ratio. The study consisted of two components: (1) Monte Carlo simulation was conducted to identify characteristics of hypothetical cost-effectiveness data sets which might lead one CI estimation technique to outperform another. These results were matched to the characteristics of an (2) extant data set derived from the National AIDS Demonstration Research (NADR) project. The methods were used to calculate (CIs) for data set. These results were then compared. The main performance criterion in the simulation study was the percentage of times the estimated (CIs) contained the “true” CE. A secondary criterion was the average width of the confidence intervals. For the bootstrap methods, bias was estimated. ^ Simulation results for Taylor and Fieller methods indicated that the CIs estimated using the Taylor series method contained the true CE more often than did those obtained using the Fieller method, but the opposite was true when the correlation was positive and the CV of effectiveness was high for each value of CV of costs. Similarly, the CIs obtained by applying the Taylor series method to the NADR data set were wider than those obtained using the Fieller method for positive correlation values and for values for which the CV of effectiveness were not equal to 30% for each value of the CV of costs. ^ The general trend for the bootstrap methods was that the percentage of times the true CE ratio was contained in CIs was higher for the percentile method for higher values of the CV of effectiveness, given the correlation between average costs and effects and the CV of effectiveness. The results for the data set indicated that the bias corrected CIs were wider than the percentile method CIs. This result was in accordance with the prediction derived from the simulation experiment. ^ Generally, the bootstrap methods are more favorable for parameter specifications investigated in this study. However, the Taylor method is preferred for low CV of effect, and the percentile method is more favorable for higher CV of effect. ^

Quantification of pleural effusion from single area measurements on CT

The objective of this study was to determine if area measurements of pleural fluid on computed tomography (CT) reflect the actual pleural fluid volume (PEvol) as measured at autopsy, to establish a formula to estimate the volume of pleural effusion (PEest), and to test the accuracy and observer reliability of PEest.132 human cadavers, with pleural effusion were divided into phase 1 (n = 32) and phase 2 (n = 100). In phase 1, PEvol was compared to area measurements on axial (axA), sagittal (sagA), and coronal (corA) CT images. Linear regression analysis was used to create a formula to calculate PEest. In phase 2, intra-class correlation (ICC) was used to assess inter-reader reliability and determine the agreement between PEest and PEvol. PEvol correlated to a higher degree to axA (r s mean = 0.738; p < 0.001) than to sagA (r s mean = 0.679, p < 0.001) and corA (r s mean = 0.709; p < 0.001). PEest can be established with the following formula: axA × 0.1 = PEest. Mean difference between PEest and PEvol was less than 40 mL (ICC = 0.837-0.874; p < 0.001). Inter-reader reliability was higher between two experienced readers (ICC = 0.984-0.987; p < 0.001) than between an inexperienced reader and both experienced readers (ICC = 0.660-0.698; p < 0.001). Pleural effusions may be quantified in a rapid, reliable, and reasonably accurate fashion using single area measurements on CT.

Identification and quantification of phytochelatins in roots of rice to long-term exposure: evidence of individual role on arsenic accumulation and translocation

Rice has the predilection to take up arsenic in the form of methylated arsenic (o-As) and inorganic arsenic species (i-As). Plants defend themselves using i-As efflux systems and the production of phytochelatins (PCs) to complex i-As. Our study focused on the identification and quantification of phytochelatins by HPLC-ICP-MS/ESI-MS, relating them to the several variables linked to As exposure. GSH, 11 PCs, and As–PC complexes from the roots of six rice cultivars (Italica Carolina, Dom Sofid, 9524, Kitrana 508, YRL-1, and Lemont) exposed to low and high levels of i-As were compared with total, i-As, and o-As in roots, shoots, and grains. Only Dom Sofid, Kitrana 508, and 9524 were found to produce higher levels of PCs even when exposed to low levels of As. PCs were only correlated to i-As in the roots (r=0.884, P <0.001). However, significant negative correlations to As transfer factors (TF) roots–grains (r= –0.739, P <0.05) and shoots–grains (r= –0.541, P <0.05), suggested that these peptides help in trapping i-As but not o-As in the roots, reducing grains’ i-As. Italica Carolina reduced i-As in grains after high exposure, where some specific PCs had a special role in this reduction. In Lemont, exposure to elevated levels of i-As did not result in higher i-As levels in the grains and there were no significant increases in PCs or thiols. Finally, the high production of PCs in Kitrana 508 and Dom Sofid in response to high As treatment did not relate to a reduction of i-As in grains, suggesting that other mechanisms such as As–PC release and transport seems to be important in determining grain As in these cultivars.

