Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth.

Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal "sweet-spot" GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which retains approximately half of the EWS portion of the fusion) showed low affinity for smaller GGAA-microsatellites but instead significantly increased its affinity at sweet-spot microsatellite lengths. Single-gene ChIP and genome-wide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.

Chronic Exertional Compartment Syndrome of the Lower Extremity: Diagnosis and Surgical Treatment.

Background: Chronic exertional compartment syndrome of the lower extremity is a condition that characteristically presents as recurrent anterior, posterior, and/or lateral lower-extremity pain on repetitive activity and physical exertion1. This condition is commonly seen in athletes, runners, and military personnel2. Open fasciotomy has been demonstrated to be a highly effective surgical treatment for patients with this condition who do not experience symptomatic relief after a thorough trial of nonoperative treatment3. Description: Diagnostic compartment pressure management is achieved through direct insertion of a compartment-pressure-measuring device into the anterior, lateral, and posterior compartments of the lower extremity4. Surgical treatment of the anterior and lateral compartments with use of open fasciotomy employs longitudinal proximal and distal incisions that are made on the lateral surface of the leg approximately 3 finger-breadths distal and proximal to the fibular flare, respectively, and 3 finger-breadths lateral to the tibial crest. Surgical treatment of the posterior compartments with use of open fasciotomy employs a single, mid-shaft incision made approximately 2.5 cm medial to the tibial ridge. Dissection is carried down to the deep fascia at both sites, beginning at the distal operative site. Care is taken to avoid transection of the superficial peroneal nerve at the distal anterolateral incision and saphenous vein and nerve at the medial incision. Once down to the deep fascia, a scalpel is utilized to incise the fascia. Metzenbaum scissors are then employed under the incision, spreading the scissors while sliding them over the muscles proximally and distally to release the muscular attachments from the fascia as well as to release the fascia itself3. This process is repeated in the anterior, lateral, and superficial posterior compartments through the proximal and distal incisions. In the deep posterior compartment, the fascia is released from the tibial ridge with a large Cobb elevator. Closure is achieved with deep dermal and superficial sutures. Alternatives: Nonoperative alternatives have been reported to include nonpharmacological modalities such as walking modification and shoe inserts, pharmacological therapy with nonsteroidal anti-inflammatory drugs, and physical therapy targeted at conditioning the lower extremity5. Nonoperative intervention has been demonstrated to increase endurance in select patients; however, most patients must either stop the activity associated with the compartment syndrome altogether or proceed to surgery for complete resolution of symptoms5. There are a few surgical alternatives that differ in their utilization of minimally invasive approaches versus a direct open approach6; however, all existing surgical treatments of this condition involve physical release of the fascial compartment. Rationale: Diagnostic compartment-pressure measurement is useful in confirming or ruling out the presence of this condition in patients with unclear symptoms4. Furthermore, diagnostic compartment-pressure management ensures accuracy in diagnosis and validates invasive treatment when patients desire surgical intervention. Surgical management of exertional compartment syndrome of the lower extremity is indicated in patients when nonoperative treatment has failed despite clinically notable symptoms and objectively elevated lower-extremity compartment pressures. Open fasciotomy has been postulated to prevent compression of local vasculature and effectively prevent ischemia; however, the definitive mechanism is unclear1. Expected Outcomes: Surgical treatment of chronic exertional compartment syndrome with use of open fasciotomy is highly successful in the civilian population. One study showed excellent return to activity/sport in 15 of 16 patients (25 of 26 limbs; 96%), with patients often reporting no symptoms postoperatively3. Military personnel have been reported to experience satisfactory results, with another study showing positive subjective feedback in 35 (76%) of 46 patients on long-term follow-up; however, only 19 patients (41%) were able to return to full active duty postoperatively7. Important Tips: Balloting the fascial compartment with ∼1 cc of saline solution can be helpful in determining successful placement of the pressure-measuring device at the time of needle entry.Identifying the course of the superficial peroneal nerve via physical examination can help avoid iatrogenic injury to this important superficial structure during the dissection leading to the distal fasciotomy.Deep posterior compartment release with use of open fasciotomy may not provide symptomatic relief; patients who demonstrate elevation of pressures in this specific compartment should be counseled accordingly. Acronyms & Abbreviations: ROM = range of motionSPN = superficial peroneal nerve.

SimTube: A National Simulation Training and Research Project.

OBJECTIVE: To test the feasibility and impact of a simulation training program for myringotomy and tube (M&T) placement. STUDY DESIGN: Prospective randomized controlled. SETTING: Multi-institutional. SUBJECTS AND METHODS: An M&T simulator was used to assess the impact of simulation training vs no simulation training on the rate of achieving competency. Novice trainees were assessed using posttest simulator Objective Structured Assessment of Technical Skills (OSATS) scores, OSATS score for initial intraoperative tube insertion, and number of procedures to obtain competency. The effect of simulation training was analyzed using χ2 tests, Wilcoxon-Mann-Whitney tests, and Cox proportional hazards regression. RESULTS: A total of 101 residents and 105 raters from 65 institutions were enrolled; however, just 63 residents had sufficient data to be analyzed due to substantial breaches in protocol. There was no difference in simulator pretest scores between intervention and control groups; however, the intervention group had better OSATS global scores on the simulator (17.4 vs 13.7, P = .0003) and OSATS task scores on the simulator (4.5 vs 3.6, P = .02). No difference in OSATS scores was observed during initial live surgery rating (P = .73 and P = .41). OSATS scores were predictive of the rate at which residents achieved competence in performing myringotomy; however, the intervention was not associated with subsequent OSATS scores during live surgeries (P = .44 and P = .91) or the rate of achieving competence (P = .16). CONCLUSIONS: A multi-institutional simulation study is feasible. Novices trained using the M&T simulator achieved higher scores on simulator but not initial intraoperative OSATS, and they did not reach competency sooner than those not trained on the simulator.