Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format

In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)₃₀ segment at the 5′ end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.     

Comparative investigation of the cleavage step in the synthesis of model peptide resins: implications for Nalpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.     

Stability and state of aggregation of aqueous fibrinogen and dipalmitoylphosphatidylcholine lipid vesicles

The stability and state of aggregation of aqueous fibrinogen (FB) and dipalmitoylphosphatidylcholine (DPPC) vesicles in water or buffer at 25 degrees C were studied with dynamic light scattering (DLS), UV-vis spectroturbidimetry (ST), and cryo-transmission electron microscopy (cryo-TEM). In water, when 1000 ppm (0.10 wt %) DPPC dispersions were prepared with a protocol including extensive sonication, they contained mostly vesicles and were quite clear, transparent, and stable for at least 30 days. FB mixtures with water (0.075 wt %) were quite unstable and biphasic. They formed large aggregates which eventually precipitated. The addition of DPPC vesicles into these unstable FB dispersions reversed FB aggregation and precipitation and produced stable translucent microdispersions. The inferred lipid/protein aggregates were limited in size, with average diameters ranging from 200 to 300 nm. In buffer, DPPC dispersions were also clear and quite stable, with average dispersed particles diameter of ca. 90 nm. FB dissolved in aqueous buffer and formed transparent and stable solutions. Adding salt to an aggregated FB dispersion in water reversed the aggregation. FB aggregated and redissolved in the presence of the citrate and after the citrate was removed. There was no effect of citrate (present in FB initially) in the FB aggregation or redissolution. FB molecules in buffer form dimers or higher aggregates. Their average aggregation number is 2, determined with Rayleigh scattering analysis of turbidity data. The average hydrodynamic diameter of FB solutions from DLS was 30 nm. Mixing a stable FB solution in buffer and a stable DPPC dispersion in buffer produced highly unstable mixtures, in which large aggregates precipitated. These results have implications in understanding the interactions of lipids and proteins in many biological applications and food processing applications.     

Exploring the Effect of Ammonium Iodide Salts Employed in Multication Perovskite Solar Cells with a Carbon Electrode

Perovskite solar cells that use carbon (C) as a replacement of the typical metal electrodes, which are most commonly employed, have received growing interest over the past years, owing to their low cost, ease of fabrication and high stability under ambient conditions. Even though Power Conversion Efficiencies (PCEs) have increased over the years, there is still room for improvement, in order to compete with metal-based devices, which exceed 25% efficiency. With the scope of increasing the PCE of Carbon based Perovskite Solar Cells (C-PSCs), in this work we have employed a series of ammonium iodides (ammonium iodide, ethylammonium iodide, tetrabutyl ammonium iodide, phenethylammonium iodide and 5-ammonium valeric acid iodide) as additives in the multiple cation-mixed halide perovskite precursor solution. This has led to a significant increase in the PCE of the corresponding devices, by having a positive impact on the photocurrent values obtained, which exhibited an increase exceeding 20%, from 19.8 mA/cm², for the reference perovskite, to 24 mA/cm², for the additive-based perovskite. At the same time, the ammonium iodide salts were used in a post-treatment method. By passivating the defects, which provide charge recombination centers, an improved performance of the C-PSCs has been achieved, with enhanced FF values reaching 59%, which is a promising result for C-PSCs, and V_oc values up to 850 mV. By combining the results of these parallel investigations, C-PSCs of the triple mesoscopic structure with a PCE exceeding 10% have been achieved, while the in-depth investigation of the effects of ammonium iodides in this PSC structure provide a fruitful insight towards the optimum exploitation of interface and bulk engineering, for high efficiency and stable C-PSCs, with a structure that is favorable for large area applications.     

MRI findings in nonalcoholic steatohepatitis: correlation with histopathology and clinical staging

Purpose

To evaluate magnetic resonance imaging (MRI) findings of nonalcoholic steatohepatitis (NASH) and to determine the correlation of MRI findings with histopathology and Mayo End-Stage Liver Disease (MELD) score.

Materials and Methods

Thirty patients (18 males, 12 females; mean age: 57±8.9 years; age range: 35–71 years) with histopathologically proven NASH who underwent MRI examinations between January 2001 and October 2005 were included in the study. Two radiologists retrospectively reviewed all magnetic resonance (MR) examinations in consensus to evaluate the presence and extent of predetermined findings of NASH including liver steatosis, early patchy liver enhancement indicating inflammation and liver fibrosis. The findings detected on MRI were correlated and compared to histopathological findings and MELD score by using nonparametric Spearman correlation coefficient and Kruskal–Wallis analysis of variance.

Results

Liver steatosis was observed in 10 of 30 patients; early patchy liver enhancement, in 8 of 30 patients and liver fibrosis in 19 of 30 patients on MR images. Liver fibrosis was reticular in all these patients. There were statistically significant moderate correlations between MRI findings of liver steatosis and histopathologic grades of steatosis (r=0.43; P<.05), and between MRI findings of fibrosis and histopathologic stages of fibrosis (r=0.61; P<.001). Early patchy enhancement did not demonstrate statistically significant correlation with inflammation (P=.28). There was no statistically significant overall correlation between MRI findings of NASH and MELD score.

Conclusion

MRI findings of liver steatosis and fibrosis in NASH showed moderate correlations with histopathologic grades of steatosis and stages of fibrosis, but MRI findings of NASH did not demonstrate any significant correlations with MELD score.     

Discreteness of space from the generalized uncertainty principle

Various approaches to Quantum Gravity (such as String Theory and Doubly Special Relativity), as well as black hole physics predict a minimum measurable length, or a maximum observable momentum, and related modifications of the Heisenberg Uncertainty Principle to a so-called Generalized Uncertainty Principle (GUP). We propose a GUP consistent with String Theory, Doubly Special Relativity and black hole physics, and show that this modifies all quantum mechanical Hamiltonians. When applied to an elementary particle, it implies that the space which confines it must be quantized. This suggests that space itself is discrete, and that all measurable lengths are quantized in units of a fundamental length (which can be the Planck length). On the one hand, this signals the breakdown of the spacetime continuum picture near that scale, and on the other hand, it can predict an upper bound on the quantum gravity parameter in the GUP, from current observations. Furthermore, such fundamental discreteness of space may have observable consequences at length scales much larger than the Planck scale.     

Modeling of electrical confined-capillary-discharge where the discharge zone is extended by an additional pipe

Plasma injectors are a source of pulsed, high momentum and temperature fluid. This fluid can serve as a very efficient reactive mixing and accelerating agent in several applications including chemical waste decomposition and hard materials coating. It can also serve as an efficient medium for synthesis of nano-particles and their deposition on various substrates. In those applications tuning the momentum and the thermodynamic properties of the plasma jet is of paramount importance as the quality of the interaction strongly depends on them. This Letter proposes a method and a model that will allow additional tuning to the thermodynamic properties of the plasma jet by adding an extension to the discharge zone. A steady state model of processes taking place in a realistic confined capillary discharge system is presented. A comparison between this system and the parameters characterizing a discharge in a “conventional” ablative system is presented. The results obtained indicate that the non-discharge zone may provide an additional degree of freedom to optimize the system's performance. It enhances the control of the plasma parameters that allows optimal and predictable momentum control over the plasma jet. The theoretical predictions for the plasma parameters agree well with experimentally obtained data.