Tandem Ubiquitin Binding Entities (TUBEs) as Tools to Explore Ubiquitin-Proteasome System and PROTAC Drug Discovery.

The ubiquitin proteasome system (UPS) is a complex pathway that involves multiple enzymes and culminates in the formation of a polyubiquitin chain on a target protein. As its importance is becoming more evident in drug discovery, there is a renewed interest in understanding the role that polyubiquitin chains play. This has been a challenge, mostly due to the lack of experimental tools for detecting the polyubiquitinated forms of a protein of interest (POI). Tandem Ubiquitin Binding Entities (TUBEs) are engineered protein domains that bind specifically to polyubiquitin chains. These polyubiquitin affinity matrices are highly sensitive as they bind to polyubiquitin chains in the nanomolar range. They exist in two forms: pan-selective TUBEs and chain-selective TUBEs. The ability of TUBEs to be conjugated to different entities is truly what makes them unique. TUBEs are used in a wide variety of experiments such as in protein pulldowns to enrich for polyubiquitinated proteins. They are an alternative to ubiquitin antibodies in Western blots. Further, TUBEs are used as capture reagents for immobilizing polyubiquitinated proteins on a microtiter plate. The use of TUBEs as components of in vitro and cell-based assays presents the unique feature of confirming and assessing the polyubiquitination of a POI in response to inhibitors, activators, or PROTAC® molecules. Therefore, TUBEs not only play a big role in studying the UPS but also have a huge potential for speeding up the drug discovery process.

Carotid Artery Angioplasty and Stenting Without Distal Embolic Protection Devices.

BACKGROUND: Embolic protection devices are used during carotid artery stenting procedures to reduce risk of distal embolization. Although this is a standard procedural recommendation, no studies have shown superiority of these devices over unprotected stenting procedures. OBJECTIVE: To assess the periprocedural outcome and durability of carotid artery stenting without embolic protection devices and poststent angioplasty. METHODS: We performed a retrospective chart review of 174 carotid angioplasty stent procedures performed at our institution. One hundred sixty-six patients underwent angioplasty and stenting without distal protection devices or poststent angioplasty. Complications related to stenting, including procedural complications, postoperative stroke and/or myocardial infarction, and stent restenosis were analyzed. RESULTS: One hundred thirty-five stents (78%) were performed in symptomatic patients, whereas 22% of stents were placed for asymptomatic internal carotid artery stenosis. The degree of stenosis was 80% or greater in 75% of patients and 90% or greater in 55% of patients. Following the stenting procedure, the 24-hour and 30-day rate of transient ischemic attack, intracranial hemorrhage, or ischemic stroke was 0. Three (2%) patients had a perioperative, non-ST elevation myocardial infarction. Five patients (2.8%) required treatment for restenosis (>50% stenosis from baseline), 1 of which was symptomatic. CONCLUSION: Our data show that carotid artery stenting without the use of embolic protection devices and without postangioplasty stenting, in experienced hands, can be performed safely. Furthermore, this technique does not result in a higher degree of in-stent restenosis than series in which poststenting angioplasty is performed.