Development and Validation of a Western Blot Method to Quantify Mini-Dystrophin in Human Skeletal Muscle Biopsies.

Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked recessive pathogenic variants in the dystrophin gene, but may also arise from de novo mutations. Disease-causing variants include nonsense, out of frame deletions or duplications that result in loss of dystrophin protein expression. There is currently no cure for DMD and the few treatment options available aim at slowing muscle degradation. New advances in gene therapy and understanding of dystrophin (DYS) expression in other muscular dystrophies have opened new opportunities for treatment. Therefore, reliable methods are needed to monitor dystrophin expression and assess the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy (BMD). Here, we describe the validation of a novel Western blot (WB) method for the quantitation of mini-dystrophin protein in human skeletal muscle tissues that is easy to adopt in most laboratory settings. This WB method was assessed through precision, accuracy, selectivity, dilution linearity, stability, and repeatability. Based on mini-DYS standard performance, the assay has a dynamic range of 0.5-15 ng protein (per 5 µg total protein per lane), precision of 3.3 to 25.5%, and accuracy of - 7.5 to 3.3%. Our stability assessment showed that the protein is stable after 4 F/T cycles, up to 2 h at RT and after 7 months at - 70°C. Furthermore, our WB method was compared to the results from our recently published LC-MS method. Workflow for our quantitative WB method to determine mini-dystrophin levels in muscle tissues (created in Biorender.com). Step 1 involves protein extraction from skeletal muscle tissue lysates from control, DMD, or BMD biospecimen. Step 2 measures total protein concentrations. Step 3 involves running gel electrophoresis with wild-type dystrophin (wt-DYS) from muscle tissue extracts alongside mini-dystrophin STD curve and mini-DYS and protein normalization with housekeeping GAPDH.

Prandial ghrelin attenuation provides evidence that des-acyl ghrelin may be an artifact of sample handling in human plasma.

BACKGROUND: Plasma acyl and des-acyl ghrelin are thought of as components of total ghrelin, but this has never been validated using ex vivo spiking experiments, human sample collection comparisons and fit-for-purpose translatable assays. RESULTS: Acyl ghrelin plasma stability was analyzed by LC-MS/MS and it revealed that acyl ghrelin is enzymatically and chemically converted to des-acyl ghrelin in the presence of active serine proteases and HCl. ELISAs with less than 30% total error were used to assess acyl ghrelin behavior in matched authentic human samples. Acyl and total ghrelin were not statistically different in 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride samples and acyl ghrelin losses in K(2)EDTA plasma were accounted for in des-acyl ghrelin formation. CONCLUSION: Acyl ghrelin is total ghrelin and des-acyl ghrelin should not be detectible in healthy human plasma under optimal sample handling and assaying conditions.

Comparison of four distinct detection platforms using multiple ligand binding assay formats.

Several detection platforms are available for ligand binding assays (LBA), each claiming superiority in sensitivity and dynamic range. However, little information exists in the literature directly comparing the various LBA platforms for quantitation. We have tested four common platforms to evaluate and compare the interchangeability of detection platforms by comparing sensitivity and dynamic range to a colorimetric LBA. The detection platforms compared are: colorimetric, chemiluminescence, time-resolved fluorescence (TRF) and electrochemiluminescence (ECL). Five different LBA protocols were tested with each of the detection endpoints. The assay protocols include the following ligand binding assay formats: direct binding, sandwich ELISA, competitive and cell based ELISA. We found that no detection platform consistently performed better than all the others and it was not possible to predict which platform would perform best for a given assay protocol. We also found surprising differences in assays (plate coating efficiency, low signal) which add to difficulty in choosing the best platform ad hoc. We propose here that in developing new assay protocols for detection of biotherapeutic agents, multiple detection platforms should be tested in order to forward the best assays possible and for the right reasons.

A practical guide for the stabilization of acylghrelin in human blood collections.

OBJECTIVE AND METHODS: To better understand acylghrelin plasma stability, human synthetic acylghrelin was spiked into plasma and tracked by liquid chromatography tandem mass spectrometry. To investigate the best method for quantifying clinical plasma acylghrelin levels, pre- and postprandial human blood was collected from healthy volunteers (n=6) using various sample collections and treatments. Plasma ghrelin levels from human blood collections were analysed by enzyme-linked immunosorbant assay (ELISA). RESULTS: Acylghrelin's half-life in plasma was approximately 45 min with the formation of des-acylghrelin approaching 50% before the end of the 60-min incubation. Loss of acylghrelin inversely correlated with an increase in des-acylghrelin (P<0.008; r(2) =0.870). Plasma pretreated with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) or protease inhibitor cocktail without acidification resulted in no detectible acylghrelin losses. Acylghrelin measurements with AEBSF-treated blood were minimally 40% higher than sodium citrate/citric acid, K(2) EDTA, aprotinin/HCl and P800 collections. HCl addition to AEBSF-treated plasma did not provide enhanced acylghrelin stability and induced deacylation at and above the 100 mM final concentration. Pre- and postprandial ghrelin attenuation was investigated using aprotinin/HCl, AEBSF, protease inhibitor cocktail and no treatment for blood and plasma preparations. Fasting samples treated with AEBSF and protease inhibitor cocktail were approximately threefold higher than aprotinin/HCl and control treatments (P<0.03). Pre- and postprandial ghrelin attenuation was approximately twofold different (P<0.04) with significant counterintuitive trends in aprotinin/HCl and K(2) EDTA groups. CONCLUSIONS: Our data suggest that AEBSF addition to K(2) EDTA blood immediately after collection without plasma acidification, processing on ice and 14-day 70 °C storage is the best treatment for accurately quantifying acylghrelin in human plasma.

Design and SAR of thienopyrimidine and thienopyridine inhibitors of VEGFR-2 kinase activity.

Novel classes of thienopyrimidines and thienopyridines have been identified as potent inhibitors of VEGFR-2 kinase. The synthesis and SAR of these compounds is presented, along with successful efforts to diminish EGFR activity present in the lead series.