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.

Aorta-innominate bypass through ministernotomy.

Atherosclerotic innominate artery occlusive disease can lead to cerebral and upper extremity ischemia. Innominate artery angioplasty and stenting can be complicated by stent fractures and restenosis; furthermore, this technique is limited in treatment of innominate artery occlusions. Ministernotomy to the second or third intercostal space can be used instead of conventional full sternotomy for open surgical revascularization of the innominate artery with excellent perioperative and long-term outcomes. This series of three consecutive patients highlights the technique of aorta-innominate artery bypass through ministernotomy.

Chromatin profiling reveals relocalization of lysine-specific demethylase 1 by an oncogenic fusion protein.

Paediatric cancers commonly harbour quiet mutational landscapes and are instead characterized by single driver events such as the mutation of critical chromatin regulators, expression of oncohistones, or expression of oncogenic fusion proteins. These events ultimately promote malignancy through disruption of normal gene regulation and development. The driver protein in Ewing sarcoma, EWS/FLI, is an oncogenic fusion and transcription factor that reshapes the enhancer landscape, resulting in widespread transcriptional dysregulation. Lysine-specific demethylase 1 (LSD1) is a critical functional partner for EWS/FLI as inhibition of LSD1 reverses the transcriptional activity of EWS/FLI. However, how LSD1 participates in fusion-directed epigenomic regulation and aberrant gene activation is unknown. We now show EWS/FLI causes dynamic rearrangement of LSD1 and we uncover a role for LSD1 in gene activation through colocalization at EWS/FLI binding sites throughout the genome. LSD1 is integral to the establishment of Ewing sarcoma super-enhancers at GGAA-microsatellites, which ubiquitously overlap non-microsatellite loci bound by EWS/FLI. Together, we show that EWS/FLI induces widespread changes to LSD1 distribution in a process that impacts the enhancer landscape throughout the genome.

How does transtrochanteric anterior rotational osteotomy change the dynamic three-dimensional intact ratio in hips with osteonecrosis of the femoral head?

BACKGROUND: The intact ratio (the ratio of the intact area of the femoral head) on a two-dimensional anteroposterior radiograph is associated with the prognosis of hips with osteonecrosis of the femoral head after transtrochanteric anterior rotational osteotomy. However, changes of the three-dimensional intact ratio during dynamic weight-bearing activity and correlation of the three-dimensional intact ratio with clinical scores are still unknown. METHODS: Kinematics of eight hips with osteonecrosis of the femoral head that underwent anterior rotational osteotomy were analyzed using image-matching techniques during chair-rising and squatting preoperatively and postoperatively. Two types of dynamic three-dimensional intact ratios were examined, including the lunate covered area (IRLC) and in vivo peak contact force vector intersected area (IRFV). The static three-dimensional intact ratio in each octant of the femoral head was also examined. FINDINGS: The mean Harris hip score significantly improved from 67 preoperatively to 90 postoperatively. During chair-rising rising/squatting, the mean IRLC and IRFV significantly increased from 42%/41% and 7%/4% preoperatively, to 66%/65% and 79%/77% postoperatively, respectively. IRLC significantly changed during the motion whereas substantial postoperative IRFV was maintained throughout the motion. Additionally, Harris hip score and the static three-dimensional intact ratio in the superolateral regions had significant positive correlations with both IRLC and IRFV. INTERPRETATION: Hip kinematics affected IRLC but not IRFV, which suggests that substantial intact bone occupies the region in which peak contact forces are applied during deep hip flexion. Additionally, improving intact ratio in the superolateral region led to improvements in both IRLC and IRFV with favorable clinical scores.

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Transcriptomic analysis functionally maps the intrinsically disordered domain of EWS/FLI and reveals novel transcriptional dependencies for oncogenesis.

