Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation.

Cullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8~Cul5(ctd)-Rbx1 and SAXS analysis of NEDD8~Cul1(ctd)-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.

Abdominopelvic MR to CT registration using a synthetic CT intermediate.

Accurate coregistration of computed tomography (CT) and magnetic resonance (MR) imaging can provide clinically relevant and complementary information and can serve to facilitate multiple clinical tasks including surgical and radiation treatment planning, and generating a virtual Positron Emission Tomography (PET)/MR for the sites that do not have a PET/MR system available. Despite the long-standing interest in multimodality co-registration, a robust, routine clinical solution remains an unmet need. Part of the challenge may be the use of mutual information (MI) maximization and local phase difference (LPD) as similarity metrics, which have limited robustness, efficiency, and are difficult to optimize. Accordingly, we propose registering MR to CT by mapping the MR to a synthetic CT intermediate (sCT) and further using it in a sCT-CT deformable image registration (DIR) that minimizes the sum of squared differences. The resultant deformation field of a sCT-CT DIR is applied to the MRI to register it with the CT. Twenty-five sets of abdominopelvic imaging data are used for evaluation. The proposed method is compared to standard MI- and LPD-based methods, and the multimodality DIR provided by a state of the art, commercially available FDA-cleared clinical software package. The results are compared using global similarity metrics, Modified Hausdorff Distance, and Dice Similarity Index on six structures. Further, four physicians visually assessed and scored registered images for their registration accuracy. As evident from both quantitative and qualitative evaluation, the proposed method achieved registration accuracy superior to LPD- and MI-based methods and can refine the results of the commercial package DIR when using its results as a starting point. Supported by these, this manuscript concludes the proposed registration method is more robust, accurate, and efficient than the MI- and LPD-based methods.

More Than a Myoma: A Review of Myomatous Erythrocytosis Syndrome.

Myomatous erythrocytosis syndrome (MES) is gynaecological condition marked by isolated erythrocytosis and a fibroid uterus. This report presents a case of MES and reviews common clinical presentations, hematological trends, and patient outcomes. This study was a combined case report and review of published cases of MES. Cases were identified using Medline and EMBASE databases. Binomial statistics were used to compare clinical characteristics among patients with MES. Kruskal-Wallis one-way analysis of variance was used to compare hematological values across time points (Canadian Task Force Classification III). A total of 57 cases of MES were reviewed. The mean age at presentation was 48.7 years. Commonly reported signs or symptoms at presentation include abdominopelvic distension or mass (93%), skin discolouration (33%), and menstrual irregularities (25%). There was no difference in parity (P = 0.42), menopausal status (P = 0.87), or hydronephrosis on imaging (P = 0.48) among patients. Preoperative phlebotomy to reduce the risk of thromboembolic complications was performed in half of all cases. On average, a 51% reduction in serum erythropoietin levels was observed following surgical resection (P = 0.004). In conclusion, patients with MES present with signs and symptoms attributed to either an abdominopelvic mass or erythrocytosis. Preoperative phlebotomy to decrease the severity of erythrocytosis has been used to mitigate the risk of thrombotic complications. Surgical resection of the offending leiomyoma is a valid approach for the treatment of MES.

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification.

Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation.

UCL Throwing Injuries in Nonprofessional Baseball Players: A 14-Year Retrospective Study.

BACKGROUND: This study evaluated treatment modality (surgical vs nonoperative) of medial ulnar collateral ligament (UCL) injuries in nonprofessional throwing baseball athletes by comparing type, severity, and location of UCL injury. HYPOTHESIS: Baseball players with closed medial epicondyle physes and concomitant throwing-related UCL injury will be more likely to undergo surgical intervention than players with open medial epicondyle physes. STUDY DESIGN: Retrospective. LEVEL OF EVIDENCE: Level 5. METHODS: A total of 119 baseball players with a mean age of 16.9 ± 2.5 years (range, 11-25 years) were included in the study. Datapoints included sex, age at time of injury, severity, and location of UCL injury, growth plate status, operative versus conservative management, and concomitant flexor forearm injury. RESULTS: A total of 75 players were treated conservatively; 43 underwent UCL reconstruction (UCL-R), and 1 had an unknown treatment outcome. No significant difference was found for age related to treatment type, UCL-R (17.2 ± 2.2) versus conservative treatment (16.8 ± 2.6). Athletes with closed medial epicondylar growth plates were more likely to undergo UCL-R than athletes with open medial epicondylar growth plates (P = 0.02). There were no significant differences between UCL injury location (42 distal, 37 proximal, 18 combined tear locations, 11 complete tears, and 11 intact UCLs with inflammation) by treatment type (P = 0.09). There was a significant difference for UCL severity (11 complete tears, 96 partial tears) by treatment type (P = 0.03). CONCLUSION: Nonprofessional athletes with closed medial epicondylar growth plates and throwing-related UCL injuries were more likely to be treated surgically. Baseball athletes with partial tears, if skeletally immature, require further long-term evaluation. CLINICAL RELEVANCE: Continued knowledge gains in this area of throwing medicine will further improve our treatment algorithms in nonprofessional baseball players.

Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity.

Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.

Telephone reminders reduce no-shows: A quality initiative at a breast imaging center.

PURPOSE: To improve the workflow and productivity at a Breast Imaging Center, primarily by decreasing the no-show rate. MATERIALS AND METHODS: Mammography clinic data were recorded and analyzed for a 6-month period prior to our intervention. Personal pre-appointment phone calls were then instituted for all patients, following which data was recorded and analyzed for a 2-month period. Analyses compared pre- and post-intervention data. RESULTS: No-shows were significantly reduced (p < 0.001) from 20.99% (907/3775) pre-intervention to 7.07% (69/976) post-intervention. Calling ahead to cancel an appointment from those who either were no-shows or canceled their appointment significantly improved (p < 0.001) from only 2.81% (22/784) calling to cancel pre-intervention to 25.00% (23/92) calling to cancel post-intervention. CONCLUSIONS: Through systematic pre-appointment phone calls and documentation, we were able to achieve a very significant decrease in the no-show rate at our Breast Imaging Center. We believe that our intervention can be useful for other radiology groups to implement in their practices to reduce no-show rates.

The role of immunologic response in fresh osteochondral allografting of the knee.

BACKGROUND: Osteochondral allografting, a restorative treatment option for articular cartilage lesions in the knee, involves transplantation of fresh osteochondral tissue with no tissue matching. Although retrieval studies have not consistently shown evidence of immunologic response, development of anti-human leukocyte antigen class I cytotoxic antibodies has been observed in allograft recipients. HYPOTHESIS: Postallograft antibody formation is related to graft size and may affect clinical outcome. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: This study retrospectively compared 42 antibody-positive postallograft patients with 42 antibody-negative patients. Groups were matched for age, sex, and body mass index but not intra-articular disease severity. Seventeen patients (20%) were lost to follow-up. Of the remaining 67 patients (33 antibody-positive and 34 antibody-negative), average follow-up time was 50.3 months (range, 24-165 months). Mean age was 38.1 years (range, 15-68 years) with 58% being male. Graft area was categorized as small (<5 cm2), medium (5-10 cm2), or large (>10 cm2). Graft survival and Knee Society function scores were used to measure clinical outcome. RESULTS: Of the 84 patients, 80 had graft area data. Of 27 patients with large graft area, 19 (70%) had positive postoperative antibody screens, compared with 1 of 16 (6%) with small graft area (P < .001). Graft survival rates in the antibody-positive and antibody-negative groups were 64% and 79%, respectively (P = .152). Mean postoperative Knee Society function scores in surviving antibody-positive and antibody-negative groups were 88.3 and 84.6 points, respectively (P = .482). CONCLUSION: Antibody development after fresh, non-tissue-matched osteochondral allograft transplants in the knee appears related to graft size. No difference was observed in clinical outcome between groups. Graft survival is multifactorial, and the effect that the immunologic response has on clinical outcome merits further investigation.

Crystal structure of UBA2(ufd)-Ubc9: insights into E1-E2 interactions in Sumo pathways.

Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2(ufd)), alone and in complex with Ubc9. The overall structures of both yeast Uba2(ufd) and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2(ufd)-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2(ufd)-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades.

Exploration of self-identified education needs of alcohol and other drug workers.

OBJECTIVE: It is well established that people who work with clients who are dependent on substances need to be well equipped to deal with challenging and complex situations. Our research explores the experience of alcohol and other drug workers (AODW) in order to identify how successful Australia's teaching institutions are at preparing AODWs to meet this challenge. DESIGN: Interviews were conducted to ascertain the perceived gaps in existing training programs and the perceived training needs of AODW. Using a qualitative research approach in-depth interviews were conducted with more than 20 counsellors working in six AODW rehabilitation centres in regional New South Wales. RESULTS: There was a perception among all workers that their training had been inadequate and that the preparation for employment in AODW settings had not meet the felt needs of grass-roots workers leaving them to learn through experience. CONCLUSION: Considering the recommendations of both national and state policies on AODWs in Australia there is an urgent need to develop courses relevant to the needs of workers as suggested by respondents to this research.