Use of GPI-anchored proteins to study biomolecular interactions by surface plasmon resonance

AbstractSurface plasmon resonance is a powerful tool to examine the kinetics of cell surface receptor-ligand interactions and requires only small amounts of protein. For these studies, one component is required in highly purified form to be coupled to the biosensor surface. The second component does not need to be purified. The human high affinity receptor for immunoglobulin G, FcγRI, presents a problem as the receptor itself cannot readily be produced in large amounts for purification and, as there are eight potential ligands for the receptor (human IgG1–4 and mouse IgG1, 2a, 2b and 3), it is difficult to immobilise the ligand. Using a previously established method for generating GPI-anchored proteins, we have produced and captured a soluble version of FcγRI and shown that it retains its affinity for human IgG1 and specificity for the different IgG subclasses. In addition, we also produced and captured a GPI-anchored version of the cell adhesion molecule CD2. This system circumvents the need for extensive receptor purification and is very rapid as solubilised receptors can be transferred from the cell surface to the sensor chip in 2 h. This system may be generally applicable for biosensor studies to other type I membrane proteins, and/or naturally occurring GPI-anchored proteins, especially where the interaction between a ligand and a panel of variant receptors is to be studied

Metformin and cardiorenal outcomes in diabetes : a reappraisal

The guidance issued to the pharmaceutical industry by the US Food and Drug Administration in 2008 has led to the publication of a series of randomised, controlled cardiovascular outcomes trials with newer therapeutic classes of glucose‐lowering medications. Several of these trials, which evaluated the newer therapeutic classes of SGLT2 inhibitors and GLP‐1 receptor agonists have reported a reduced incidence of major adverse cardiovascular and/or renal outcomes, usually relative to placebo and standard of care. Metformin was the first glucose‐lowering agent reported to improve cardiovascular outcomes in the UK Prospective diabetes Study (UKPDS) and thus became the foundation of standard care. However, as this clinical trial reported more than 20 years ago, differences from current standards of trial design and evaluation complicate comparison of the cardiovascular profiles of older and newer agents. Our article revisits the evidence for cardiovascular protection with metformin and reviews its effects on the kidney.Publisher PDFPeer reviewe

Bed-load effects on hydrodynamics of rough-bed open-channel flows

Peer reviewedPublisher PD

Alice in recidivism land: The Queens logic and child protection workers\u27 assessment of sexual dangerousness

This article is based on a case that highlights the dearth of cogent and pertinent risk assessment information in the reports and testimonies of many involved in assessing risks to children in real life problematic circumstances. In the case, the decision to exclude an intrafamilial sexual offender from the family home was made in large measure on the basis that the offender\u27s wife accepted the offender\u27s "denial of guilt" to accusations previously made against him. Keeping families apart should not be entertained without reliable and valid evidence pointing to that decision. However, as will be seen, the evidence often relied upon by child protection workers, albeit in good faith, is neither. The assessment processes described in the case point directly to what appears to be a wider lack of knowledge specific to assessment of recidivism, to misrepresentation of risk assessment information, and to overarching epistemological issues that appear to be widely ignored and/or misunderstood within the overall field of risk assessment and threat management. The purpose of this article is to describe how the information used to decide upon exclusion in the case cannot be considered probative and to iterate the methodological processes that must be considered in such cases if miscarriages of justice are to be avoided

Stretched exponential behavior and random walks on diluted hypercubic lattices

Diffusion on a diluted hypercube has been proposed as a model for glassy relaxation and is an example of the more general class of stochastic processes on graphs. In this article we determine numerically through large scale simulations the eigenvalue spectra for this stochastic process and calculate explicitly the time evolution for the autocorrelation function and for the return probability, all at criticality, with hypercube dimensions $N$ up to N=28. We show that at long times both relaxation functions can be described by stretched exponentials with exponent 1/3 and a characteristic relaxation time which grows exponentially with dimension $N$. The numerical eigenvalue spectra are consistent with analytic predictions for a generic sparse network model.Comment: 16 pages, 7 figure

