Allergen Micro-Bead Array for IgE Detection: A Feasibility Study Using Allergenic Molecules Tested on a Flexible Multiplex Flow Cytometric Immunoassay

Background: Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing. Objective and Methods: To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Moleculebased micro-bead Array system (ABA) was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems. Results: Successful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE t

Clusters of Basic Amino Acids Contribute to RNA Binding and Nucleolar Localization of Ribosomal Protein L22

The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80–93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80–93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA

Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs): Influence of Protein Expression on Drug–Drug Interactions

ABSTRACT: The hepatic organic anion transporting poly-peptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug−drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1. The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors

Augmented lagrangian and mass-orthogonal projection methods for constrained multibody dynamics

This paper presents a new method for the integration of the equations of motion of constrained multibody systems in descriptor form. The method is based on the penalty-Augmented Lagrangian formulation and uses massorthogonal projections for the solution to satisfy the kinematic constraint conditions. The number of equations being solved is equal to the number of states, and does not depend on the number of constraint conditions. Therefore, the method is particularly suitable for systems with redundant constraints, singular configurations or topology changes. The major advantage of the new method relies on the fact that for a low computational cost, the constraints in positions, velocities and accelerations are satisfied to machine precision during the numerical integration. This process is efficiently done by means of a mass-orthogonal projection without the need for coordinate partitioning or reduction to a minimum set of coordinates. The projection scheme allows for a more accurate and robust integration of the equations of motion since constraint violations constitute one of the primary sources of numerical errors and instabilities during the integration process. The proposed projection is also applied to the classical Lagrangian approach, thus eliminating the need for further stabilization as well as the selection of parameters in Baumgarte's method.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43334/1/11071_2005_Article_BF01833296.pd

The Dyslexia Candidate Locus on 2p12 Is Associated with General Cognitive Ability and White Matter Structure

Peer reviewe

Elucidation of reaction mechanisms using kinetic isotope effects of short-lived radionuclides

The use of the short-lived radionuclides 11C and 18F for the elucidation of organic reaction mechanisms is described. Examples of the different mechanistic problems that are discussed include concerted vs. stepwise base catalysed elimination (E2 or E1cB) and finding the rate limiting step for nucleophilic aromatic substitution (SNAr). The use of radionuclides to learn details about transition state structure for concerted nucleophilic aliphatic substitution (SN2) is also described