A new sensitive method for the quantification of glyoxal and methylglyoxal in snow and ice by stir bar sorptive extraction and liquid desorption-HPLC-ESI-MS

In this study, the development of a new sensitive method for the analysis of alpha-dicarbonyls glyoxal (G) and methylglyoxal (MG) in environmental ice and snow is presented. Stir bar sorptive extraction with in situ derivatization and liquid desorption (SBSE-LD) was used for sample extraction, enrichment, and derivatization. Measurements were carried out using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As part of the method development, SBSE-LD parameters such as extraction time, derivatization reagent, desorption time and solvent, and the effect of NaCl addition on the SBSE efficiency as well as measurement parameters of HPLC-ESI-MS/MS were evaluated. Calibration was performed in the range of 1–60 ng/mL using spiked ultrapure water samples, thus incorporating the complete SBSE and derivatization process. 4-Fluorobenzaldehyde was applied as internal standard. Inter-batch precision was <12 % RSD. Recoveries were determined by means of spiked snow samples and were 78.9 ± 5.6 % for G and 82.7 ± 7.5 % for MG, respectively. Instrumental detection limits of 0.242 and 0.213 ng/mL for G and MG were achieved using the multiple reaction monitoring mode. Relative detection limits referred to a sample volume of 15 mL were 0.016 ng/mL for G and 0.014 ng/mL for MG. The optimized method was applied for the analysis of snow samples from Mount Hohenpeissenberg (close to the Meteorological Observatory Hohenpeissenberg, Germany) and samples from an ice core from Upper Grenzgletscher (Monte Rosa massif, Switzerland). Resulting concentrations were 0.085–16.3 ng/mL for G and 0.126–3.6 ng/mL for MG. Concentrations of G and MG in snow were 1–2 orders of magnitude higher than in ice core samples. The described method represents a simple, green, and sensitive analytical approach to measure G and MG in aqueous environmental samples.

The influence of sampling design on tree-ring-based quantification of forest growth

Tree-rings offer one of the few possibilities to empirically quantify and reconstruct forest growth dynamics over years to millennia. Contemporaneously with the growing scientific community employing tree-ring parameters, recent research has suggested that commonly applied sampling designs (i.e. how and which trees are selected for dendrochronological sampling) may introduce considerable biases in quantifications of forest responses to environmental change. To date, a systematic assessment of the consequences of sampling design on dendroecological and-climatological conclusions has not yet been performed. Here, we investigate potential biases by sampling a large population of trees and replicating diverse sampling designs. This is achieved by retroactively subsetting the population and specifically testing for biases emerging for climate reconstruction, growth response to climate variability, long-term growth trends, and quantification of forest productivity. We find that commonly applied sampling designs can impart systematic biases of varying magnitude to any type of tree-ring-based investigations, independent of the total number of samples considered. Quantifications of forest growth and productivity are particularly susceptible to biases, whereas growth responses to short-term climate variability are less affected by the choice of sampling design. The world's most frequently applied sampling design, focusing on dominant trees only, can bias absolute growth rates by up to 459% and trends in excess of 200%. Our findings challenge paradigms, where a subset of samples is typically considered to be representative for the entire population. The only two sampling strategies meeting the requirements for all types of investigations are the (i) sampling of all individuals within a fixed area; and (ii) fully randomized selection of trees. This result advertises the consistent implementation of a widely applicable sampling design to simultaneously reduce uncertainties in tree-ring-based quantifications of forest growth and increase the comparability of datasets beyond individual studies, investigators, laboratories, and geographical boundaries.

