Comparison of Interobserver Agreement of Four Classification Systems for Lateral Clavicle Fractures between Two Groups of Surgeons: A Multicenter Study.

OBJECTIVE: Distal clavicle fracture classification directly affects the treatment decisions. It is unclear whether the classification systems implemented differ depending on surgeons' backgrounds. This study aimed to compare the interobserver agreement of four classification systems used for lateral clavicle fractures by shoulder specialists and general trauma surgeons. METHODS: Radiographs of 20 lateral clavicle fractures representing a full spectrum of adult fracture patterns were analyzed by eight experienced shoulder specialists and eight general trauma surgeons from 10 different hospitals. All cases were graded according to the Orthopedic Trauma Association (OTA), Neer, Jäger/Breitner, and Gongji classification systems. To measure observer agreement, Fleiss' kappa coefficient (κ) was applied and assessed. RESULTS: When only X-ray films were presented, both groups achieved fair agreement. However, when the 3D-CT scan images were provided, improved interobserver agreement was found in the specialist group when the OTA, Jäger/Breitner, and Gongji classification systems were used. In the generalist groups, improved agreement was found when using the Gongji classification system. In terms of interobserver reliability, the OTA, Neer, and Jäger/Breitner classification systems showed better agreement among shoulder specialists, while a slightly lower level of agreement was found using the Gongji classification system. For the OTA classification system, interobserver agreement had a mean kappa value of 0.418, ranging from 0.446 (specialist group) to 0.402 (generalist group). For the Neer classification system, interobserver agreement had a mean kappa value of 0.368, ranging from 0.402 (specialist group) to 0.390 (generalist group). For the Jäger/Breitner classification system, the inter-observer agreement had a mean kappa value of 0.380, ranging from 0.413 (specialist group) to 0.404 (generalist group). For the Gongji classification system, interobserver agreement had a mean kappa value of 0.455, ranging from 0.480 (specialist group) to 0.485 (generalist group). CONCLUSION: Generally speaking, 3D-CT scans provide a richer experience that can lead to better results in most classification systems of lateral clavicle fractures, highlighting the value of digitization and specialization in diagnosis and treatment. Competitive interobserver agreement was exhibited in the generalist group using the Gongji classification system, suggesting that the Gongji classification is suitable for general trauma surgeons who are not highly experienced in the shoulder field.

Perioperative Protocol of Ankle Fracture and Distal Radius Fracture Based on Enhanced Recovery after Surgery Program: A Multicenter Prospective Clinical Controlled study.

Background: The enhanced recovery after surgery (ERAS) program is aimed to shorten patients' recovery process and improve clinical outcomes. This study aimed to compare the outcomes between the ERAS program and the traditional pathway among patients with ankle fracture and distal radius fracture. Methods: This is a multicenter prospective clinical controlled study consisting of 323 consecutive adults with ankle fracture from 12 centers and 323 consecutive adults with distal radial fracture from 13 centers scheduled for open reduction and internal fixation between January 2017 and December 2018. According to the perioperative protocol, patients were divided into two groups: the ERAS group and the traditional group. The primary outcome was the patients' satisfaction of the whole treatment on discharge and at 6 months postoperatively. The secondary outcomes include delapsed time between admission and surgery, length of hospital stay, postoperative complications, functional score, and the MOS item short form health survey-36. Results: Data describing 772 patients with ankle fracture and 658 patients with distal radius fracture were collected, of which 323 patients with ankle fracture and 323 patients with distal radial fracture were included for analysis. The patients in the ERAS group showed higher satisfaction levels on discharge and at 6 months postoperatively than in the traditional group (P < 0.001). In the subgroup analysis, patients with distal radial fracture in the ERAS group were more satisfied with the treatment (P=0.001). Furthermore, patients with ankle fracture had less time in bed (P < 0.001) and shorter hospital stay (P < 0.001) and patients with distal radial fracture received surgery quickly after being admitted into the ward in the ERAS group than in the traditional group (P=0.001). Conclusions: Perioperative protocol based on the ERAS program was associated with high satisfaction levels, less time in bed, and short hospital stay without increased complication rate and decreased functional outcomes.

Difference Between Posterior Monteggia Fractures and Posterior Fracture-Dislocation of Proximal Ulna in Adults.