Evaluating implicit feedback models using searcher simulations

In this article we describe an evaluation of relevance feedback (RF) algorithms using searcher simulations. Since these algorithms select additional terms for query modification based on inferences made from searcher interaction, not on relevance information searchers explicitly provide (as in traditional RF), we refer to them as implicit feedback models. We introduce six different models that base their decisions on the interactions of searchers and use different approaches to rank query modification terms. The aim of this article is to determine which of these models should be used to assist searchers in the systems we develop. To evaluate these models we used searcher simulations that afforded us more control over the experimental conditions than experiments with human subjects and allowed complex interaction to be modeled without the need for costly human experimentation. The simulation-based evaluation methodology measures how well the models learn the distribution of terms across relevant documents (i.e., learn what information is relevant) and how well they improve search effectiveness (i.e., create effective search queries). Our findings show that an implicit feedback model based on Jeffrey's rule of conditioning outperformed other models under investigation

Using mobile phones in pub talk

We present the findings from a study of how people interleave mobile phone use with conversation in pubs. Our findings, informed by ethnomethodology and conversation analysis, unpack the interactional methods through which groups of people in pubs occasioned, sustained, and disengaged from mobile device use during conversation with friends. Fundamentally, the work that is done consists of various methods of accounting for mobile device use, and displaying involvement in social interaction while the device is used. We highlight multiple examples of the nuanced ways in which interleaving is problematic in interaction, and relate our findings to the CSCW and HCI literature on collocated interaction. We conclude by considering avenues for future research, and discuss how we may support or disrupt interleaving practices through design to overcome the highlighted interactional troubles

Reducing uncertainty in the assessment of the Australian spanner crab fishery

In collaboration with the New South Wales Department of Primary Industries we compared the effectiveness of the spanner crab monitoring systems used by New South Wales and Queensland and developed a fishery-independent survey protocol acceptable to both states. The objectives of this project were to: 1. Determine the age at which spanner crabs (Ranina ranina) recruit to the fishery 2. Develop a common methodology for monitoring and assessing the Australian spanner crab stock 3. Investigate sources of variability in apparent population density

Polarons as Nucleation Droplets in Non-Degenerate Polymers

We present a study of the nucleation mechanism that allows the decay of the metastable phase (trans-cisoid) to the stable phase (cis-transoid) in quasi one-dimensional non-degenerate polymers within the continuum electron-phonon model. The electron-phonon configurations that lead to the decay, i.e. the critical droplets (or transition state), are identified as polarons of the metastable phase. We obtain an estimate for the decay rate via thermal activation within a range of parameters consistent with experimental values for the gap of the cis-configuration. It is pointed out that, upon doping, the activation barriers of the excited states are quite smaller and the decay rate is greatly enhanced. Typical activation energies for electron or hole polarons are $\approx 0.1$ eV and the typical size for a critical droplet (polaron) is about $20 \AA$. Decay via quantum nucleation is also studied and it is found that the crossover temperature between quantum nucleation and thermal activation is of order $T_c \leq 40 ^oK$. Metastable configurations of non-degenerate polymers may provide examples for mesoscopic quantum tunneling.Comment: REVTEX 3.0, 28 PAGES, 3 FIGURES AVAILABLE UPON REQUEST, PITT 94-0

Localization and Functional Characterization of the Alternative Oxidase in Naegleria

The Alternative oxidase (AOX) is a protein involved in maintaining the Krebs cycle in instances where the respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have disperse distribution and are found in plants, fungi and a few protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates, and include the pathogenic species of N. fowleri, the so-called brain eating amoeba. Using a multidisciplinary approach, we aimed to understand the evolution, localization and function of AOX and the role that plays in Naegleria’s biology. Our analyses suggest that the protein was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using a combination of cellular and biochemical techniques, we also functionally characterize N. gruberi’s AOX in its mitochondria and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism and evolution of Naegleria and other free-living relatives