Vascular endothelial growth factor (VEGF) induces rapid prourokinase (pro-uPA) activation on the surface of endothelial cells

Vascular endothelial growth factor (VEGF) is the pivotal angiogenic growth factor activating endothelial cells to migrate, proliferate, and form capillary tubes. For an ordered endothelial cell migration, tissue invasion, and degradation of the extracellular matrix, proteolytic machinery is indispensable. Such machinery, suitable for localized proteolysis, is provided by the prourokinase-urokinase-plasmin system. Prourokinase (pro-uPA), the initial component of this system, is, however, synthesized in its inactive precursor form and as such bound to its cellular receptor uPAR. Here we identify a mechanism via which VEGF(165) interacting with its receptor VEGFR-2 rapidly induces prourokinase activation that is dependent on a change in integrin affinity, activation of matrix metalloproteinase 2 (MMP-2), and pro-uPA being bound to its surface receptor uPAR. This VEGF-induced pro-uPA activation on endothelial cells is responsible for VEGF-dependent local fibrinolytic activity and might be one of the initial steps in the angiogenic process.     

Baseball implant. A method of secondary insertion of an intraorbital implant

A surgical technique for secondary emplacement of an orbital implant is described in which a spherical implant encased in a scleral homograft is placed in the orbit through a transconjuctival incision and sutured to the superior part of the periosteum.     

Clinical and Genetic Re-Evaluation of Inherited Retinal Degeneration Pedigrees following Initial Negative Findings on Panel-Based Next Generation Sequencing

Although rare, inherited retinal degenerations (IRDs) are the most common reason for blind registration in the working age population. They are highly genetically heterogeneous (>300 known genetic loci), and confirmation of a molecular diagnosis is a prerequisite for many therapeutic clinical trials and approved treatments. First-tier genetic testing of IRDs with panel-based next-generation sequencing (pNGS) has a diagnostic yield of ≈70–80%, leaving the remaining more challenging cases to be resolved by second-tier testing methods. This study describes the phenotypic reassessment of patients with a negative result from first-tier pNGS and the rationale, outcomes, and cost of second-tier genetic testing approaches. Removing non-IRD cases from consideration and utilizing case-appropriate second-tier genetic testing techniques, we genetically resolved 56% of previously unresolved pedigrees, bringing the overall resolve rate to 92% (388/423). At present, pNGS remains the most cost-effective first-tier approach for the molecular assessment of diverse IRD populations Second-tier genetic testing should be guided by clinical (i.e., reassessment, multimodal imaging, electrophysiology), and genetic (i.e., single alleles in autosomal recessive disease) indications to achieve a genetic diagnosis in the most cost-effective manner.     

Magnetic resonance imaging of coronary arteries and coronary stenosis

Coronary artery imaging is an important investigation for the management of coronary artery disease. Alternative noninvasive imaging would be useful, but the small caliber and tortuosity of the coronary vessels and cardiac and respiratory motion create formidable imaging problems. We first studied 21 normal subjects and 5 with coronary artery disease established by X-ray contrast angiography, of whom 2 had undergone bypass grafting. Of these, 22 were imaged successfully. Identification of the artery was possible for the left main stem, left anterior descending, right coronary, and left circumflex arteries respectively in 95%, 91%, 95%, and 76%. The arterial diameter at the origin could be measured in 77%, 77%, 81%, and 63%. The mean ±SD arterial diameter in each case (4.8±0.8, 3.7±0.5, 3.9±0.9, and 2.9±0.6 mm) was not significantly different from reference values (allp=ns). The mean length of artery visualized was 10.4±5.2,46.7±22.8,53.7±27.9, and 26.3±17.5 mm. In 12 normal males, the total coronary area was 30.9±9.2 mm² and the ratio compared with body surface area was 16.4±4.4 mm² m^–2 (bothp=ns compared with reference values). In seven patients, with X-ray contrast coronary angiography, the proximal arterial diameter measured by magnetic resonance was 3.9±1.1 mm, and by X-ray contrast angiography 3.7±1.0 mm (p=ns). We then studied 17 patients with angina. Imaging of just the relevant artery was performed and analysis was blinded to the X-ray angiography results. Stenosis was identified on the magnetic resonance (MR) images by localized reduction in vessel signal intensity. Stenosis location by MR was assessed by measurement of its distance from a reference vessel, with correlation to the X-ray findings. X-ray coronary angiography showed 23 stenoses of which 15 (65%) were correctly located by blind assessment of the MR images. Of the eight remaining stenoses, a further 5 (63%) were correctly located on the MR images after retrospective comparison (overall sensitivity 87%). There were three lesions thought to represent stenosis by MR, which on review of the X-ray angiogram proved to be a minor stenosis <50% (two cases) or a tortuous vessel (one case). Greater signal loss was seen in the more severe stenoses. The stenosis length by MRI was greater than by X-ray (8.4 versus 5.1 mm,p<0.001). The overestimation of stenosis length may be due to turbulence.     

Functional evaluation in congenital and acquired heart disease

Conclusions Functional evaluation of the cardiovascular system using rapid imaging (spiral echo planar), chemical shift, and velocity mapping is capable of making the diagnosis in congenital and acquired cardiovascular disease which, between them, cause the largest number of deaths of any disease in the western world and massive morbidity and suffering. Furthermore, for the first time in the history of medicine, there is the opportunity to apply preventive measures to eardicate the epidemic of preventable arterial disease. There needs to be a change of emphasis and a switching of resources to apply to the most common diseases rather than to those which are most easily studied. There also needs to be proper training in cardiovascular MR, not so much for imagers as for cardiologists and experts in vascular disease.     

Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.