OBJECTIVE: To figure out the difference between patients with posterior Monteggia fractures which were concomitant with proximal radioulnar joint (PRUJ) dislocation and posterior fracture-dislocation of the proximal ulna that were not concomitant with PRUJ. METHODS: From January 2016 to January 2019, 37 consecutive adult patients who had posterior fracture-dislocation of proximal ulna (no PRUJ dislocation, n = 16) and posterior Monteggia fractures (PRUJ dislocation, n = 21) were included. All patients had intraoperative fluoroscopy, computed tomography (CT) scans, and standard radiography (anteroposterior view and lateral view). The mechanism of injury, the cases with open fracture, sustained multiple injuries and classification of fracture was recorded. The clinical details of the patients such as the final range of motion (ROM) and the Broberg-Morrey scores were described. RESULTS: Patients with PRUJ dislocation (ten type A, five type B, and six type D) and those without concomitant PRUJ dislocation (fifteen type A and one type C) exhibited an obvious difference according to the classifications of Jupiter et al. (P = 0.010). Ninety-five percent of patients who had PRUJ dislocation were accompanied by a metaphyseal fracture, while only 50% of the patients who did not have PRUJ dislocation were accompanied by a metaphyseal fracture (P = 0.002). Meanwhile, 16 of 20 metaphyseal fractures had more than one fragment in the group of dislocations, but five of eight metaphyseal fractures were comminuted in the control group. The two groups exhibited an obvious difference (P = 0.009). The 21 patients who sustained a radioulnar dislocation had less mean arc of flexion, pronation, and Broberg-Morrey scores were significantly less than the patients of the control group (flexion: 117.38 ± 14.46 vs 127.50 ± 13.416, P = 0.035; pronation: 59.76 ± 11.88 vs 67.50 ± 6.58, P = 0.017; Broberg-Morrey: 80.48 ± 12.17 vs 88.19 ± 10.28, P = 0.040). CONCLUSIONS: Patients suffering posterior Monteggia fractures had more metaphyseal fractures, more comminuted fractures of the metaphysis, and worse ultimate ulnohumeral motion than patients of posterior fracture-dislocation of proximal ulna.

Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.

Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds ß integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to ß integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin ß1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome.

MiRNA-15a inhibits proliferation, migration and invasion by targeting TNFAIP1 in human osteosarcoma cells.

Recent data strongly suggest the important role of miRNAs in various cancer-related processes. Osteosarcoma is the most common type of primary malignant bone tumor and is characterized by complex genetic changes and resistance to conventional treatments. In this study, the role of miRNA-15a (miR-15a) in the progression and metastasis of osteosarcoma was investigated. The result demonstrated that the expression of miR-15a was down-regulated in osteosarcoma tissues and cell lines as compared with that in adjacent non-neoplastic bone tissues and the osteoblastic cell line. In functional assays, miR-15a inhibited cell proliferation, migration and invasion in U2OS and MG-63 cells. Meanwhile, bioinformatic analysis combined with experimental confirmation demonstrated that tumor necrosis factor; α-induced protein 1 (TNFAIP1) gene is a potential target of miR-15a and can be directly regulated by miR-15a. Down-regulation of TNFAIP1 induced effects on osteosarcoma cell lines similar to those induced by miR-15a. Taken together, these data suggest that miR-15a may act as a tumor suppressor, which is commonly down-regulated in both osteosarcoma tissues and cells. TNFAIP1 plays an important role in mediating miR-15a dependent biological functions in osteosarcoma. Reintroduction of miR-15a may be a novel therapeutic strategy by down-regulating TNFAIP1 expression.

A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow.

Actomyosin retrograde flow underlies the contraction essential for cell motility. Retrograde flow in both lamellipodia and lamella is required for membrane protrusion and for force generation by coupling to cell adhesion. We report that the Rac/Cdc42-binding kinase MRCK and myosin II-related MYO18A linked by the adaptor protein LRAP35a form a functional tripartite complex, which is responsible for the assembly of lamellar actomyosin bundles and of a subnuclear actomyosin network. LRAP35a binds independently to MYO18A and MRCK. This binding leads to MRCK activation and its phosphorylation of MYO18A, independently of ROK and MLCK. The MRCK complex moves in concert with the retrograde flow of actomyosin bundles, with MRCK being able to influence other flow components such as MYO2A. The promotion of persistent protrusive activity and inhibition of cell motility by the respective expression of wild-type and dominant-negative mutant components of the MRCK complex show it to be crucial to cell protrusion and migration.

Bcl-xL and UVRAG cause a monomer-dimer switch in Beclin1.

Beclin1 has a key regulatory role in the initiation of autophagy and is a tumor suppressor. We have examined the interplay between viral or human Bcl-2-like proteins and UVRAG and their opposite effects on Beclin1. We show that Beclin1 forms a dimer in solution via its coiled-coil domain both in vivo and in vitro. Viral Bcl-2 binds independently to two sites on the Beclin1 dimer, one with high affinity and one with lower affinity, whereas human Bcl-x(L) binds both sites equally with relatively low affinity. UVRAG disrupts the Beclin1-dimer interface, forming a heterodimer with Beclin1, suggesting that this is how UVRAG causes its effects on Beclin1 to activate autophagy. Both Bcl-2-like proteins reduce the affinity of UVRAG for Beclin1 approximately 4-fold, suggesting that they stabilize the Beclin1 dimer. Moreover, coimmunoprecipitation assays show that UVRAG substantially reduces Beclin1 dimerization in vivo. These data explain the concentration-dependent interplay between Bcl-2, UVRAG, and Beclin1, as both tumor suppressors, UVRAG and Beclin1, have single-copy mutations in human cancers. Furthermore, our data suggest that an alternative strategy for developing anti-cancer compounds would be to disrupt the Beclin1-dimer interface.

PRL-3 down-regulates PTEN expression and signals through PI3K to promote epithelial-mesenchymal transition.

PRL-3 is a metastasis-associated phosphatase. We and others have shown that its overexpression increases cell motility and invasiveness. These phenotypic changes are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development and oncogenesis. The EMT is a complex process that converts epithelia into migratory mesenchymal cells. We here attempt to unravel the underlying mechanistic basis of these phenomena. HeLa cells transiently expressing EGFP-PRL-3 (HeLa-PRL-3) exhibit reduced levels of paxillin. Similarly, Chinese hamster ovary cells stably expressing myc-PRL-3 (CHO-PRL-3) also show marked reductions in paxillin, phosphorylated paxillin-Tyr(31), and vinculin at focal adhesion complexes and notable reductions in the levels of RhoA-GTP, Rac1-GTP, and filamentous-actin filaments. DLD-1 human colorectal cancer cells engineered to express EGFP-PRL-3 (DLD-1-PRL-3) underwent changes consistent with EMT. In these cells, PRL-3 activates Akt and inactivates glycogen synthase kinase-3beta as assessed by phosphospecific antibodies. PRL-3 up-regulates mesenchymal markers fibronectin and Snail and down-regulates epithelial markers E-cadherin, gamma-catenin (plakoglobin), and integrin beta(3), which are major effectors in the EMT pathway. The changes in these EMT characteristics brought about by PRL-3 can be abrogated by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, implying that PRL-3 acts upstream of PI3K and could play an initiating role to trigger the EMT switch during cancer metastasis. In addition, PRL-3 can down-regulate phosphatase and tensin homologue deleted on chromosome 10, which is an important antagonist of PI3K, further reinforcing PI3K/Akt function in PRL-3-triggered EMT. Catalytically inactive PRL-3 (C104S) was impaired in the above PRL-3-mediated events, indicating that these properties require phosphatase activity. Targeting PRL-3 may thus be a useful strategy to impede cancer cell invasion and metastasis.

Outgrowth of neurites from NIE-115 neuroblastoma cells is prevented on repulsive substrates through the action of PAK.

In the central nervous system (CNS), damaged axons are inhibited from regeneration by glial scars, where secreted chondroitin sulfate proteoglycan (CSPG) and tenascin repulse outgrowth of neurites, the forerunners of axons and dendrites. During differentiation, these molecules are thought to form boundaries for guiding neurons to their correct targets. In neuroblastoma NIE-115 cells, outgrowth of neurites on laminin could be induced by serum starvation or inhibition of RhoA by Clostridium botulinum C3 toxin. The outgrowing neurites avoided crossing onto the repulsive substrate CSPG or tenascin. This avoidance response was partially overcome on expression of membrane-targeted and kinase-inactive forms of PAK. In these cells, the endogenous PAK isoforms colocalized with actin in distinctive sites, alphaPAK in the cell center as small clusters and along the neurite shaft and betaPAK and gammaPAK in areas with membrane ruffles and filopodia, respectively. When isoform-specific N-terminal PAK sequences were introduced to interfere with PAK function, substantially more neurites crossed onto CSPG when cells contained a gammaPAK-derived peptide but not the corresponding alphaPAK- or betaPAK-derived peptide. Thus, while neurite outgrowth can be promoted by RhoA inhibition, overcoming the accompanying repulsive guidance response will require modulation of PAK activity. These results have therapeutic implications for CNS repair processes.

PRL-3 and PRL-1 promote cell migration, invasion, and metastasis.

We demonstrate here that Chinese hamster ovary cells stably expressing PRL-3, a M(r) 20000 prenylated protein tyrosine phosphatase, or its relative, PRL-1, exhibit enhanced motility and invasive activity. A catalytically inactive PRL-3 mutant has significantly reduced migration-promoting activity. We observe that PRL-3 is associated with diverse membrane structures involved in cell movement. Furthermore, we show that PRL-3- and -1-expressing cells, but not control cells, induce metastatic tumor formation in mice. Thus, our results deliver the first evidence for a causative role of PRL-3 and -1 in promoting cell motility, invasion activity, and metastasis.

Cdc42 antagonizes inductive action of cAMP on cell shape, via effects of the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) on myosin light chain phosphorylation.

Rho GTPases play pivotal roles in regulating cell morphology. We previously showed that RhoA acts via ROKalpha to counteract the effects of the classical second messenger cyclic AMP on cell shape changes. Here we show that active Cdc42V12 also competes against the cAMP-induced stellate morphology in SH-EP cells. This Cdc42 effect is not mediated by the RhoA/ ROK pathway but rather the related MRCKalpha, a myotonic dystrophy kinase-related Cdc42-binding kinase. Co-expression of a dominant inhibitory MRCKalpha mutant with Cdc42V12 blocks the ability of the GTPase to counteract cAMP, suggesting that MRCK acts downstream of Cdc42 in this process. Cdc42V12 enhances the phosphorylation of myosin light chain (MLC) at the cell periphery and sustains focal adhesion complexes, while MLC kinase inhibitors destroy focal adhesion complexes and impair the Cdc42V12 protective effect. The data suggest that the maintenance of focal adhesion complexes via the regulation of myosin II activity underlies the ability of Cdc42 to protect against the effect of elevated cAMP.