A profiling platform for the identification of selective metalloprotease inhibitors

Although proteases represent an estimated 5% to 10% of potential drug targets, inhibitors for metalloproteases (MPs) account for only a small proportion of all approved drugs, failures of which have typically been associated with lack of selectivity. In this study, the authors describe a novel and universal binding assay based on an actinonin derivative and show its binding activities for several MPs and its lack of activity toward all the non-MPs tested. This newly developed assay would allow for the rapid screening for inhibitors of a given MP and for the selectivity profiling of the resulting hits. The assay has successfully enabled for the first time simultaneous profiling of 8 well-known inhibitors against a panel of selected MPs. Previously published activities for these inhibitors were confirmed, and the authors have also discovered new molecular targets for some of them. The authors conclude that their profiling platform provides a generic assay solution for the identification of novel metalloprotease inhibitors as well as their selectivity profiling using a simple and homogeneous assay.     

Comparative evaluation of antioxidant reactivity within obstructed and control rabbit urinary bladder tissue using FRAP and CUPRAC assays

Partial urinary bladder outlet obstruction mediates cyclic ischemia and reperfusion resulting in the generation of both reactive oxygen species and reactive nitrogen species. It is theorized that with an increase in the level of free radicals, the level of protective antioxidants should decrease. To test this hypothesis, two electron transfer assays, the FRAP method and the CUPRAC method, were used to determine the level of antioxidant reactivity of obstructed and control bladder tissue. The results showed that the CUPRAC assay detected a significant decrease in the reactivity of antioxidants found within the obstructed bladder tissue as compared to the control bladder tissue in both the muscle and mucosa. The FRAP assay did not detect any difference between the muscle and mucosa of the obstructed and control bladder tissue.     

Self-assembling diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] and a beta-sheet peptide

The self-assembly of hybrid diblock copolymers composed of poly(HPMA) and beta-sheet peptide P11 (CH(3)CO-QQRFQWQFEQQ-NH(2)) blocks was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified beta-sheet peptide. As expected, CD and CR binding studies showed that the peptide block imposed its beta-sheet structural arrangement on the structure of diblock copolymers. TEM and AFM proved that peptide and these copolymers had the ability to self-assemble into fibrils.     

The insulin polymorphism -23Hph increases the risk for type 1 diabetes mellitus in the Romanian population

The insulin -23Hph and IGF2 Apa polymorphisms were genotyped in Romanian patients with T1DM (n = 204), T2DM (n = 215) or obesity (n = 200) and normoponderal healthy subjects (n = 750). The genotypes of both polymorphisms were distributed in concordance with Hardy-Weinberg equilibrium in all groups. The -23Hph AA genotype increased the risk for T1DM (OR: 3.22, 95%CI: 2.09-4.98, p < 0,0001), especially in patients without macroalbuminuria (OR: 4.32, 95%CI: 2.54-7.45, p < 0,0001). No other significant association between the alleles or genotypes of insulin -23Hph and IGF2 Apa and diabetes or obesity was identified.     

Dependence Calibration in Conditional Copulas: A Nonparametric Approach

Summary The study of dependence between random variables is a mainstay in statistics. In many cases, the strength of dependence between two or more random variables varies according to the values of a measured covariate. We propose inference for this type of variation using a conditional copula model where the copula function belongs to a parametric copula family and the copula parameter varies with the covariate. In order to estimate the functional relationship between the copula parameter and the covariate, we propose a nonparametric approach based on local likelihood. Of importance is also the choice of the copula family that best represents a given set of data. The proposed framework naturally leads to a novel copula selection method based on cross‐validated prediction errors. We derive the asymptotic bias and variance of the resulting local polynomial estimator, and outline how to construct pointwise confidence intervals. The finite‐sample performance of our method is investigated using simulation studies and is illustrated using a subset of the Matched Multiple Birth data.     

