Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined. RESULTS: Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur. CONCLUSIONS: We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients

Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase

BACKGROUND: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. METHODS: Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen. RESULTS: No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity. CONCLUSIONS: Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space

Validation and analysis of forward osmosis CFD model in complex 3D geometries

In forward osmosis (FO), an osmotic pressure gradient generated across a semi-permeable membrane is used to generate water transport from a dilute feed solution into a concentrated draw solution. This principle has shown great promise in the areas of water purification, wastewater treatment, seawater desalination and power generation. To ease optimization and increase understanding of membrane systems, it is desirable to have a comprehensive model that allows for easy investigation of all the major parameters in the separation process. Here we present experimental validation of a computational fluid dynamics (CFD) model developed to simulate FO experiments with asymmetric membranes. Simulations are compared with experimental results obtained from using two distinctly different complex three-dimensional membrane chambers. It is found that the CFD model accurately describes the solute separation process and water permeation through membranes under various flow conditions. It is furthermore demonstrated how the CFD model can be used to optimize membrane geometry in such as way as to promote the mass transfer

Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer

The burden of colorectal cancer (CRC) is considerable—approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level (p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3

Conformational dynamics of the human serotonin transporter during substrate and drug binding

The serotonin transporter (SERT) is responsible for re-uptake of serotonin into the presynaptic neuron and plays a key role in synaptic transmission. Here, the authors use hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs

Superconducting p-branes and Extremal Black Holes

In Einstein-Maxwell theory, magnetic flux lines are `expelled' from a black hole as extremality is approached, in the sense that the component of the field strength normal to the horizon goes to zero. Thus, extremal black holes are found to exhibit the sort of `Meissner effect' which is characteristic of superconducting media. We review some of the evidence for this effect, and do present new evidence for it using recently found black hole solutions in string theory and Kaluza-Klein theory. We also present some new solutions, which arise naturally in string theory, which are non-superconducting extremal black holes. We present a nice geometrical interpretation of these effects derived by looking carefully at the higher dimensional configurations from which the lower dimensional black hole solutions are obtained. We show that other extremal solitonic objects in string theory (such as p-branes) can also display superconducting properties. In particular, we argue that the relativistic London equation will hold on the worldvolume of `light' superconducting p-branes (which are embedded in flat space), and that minimally coupled zero modes will propagate in the adS factor of the near-horizon geometries of `heavy', or gravitating, superconducting p-branes.Comment: 22 pages, 2 figure