Single hadron response measurement and calorimeter jet energy scale uncertainty with the ATLAS detector at the LHC

The uncertainty on the calorimeter energy response to jets of particles is derived for the ATLAS experiment at the Large Hadron Collider (LHC). First, the calorimeter response to single isolated charged hadrons is measured and compared to the Monte Carlo simulation using proton-proton collisions at centre-of-mass energies of root s = 900 GeV and 7 TeV collected during 2009 and 2010. Then, using the decay of K-s and Lambda particles, the calorimeter response to specific types of particles (positively and negatively charged pions, protons, and anti-protons) is measured and compared to the Monte Carlo predictions. Finally, the jet energy scale uncertainty is determined by propagating the response uncertainty for single charged and neutral particles to jets. The response uncertainty is 2-5 % for central isolated hadrons and 1-3 % for the final calorimeter jet energy scale.

Simultaneous quantification of delta-9-THC, THC-acid A, CBN and CBD in seized drugs using HPLC-DAD

An HPLC-DAD method for the quantitative analysis of Δ(9)-tetrahydrocannabinol (THC), Δ(9)-tetrahydrocannabinolic acid-A (THCA-A), cannabidiol (CBD), and cannabinol (CBN) in confiscated cannabis products has been developed, fully validated and applied to analyse seized cannabis products. For determination of the THC content of plant material, this method combines quantitation of THCA-A, which is the inactive precursor of THC, and free THC. Plant material was dried, homogenized and extracted with methanol by ultrasonication. Chromatographic separation was achieved with a Waters Alliance 2695 HPLC equipped with a Merck LiChrospher 60 RP-Select B (5μm) precolumn and a Merck LiChroCart 125-4 LiChrospher 60 RP-Select B (5μm) analytical column. Analytes were detected and quantified using a Waters 2996 photo diode array detector. This method has been accepted by the public authorities of Switzerland (Bundesamt für Gesundheit, Federal Office of Public Health), and has been used to analyse 9092 samples since 2000. Since no thermal decarboxylation of THCA-A occurs, the method is highly reproducible for different cannabis materials. Two calibration ranges are used, a lower one for THC, CBN and CBD, and a higher one for THCA-A, due to its dominant presence in fresh plant material. As provider of the Swiss proficiency test, the robustness of this method has been tested over several years, and homogeneity tests even in the low calibration range (1%) show high precision (RSD≤4.3%, except CBD) and accuracy (bias≤4.1%, except CBN).

Detection and quantification of leptospires in urine of dogs: a maintenance host for the zoonotic disease leptospirosis.

Leptospirosis is a global zoonotic disease. Pathogenic Leptospira species, the causative agent of leptospirosis, colonize the renal tubules of chronically infected maintenance hosts such as dogs, rats and cattle. Maintenance hosts typically remain clinically asymptomatic and shed leptospires into the environment via urine. In contrast, accidental hosts such as humans can suffer severe acute forms of the disease. Infection results from direct contact with infected urine or indirectly, through contaminated water sources. In this study, a quantitative real-time PCR specific for lipL32 was designed to detect the urinary shedding of leptospires from dogs. The sensitivity and specificity of the assay was evaluated using both a panel of pathogenic Leptospira species and clinical microbial isolates, and samples of urine collected from experimentally infected rats and non-infected controls. The lower limit of detection was approximately 3 genome equivalents per reaction. The assay was applied to canine urine samples collected from local dog sanctuaries and the University Veterinary Hospital (UVH) at University College Dublin. Of 525 canine urine samples assayed, 37 were positive, indicating a prevalence of urinary shedding of leptospires of 7.05%. These results highlight the need to provide effective canine vaccination strategies and raise public health awareness.