Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer.

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.

Layer-specific analysis of myocardial function for accurate prediction of reversible ischaemic dysfunction in intermediate viability defined by contrast-enhanced MRI.

BACKGROUND: Contrast-enhanced MRI (ceMRI) has impaired accuracy in the prediction of functional recovery after revascularisation in cases of intermediate myocardial viability. OBJECTIVE: To evaluate the predictive value of layer-specific myocardial deformation analysis for improvement in ischaemic dysfunction after revascularisation. METHODS: In 132 patients with ischaemic left ventricular dysfunction undergoing revascularisation, myocardial viability was assessed by pixel-tracking-derived myocardial deformation imaging and ceMRI. Peak systolic circumferential strain was determined for total wall thickness and for three myocardial layers (endocardial, mid-myocardial and epicardial) in a 16-segment model. Analysis to predict recovery of function at 8±2 months after revascularisation was performed considering all dysfunctional segments or only segments with intermediate viability by ceMRI (hyperenhancement 25-75%, N=735 segments). RESULTS: Segments with functional recovery (N=568) had higher circumferential strain in all myocardial layers and a smaller degree of hyperenhancement than segments without functional recovery (N=433). Analysis of all dysfunctional segments showed that the predictive accuracy for functional recovery was high for endocardial strain, total wall thickness strain and hyperenhancement by ceMRI (area under the curve (AUC) 0.883, 0.782 and 0.834, respectively). Considering only segments with intermediate viability by ceMRI, endocardial circumferential strain allowed prediction of functional recovery with higher accuracy (specificity 75%, sensitivity 78%, AUC=0.811, 95% CI 0.776 to 0.851) than hyperenhancement analysis (specificity 59%, sensitivity 72%, AUC=0.705, 95% CI 0.659 to 0.747, p<0.05). CONCLUSION: Analysis of layer-specific myocardial function using deformation imaging allows accurate identification of reversible myocardial dysfunction. In segments with intermediate viability analysis of layer-specific deformation may have special advantages for prediction of functional recovery.

Surface supercharged human enteropeptidase light chain shows improved solubility and refolding yield.

Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Specifically, protein surface supercharging (N6D, G21D, G22D, N141D, K209E) of the protein increased the solubility more than 100-fold. Replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.

Myocardial rotation but not circumferential strain is transducer angle dependent: a speckle tracking echocardiography study.

BACKGROUND: Doppler derived strain analysis has been shown to be angle dependent. Speckle tracking analysis using 2D echocardiographic images is thought to provide angle independent parameters of regional and global myocardial function. This study sought to evaluate whether myocardial circumferential strain and rotation derived from automatic frame-by-frame tracking of natural acoustic markers is dependent on angulation of the transducer. METHODS: In 48 healthy volunteers (mean age 36 ± 3 years, 20 male) parasternal short-axis views at apical level were obtained as follows: at the standard parasternal position (5th intercostal space) with a most possible circular short-axis image of the left ventricle (angulation 1), at an angulation of the transducer by 20° from this standard position to the apex (angulation 2) and at an angulation of the transducer by 20° to the base of the left ventricle (angulation 3). Using an automatic frame-by-frame tracking system of natural acoustic echocardiographic markers (EchoPAC, GE Ultrasound, Horton, Norway) circumferential strain and rotation were calculated for six segments within a short-axis circumference. RESULTS: Image quality was sufficient for acquisition and analysis of images at all three-transducer angulation in 90% of the analyzed segments. Rotation was measured to be 7.7 ± 1.2° at angulation 1, 2.7 ± 0.9° at angulation 2 and 4.3 ± 1.1° at angulation 3 (p < 0.05). Average circumferential strain data was found to be -27.2 ± 5.1% at angulation 1, -26.5 ± 3.8% at angulation 2 and 28.9 ± 4.4% at angulation 3 (p = 0.287). CONCLUSION: Circumferential strain analysis is not dependent on transducer angulation. In contrast, determination of myocardial rotation is dependent on transducer angulation. Therefore, accurate transducer angulation has to be taken care of if rotation measurements are performed.

Highly sensitive protein detection based on lanthanide chelates with antenna ligands providing a linear range of five orders of magnitude.

Protein detection is an important task for pharmaceutical and clinical research today. Numerous protein staining techniques exist but are limited regarding their sensitivity and often narrow linear quantification ranges. To the best of our knowledge, this is the first description of a novel class of lanthanide chelatators, which absorb in the lower energy region at 360 nm. The new compound (6,9-dicarboxymethyl-3-{4-([1,10]-phenanthrol-2-ylethinylphenyl-carbamoyl)-methyl}-3,6,9-triaza-)-undeca-1,11-dicarboxylic acid) was coupled to different proteins and showed highly sensitive protein detection limits in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1.5 fmol of bovine serum albumin) as well as Dot Blot (100 amol of lysozyme). Furthermore, the novel compound shows an exceptionally broad linear quantification range over 5 orders of magnitude allowing applications that require the highest sensitivity alongside standard sensitivity. In addition, the new compound offers multiplexing capabilities.