EWS/FLI is the pathognomic fusion oncoprotein that drives Ewing sarcoma. The amino-terminal EWS portion coordinates transcriptional regulation and the carboxy-terminal FLI portion contains an ETS DNA-binding domain. EWS/FLI acts as an aberrant transcription factor, orchestrating a complex mix of gene activation and repression, from both high affinity ETS motifs and repetitive GGAA-microsatellites. Our overarching hypothesis is that executing multi-faceted transcriptional regulation requires EWS/FLI to use distinct molecular mechanisms at different loci. Many attempts have been made to map distinct functions to specific features of the EWS domain, but described deletion mutants are either fully active or completely "dead" and other approaches have been limited by the repetitive and disordered nature of the EWS domain. Here, we use transcriptomic approaches to show an EWS/FLI mutant, called DAF, previously thought to be nonfunctional, displays context-dependent and partial transcriptional activity but lacks transforming capacity. Using transcriptomic and phenotypic anchorage-independent growth profiles of other EWS/FLI mutants coupled with reported EWS/FLI localization data, we have mapped the critical structure-function requirements of the EWS domain for EWS/FLI-mediated oncogenesis. This approach defined unique classes of EWS/FLI response elements and revealed novel structure-function relationships required for EWS/FLI activation at these response elements.

Reliable complex abdominal wall hernia repairs with a narrow, well-fixed retrorectus polypropylene mesh: A review of over 100 consecutive cases.

BACKGROUND: Our objective was to determine outcomes for complex ventral hernia repairs in a large cohort of patients utilizing an operative construct employing retrorectus placement of a narrow, macroporous polypropylene mesh with up to 45 suture fixation points for force distribution. No consensus exists on the optimal technique for repair of complex ventral hernias. Current trends emphasize large meshes with wide overlaps and minimal suture fixation, though reported complications and recurrence remain problematic. METHODS: A retrospective review was performed for all patients undergoing ventral hernia repair with retrorectus placement of midweight, uncoated, soft polypropylene mesh by a single surgeon (GAD) between the years of 2010 and 2015. Patient characteristics, operative history, operative data, and postoperative course were reviewed. RESULTS: A total of 101 patients with a mean age of 56 years and a mean body mass index of 29 m/kg2 (range 18-51 m/kg2) underwent hernia repair. Patients had a median of 3 prior abdominal operations (range 0-9), with 44 patients presenting with recurrent hernias. A total of 42 patients were Ventral Hernia Working Group grade 1, 40 grade 2, 17 grade 3, and 2 grade 4. There were no recurrences at a mean follow-up of almost 400 days for the 93 patients with long-term follow-up. The surgical site occurrence rate was 7.9% (3 surgical site infections, 2 seromas, 2 hematomas, and 4 instances of delayed wound healing in 8 patients). One patient required reoperation for hematoma drainage; 5 patients required readmission within 30 days. CONCLUSION: An operative construct employing a retrorectus placement of a narrow, macroporous polypropylene mesh with up to 45 suture fixation points for force distribution can achieve significantly better outcomes across a spectrum of Ventral Hernia Working Group grade, risk-stratified patients compared to rates reported in the literature for current strategies that employ wide meshes with minimal fixation.

Mesh Sutured Repairs of Abdominal Wall Defects.

A new closure technique is introduced, which uses strips of macroporous polypropylene mesh as a suture for closure of abdominal wall defects due to failures of standard sutures and difficulties with planar meshes. METHODS: Strips of macroporous polypropylene mesh of 2 cm width were passed through the abdominal wall and tied as simple interrupted sutures. The surgical technique and surgical outcomes are presented. RESULTS: One hundred and seven patients underwent a mesh sutured abdominal wall closure. Seventy-six patients had preoperative hernias, and the mean hernia width by CT scan for those with scans was 9.1 cm. Forty-nine surgical fields were clean-contaminated, contaminated, or dirty. Five patients had infections within the first 30 days. Only one knot was removed as an office procedure. Mean follow-up at 234 days revealed 4 recurrent hernias. CONCLUSIONS: Mesh sutured repairs reliably appose tissue under tension using concepts of force distribution and resistance to suture pull-through. The technique reduces the amount of foreign material required in comparison to sheet meshes, and avoids the shortcomings of monofilament sutures. Mesh sutured closures seem to be tolerant of bacterial contamination with low hernia recurrence rates and have replaced our routine use of mesh sheets and bioprosthetic grafts.