Blood pressure and renal hemodynamic responses to acute angiotensin II infusion are enhanced in a female mouse model of systemic lupus erythematosus

Inflammation and immune system dysfunction contributes to the development of cardiovascular and renal disease. Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disorder that carries a high risk for both renal and cardiovascular disease. While hemodynamic changes that may contribute to increased cardiovascular risk have been reported in humans and animal models of SLE, renal hemodynamics have not been widely studied. The renin-angiotensin system (RAS) plays a central role in renal hemodynamic control, and although RAS blockade is a common therapeutic strategy, the role of RAS in hemodynamic function during SLE is not clear. This study tested whether mean arterial pressure (MAP) and renal hemodynamic responses to acute infusions of ANG II in anesthetized animals were enhanced in an established female mouse model of SLE (NZBWF1). Baseline MAP was not different between anesthetized SLE and control (NZWLacJ) mice, while renal blood flow (RBF) was significantly lower in mice with SLE. SLE mice exhibited an enhanced pressor response and greater reduction in RBF after ANG II infusion. An acute infusion of the ANG II receptor blocker losartan increased RBF in control mice but not in mice with SLE. Renin and ANG II type 1 receptor expression was significantly lower, and ANG II type 2 receptor expression was increased in the renal cortex from SLE mice compared with controls. These data suggest that there are fewer ANG II receptors in the kidneys from mice with SLE but that the existing receptors exhibit an enhanced sensitivity to ANG II.     

Multi-objective workflow scheduling in Amazon EC2

Nowadays, scientists and companies are confronted with multiple competing goals such as makespan in high-performance computing and economic cost in Clouds that have to be simultaneously optimised. Multi-objective scheduling of scientific applications in these systems is therefore receiving increasing research attention. Most existing approaches typically aggregate all objectives in a single function, defined a-priori without any knowledge about the problem being solved, which negatively impacts the quality of the solutions. In contrast, Pareto-based approaches having as outcome a set of (nearly) optimal solutions that represent a tradeoff among the different objectives, have been scarcely studied. In this paper, we analyse MOHEFT, a Pareto-based list scheduling heuristic that provides the user with a set of tradeoff optimal solutions from which the one that better suits the user requirements can be manually selected. We demonstrate the potential of our method for multi-objective workflow scheduling on the commercial Amazon EC2 Cloud. We compare the quality of the MOHEFT tradeoff solutions with two state-of-the-art approaches using different synthetic and real-world workflows: the classical HEFT algorithm for single-objective scheduling and the SPEA2* genetic algorithm used in multi-objective optimisation problems. The results demonstrate that our approach is able to compute solutions of higher quality than SPEA2*. In addition, we show that MOHEFT is more suitable than SPEA2* for workflow scheduling in the context of commercial Clouds, since the genetic-based approach is unable of dealing with some of the constraints imposed by these systems.     

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.KEY WORDS: Ribosomal protein deficiency, Rps19, Rpl11, p53, ATR, RNR, Chk1, ATP, AMPK, Exogenous nucleosides     

Coordinate Based Meta-Analysis of Functional Neuroimaging Data Using Activation Likelihood Estimation; Full Width Half Max and Group Comparisons

Coordinate based meta-analysis (CBMA) is used to find regions of consistent activation across fMRI and PET studies selected for their functional relevance to a hypothesis. Results are clusters of foci where multiple studies report in the same spatial region, indicating functional relevance. Contrast meta-analysis finds regions where there are consistent differences in activation pattern between two groups. The activation likelihood estimate methods tackle these problems, but require a specification of uncertainty in foci location: the full width half max (FWHM). Results are sensitive to FWHM. Furthermore, contrast meta-analysis requires correction for multiple statistical tests. Consequently it is sensitive only to very significant localised differences that produce very small p-values, which remain significant after correction; subtle diffuse differences between the groups can be overlooked. In this report we redefine the FWHM parameter, by analogy with a density clustering algorithm, and provide a method to estimate it. The FWHM is modified to account for the number of studies in the analysis, and represents a substantial change to the CBMA philosophy that can be applied to the current algorithms. Consequently we observe more reliable detection of clusters when there are few studies in the CBMA, and a decreasing false positive rate with larger study numbers. By contrast the standard definition (FWHM independent of the number of studies) is demonstrated to paradoxically increase the false positive rate as the number of studies increases, while reducing ability to detect true clusters for small numbers of studies. We also provide an algorithm for contrast meta-analysis, which includes a correction for multiple correlated tests that controls for the proportion of false clusters expected under the null hypothesis. Furthermore, we detail an omnibus test of difference between groups that is more sensitive than contrast meta-analysis when differences are diffuse. This test is useful where contrast meta-analysis is unrevealing.     

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.