Phylogeny and expression analysis of C-reactive protein (CRP) and serum amyloid-P (SAP) like genes reveal two distinct groups in fish

This work was funded by British Society of Animal Science/Genesis Faraday to both SAM and SB Immune control of energy reallocation in fish and a BBSRC Research Experience Placements (2010).Peer reviewedPublisher PD

A new nanocrystalline diamond-based biosensor for the detection of cardiovascular risk markers

In this paper, a new method to probe associative interactions of C-reactive protein (CRP) antigen with CRP antibody immobilized on a gold-interdigitated diamond electrodes was investigated. The CRP antigen detection was performed by capacitive/dielectric-constant measurements. Our results showed that the dynamic detection range using optimized conditions for a given antibody concentration (100 μg/ml) was found to be in the range 25-800 ng/ml of CRP-antigen. Biosensor developed in this study can be potentially used for detection of elevated CRP levels in suspected subjects for early diagnosis

Comparative study of the stabilities of synthetic in vitro and natural ex vivo transthyretin amyloid fibrils

Systemic amyloidosis caused by extracellular deposition of insoluble fibrils derived from the pathological aggregation of circulating proteins, such as transthyretin, is a severe and usually fatal condition. Elucidation of the molecular pathogenic mechanism of the disease and discovery of effective therapies still represents a challenging medical issue. The in vitro preparation of amyloid fibrils that exhibit structural and biochemical properties closely similar to those of natural fibrils is central to improving our understanding of the biophysical basis of amyloid formation in vivo and may offer an important tool for drug discovery. Here, we compared the morphology and thermodynamic stability of natural transthyretin fibrils with those of fibrils generated in vitro using either the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were -12.36 kcal mol-1, -8.10 kcal mol-1 and -10.61 kcal mol-1, respectively. The fibrils generated via plasmin cleavage were more stable than those prepared at low pH and were thermodynamically and morphologically similar to natural fibrils extracted from human amyloidotic tissue. Determination of thermodynamic stability is an important tool that is complementary to other methods for structural comparison between ex vivo fibrils and fibrils generated in vitro Our finding that fibrils created via an in vitro amyloidogenic pathway are structurally similar to ex vivo human amyloid fibrils does not necessarily establish that the fibrillogenic pathway is the same for both, but it narrows the current knowledge gap between in vitro models and in vivo pathophysiology

Evaluation of an in-clinic Serum Amyloid A (SAA) assay and assessment of the effects of storage on SAA samples

<p>Abstract</p> <p>Background</p> <p>An in-clinic assay for equine serum amyloid A (SAA) analysis, Equinostic EVA1, was evaluated for use in a clinical setting. Stability of SAA in serum samples was determined.</p> <p>Methods</p> <p>Intra- and inter- assay variation of the in-clinic method was determined. The in-clinic method (EVA1) results were compared to a reference method (Eiken LZ SAA) with 62 patient samples. For samples with SAA concentrations within the assay range of EVA1 (10-270 mg/L), differences between the methods were evaluated in a difference plot. Linearity under dilution was evaluated in two samples. Stability of SAA in three serum pools stored at 4°C and approximately 22°C was evaluated with the reference method day 0, 1, 2, 4, 7, 17 and analysed with a two-way ANOVA.</p> <p>Results</p> <p>The imprecision (coefficient of variation, CV) for the in-clinic method was acceptable at higher SAA concentrations with CV values of 7,3-12%, but poor at low SAA concentrations with CV values of 27% and 37% for intra- and inter-assay variation respectively. Recovery after dilution was 50-138%. The in-clinic assay and the reference method identified equally well horses with low (<10 mg/L) and high (>270 mg/L) SAA concentrations. Within the assay range of the in-clinic method, 10-270 mg/L, the difference between the two methods was slightly higher than could be explained by the inherent imprecision of the assays. There were no significant changes of serum SAA concentrations during storage.</p> <p>Conclusions</p> <p>The in-clinic assay identified horses with SAA concentrations of <10 mg/L and >270 mg/L in a similar way as the reference method, and provided an estimate of the SAA concentration in the range of 10-270 mg/L. The imprecision of the in-clinic method was acceptable at high SAA concentrations but not at low concentrations. Dilution of samples gave inconsistent results. SAA was stable both at room temperature and refrigerated, and thus samples may be stored before analysis with the reference method.</p

Plasminogen activation triggers transthyretin amyloidogenesis in vitro

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments