Detection of vascular morphology by high frequency intravascular ultrasonic imaging

This study was designed to validate the potential clinical utility of intravascular ultrasonic imaging in vitro and in vivo. In vitro studies were performed to assess the accuracy of dimensional and morphological information. In vitro images of human vessels (n = 75) demonstrated that lesion thickness determined echographically closely related with histological samples (r = 0.83). Morphologically, muscular and elastic arteries could be distinguished echographically based on the echogenicity of the arterial media. Close relation was also found in the morphological subtypes of atherosclerosis. Subsequently, intravascular ultrasound was used percutaneously in vivo in 20 patients to obtain images of the iliac and superficial femoral artery. High quality real-time images were obtained. Normal vessels were seen showing pulsatile circular images with a hypoechoic muscular media resulting in a typical three-layered appearance. Diseased arteries revealed non-obstructive and obstructive lumen. At the site of obstruction thinning of the muscular media was evident. Pulsation was not always present. Following dilatation of the obstructive lesion using balloon angioplasty the ultrasonic cross-sections changed drastically revealing plaque rupture, dissection, plaque-free wall rupture, rest stenosis and oedema. We conclude that intravascular ultrasonic imaging is a promising technique to document accurate dimensional and morphological characteristics of human vascular disease for guidance of therapeutic interventions

Validation of quantitative analysis of intravascular ultrasound images

This study investigated the accuracy and reproducibility of a computer-aided method for quantification of intravascular ultrasound. The computer analysis system was developed on an IBM compatible PC/AT equipped with a framegrabber. The quantitative assessment of lumen area, lesion area and percent area obstruction was performed by tracing the boundaries of the free lumen and original lumen. Accuracy of the analysis system was tested in a phantom study. Echographic measurements of lumen and lesion area derived from 16 arterial specimens were compared with data obtained by histology. The differences in lesion area measurements between histology and ultrasound were minimal (mean ± SD: -0.27±1.79 mm2, p>0.05). Lumen area measurements from histology were significantly smaller than those with ultrasound due to mechanical deformation of histologic specimens (-5.38±5.09 mm2, p0.05). Finally, intra- and interobserver variability of our quantitative method was evaluated in measurements of 100 in vivo ultrasound images. The results showed that variations in lumen area measurements were low (5%) whereas variations in lesion area and percent area obstruction were relatively high (13%, 10%, respectively). Results of this study indicate that our quantitative method provides accurate and reproducible measurements of lumen and lesion area. Thus, intravascular ultrasound can be used for clinical investigation, including assessment of vascular stenosis and evaluation of therapeutic intervention

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases

Activity-Based Protein Profiling Reveals Dynamic Substrate-Specific Cellulase Secretion by Saprotrophic Basidiomycetes

BACKGROUND: Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases. RESULTS: Here we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity. CONCLUSION: Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-022-02107-z