RESUMO
Fluoroquinolones (FQs) are potent antibiotics of clinical significance, known for their unique mechanism of action as gyrase poisons, which stabilize gyrase-DNA cleavage complexes and convert gyrase into a DNA-damaging machinery. Unfortunately, FQ resistance has emerged, and these antibiotics can cause severe side effects. Therefore, discovering novel gyrase poisons with different chemical scaffolds is essential. The challenge lies in efficiently identifying them from compound libraries containing thousands or millions of drug-like compounds, as high-throughput screening (HTS) assays are currently unavailable. Here we report a novel fluorescence-based, T5 exonuclease-amplified DNA cleavage assay for gyrase poison discovery. This assay capitalizes on recent findings showing that multiple gyrase molecules can simultaneously bind to a plasmid DNA molecule, forming multiple gyrase-DNA cleavage complexes on the same plasmid. These gyrase-DNA cleavage complexes, stabilized by a gyrase poison, can be captured using sarkosyl. Proteinase K digestion results in producing small DNA fragments. T5 exonuclease, selectively digesting linear and nicked DNA, can fully digest the fragmented linear DNA molecules and, thus, "amplify" the decrease in fluorescence signal of the DNA cleavage products after SYBR Green staining. This fluorescence-based, T5 exonuclease-amplified DNA cleavage HTS assay is validated using a 50-compound library, making it suitable for screening large compound libraries.
RESUMO
OBJECTIVES: This study sought to investigate the frequency, predictors, and detailed qualitative and quantitative assessment of optical coherence tomography (OCT)-detected stent edge dissections. Its impact on subsequent management and clinical outcomes were also investigated. BACKGROUND: OCT is a high-resolution imaging modality that can lead to more frequent recognition and accurate assessment of vascular injuries during percutaneous coronary intervention (PCI). METHODS: From September 2010 to June 2011, all patients with OCT post-PCI were enrolled. Edge dissections were defined as disruptions of the arterial lumen surface in both the 5-mm distal and proximal stent edges. Qualitative and quantitative analyses of all edges were performed at 0.2-mm intervals. RESULTS: In total, 395 edges (249 lesions in 230 patients) were analyzed. The overall incidence of OCT-detected edge dissection was 37.8%, and most (84%) were not apparent on angiography. Independent predictors for OCT-detected dissections were presence of atherosclerotic plaque at stent edges, calcification angle, minimum fibrous cap thickness, thin-cap fibroatheromas, stent/lumen eccentricity, and vessel overstretching. Mean dissection length measured 2.04 ± 1.60 mm, 96.2% appeared as flaps, and 52.8% extended beyond the intima/atheroma layer. Additional stenting was performed in 22.6% of all dissections, which were longer, had bigger dimensions, and promoted deeper vascular injury. The 12-month major adverse cardiac event rate was similar between patients with (7.95%) and without (5.69%, p = 0.581) dissections. CONCLUSIONS: High rates of stent edge dissections were detected by OCT, usually related to the presence of atherosclerosis at stent edges and to PCI technique. Detailed OCT assessment of dissection severity was possible and affected the subsequent management of this complication. Non-flow-limiting, small, and superficial dissections left untreated proved benign.
