Formation and removal of alkylthiolate self-assembled monolayers on gold in aqueous solutions

We report the development of novel reagents and approaches for generating recyclable biosensors. The use of aqueous media for the formation of protein binding alkylthiolate monolayers on Au surfaces results in accelerated alkylthiolate monolayer formation and improvement in monolayer integrity as visualized by fluorescence microscopy and CV techniques. We have also developed an electrocleaning protocol that is compatible with microfluidics devices, and this technique serves as an on-chip method for cleaning Au substrates both before and after monolayer formation. The techniques for the formation and dissociation of biotinylated SAMs from aqueous solvents reported here may be applied towards the development of Au-based sensor devices and microfluidics chips in the future. A potential use of these devices includes the specific capture and triggered release of target cells, proteins, or small molecules from liquid samples

Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels

Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val381) and RGK5 (His401) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr379 in RBK1 increased its affinity, (ii) protonation of His401 in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected by changes in ionic strength. The results suggest an explanation for the marked differences in TEA sensitivity that have been observed among naturally occurring and cloned potassium channels and indicate that the amino acid corresponding to residue 379 in RBK1 lies within the external mouth of the ion channel

Interaction of Cryptococcus neoformans Rim101 and Protein Kinase A Regulates Capsule

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101Δ mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101Δ strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101Δ mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions

Bayesian parentage analysis with systematic accountability of genotyping error, missing data, and false matching

Motivation: The goal of any parentage analysis is to identify as many parent-offspring relationships as possible, while minimizing incorrect assignments. Existing methods can achieve these ends, but require additional information in the form of demographic data, thousands of markers, and/or estimates of genotyping error rates. For many non-model systems, it is simply not practical, cost-effective, or logistically feasible to obtain this information. Here, we develop a Bayesian parentage method that only requires the sampled genotypes in order to account for genotyping error, missing data, and false matches. Results: Extensive testing with microsatellite and SNP data sets reveals that our Bayesian parentage method reliably controls for the number of false assignments, irrespective of the genotyping error rate. When the number of loci is limiting, our approach maximizes the number of correct assignments by accounting for the frequencies of shared alleles. Comparisons with exclusion and likelihood-based methods on an empirical salmon data set revealed that our Bayesian method had the highest ratio of correct to incorrect assignments. Availability: Our program SOLOMON is available as an R package from the CRAN website. SOLOMON comes with a fully functional graphical user interface, requiring no user knowledge about the R programming environment. In addition to performing Bayesian parentage analysis, SOLOMON includes Mendelian exclusion and a priori power analysis modules. Further information and user support can be found at https://sites.google.com/site/parentagemethods/.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Oxford University Press and can be found at: http://bioinformatics.oxfordjournals.org/

Two Distinctly HLA-Associated Contiguous Linear Epitopes Uniquely Expressed Within the Islet Antigen 2 Molecule Are Major Autoantibody Epitopes of the Diabetes-Specific Tyrosine Phosphatase-Like Protein Autoantigens

AbstractThe related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2β are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2β to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2β and IA-2Δ 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611–620 (epitope JM1) and 621–630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules

Isolation of human monoclonal autoantibodies derived from pancreatic lymph node and peripheral blood B cells of islet autoantibody-positive patients

Aims/hypothesis Autoantibodies against pancreatic islets and infections by enteroviruses are associated with type 1 diabetes, but the specificity of immune responses within the type 1 diabetic pancreas is poorly characterised. We investigated whether pancreatic lymph nodes could provide a source of antigen-specific B cells for analysis of immune responses within the (pre)diabetic pancreas. Methods Human IgG antibodies were cloned from single B lymphocytes sorted from pancreatic lymph node cells of three organ donors positive for islet autoantibodies, and from the peripheral blood of a patient with type 1 diabetes. Antibodies to insulinoma-associated antigen 2 (IA-2), GAD65, zinc trans- porter 8 (ZnT8) and Coxsackie B virus proteins were assayed by immunoprecipitation and by immunofluorescence on pan- creatic sections. Results Human IgG antibodies (863) were successfully cloned and produced from 4,092 single B cells from lymph nodes and peripheral blood. Reactivity to the protein tyrosine phosphatase domain of the IA-2 autoantigen was detected in two cloned antibodies: one derived from a pancreatic lymph node and one from peripheral blood. Epitopes for these two antibodies were similar to each other and to those for circulat- ing antibodies in type 1 diabetes. The remaining 861 antibod- ies were negative for reactivity to IA-2, GAD65 or ZnT8 by both assays tested. Reactivity to a Coxsackie viral protein 2 was detected in one antibody derived from a peripheral blood B cell, but not from lymph nodes. Conclusions/interpretation We show evidence for the infre- quent presence of autoantigen-specific IgG+ B lymphocytes in the pancreatic-draining lymph nodes of islet autoantibody- positive individuals