Preventing the Slide in High-Energy Intertrochanteric Fractures With Intramedullary Nails.

LEVEL OF EVIDENCE: Therapeutic Level V.

Rational Design, Synthesis, and Anti-Proliferative Evaluation of Novel 4-Aryl-3,4-Dihydro-2H-1,4-Benzoxazines.

A synthetic pathway to a novel 4-aryl-3,4-dihydro-2H-1,4-benzoxazine scaffold was developed and a series of compounds based on the scaffold were synthesised as potential anticancer agents. The 4-aryl-substituted compounds were prepared via Buchwald-Hartwig cross-coupling between substituted bromobenzenes and various 1,4-benzoxazines, which in turn were generated from a cascade hydrogenation and reductive amination one-pot reaction. These analogues exhibited moderate to good potency against various cancer cell lines. Structure-activity relationship analysis indicated that the inclusion of hydroxyl groups on ring A and ring B was beneficial to biological activity, while having a para-amino group on ring C significantly enhanced potency. Molecule 14f displayed the most potent anticancer activity (IC50 = 7.84-16.2 µM against PC-3, NHDF, MDA-MB-231, MIA PaCa-2, and U-87 MG cancer cell lines), indicating its potential as a lead compound for further structural optimisation. All the synthesised compounds were fully characterised with NMR, HMRS, and IR. The novel benzoxazine scaffold described in this study holds promise and deserves further in-depth studies.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

Development of Inhibitors against Mycobacterium abscessus tRNA (m1G37) Methyltransferase (TrmD) Using Fragment-Based Approaches.

Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.

Dihydropyrrolones as bacterial quorum sensing inhibitors.

Bacteria regulate their pathogenicity and biofilm formation through quorum sensing (QS), which is an intercellular communication system mediated by the binding of signaling molecules to QS receptors such as LasR. In this study, a range of dihydropyrrolone (DHP) analogues were synthesized via the lactone-lactam conversion of lactone intermediates. The synthesized compounds were tested for their ability to inhibit QS, biofilm formation and bacterial growth of Pseudomonas aeruginosa. The compounds were also docked into a LasR crystal structure to rationalize the observed structure-activity relationships. The most active compound identified in this study was compound 9i, which showed 63.1% QS inhibition of at 31.25 µM and 60% biofilm reduction at 250 µM with only moderate toxicity towards bacterial cell growth.

Femoral Neck Fractures in Young Patients: State of the Art.

Femoral neck fractures in young patients are uncommon but are often associated with surgical challenges and complications. The quality of reduction, more than time to surgery, has the most impact on optimizing outcomes and function. There is no consensus in the best fixation construct for these fractures. Neck shortening and varus collapse are the most common challenges of current fixation options. Use of newer implants is being reported with cautious optimism, and further studies are needed. LEVEL OF EVIDENCE:: Therapeutic Level V.

Association Between 6-Week Postdischarge Risk Classification and 12-Month Outcomes After Orthopedic Trauma.

Importance: Numerous studies have demonstrated that long-term outcomes after orthopedic trauma are associated with psychosocial and behavioral health factors evident early in the patient's recovery. Little is known about how to identify clinically actionable subgroups within this population. Objectives: To examine whether risk and protective factors measured at 6 weeks after injury could classify individuals into risk clusters and evaluate whether these clusters explain variations in 12-month outcomes. Design, Setting, and Participants: A prospective observational study was conducted between July 16, 2013, and January 15, 2016, among 352 patients with severe orthopedic injuries at 6 US level I trauma centers. Statistical analysis was conducted from October 9, 2017, to July 13, 2018. Main Outcomes and Measures: At 6 weeks after discharge, patients completed standardized measures for 5 risk factors (pain intensity, depression, posttraumatic stress disorder, alcohol abuse, and tobacco use) and 4 protective factors (resilience, social support, self-efficacy for return to usual activity, and self-efficacy for managing the financial demands of recovery). Latent class analysis was used to classify participants into clusters, which were evaluated against measures of function, depression, posttraumatic stress disorder, and self-rated health collected at 12 months. Results: Among the 352 patients (121 women and 231 men; mean [SD] age, 37.6 [12.5] years), latent class analysis identified 6 distinct patient clusters as the optimal solution. For clinical use, these clusters can be collapsed into 4 groups, sorted from low risk and high protection (best) to high risk and low protection (worst). All outcomes worsened across the 4 clinical groupings. Bayesian analysis shows that the mean Short Musculoskeletal Function Assessment dysfunction scores at 12 months differed by 7.8 points (95% CI, 3.0-12.6) between the best and second groups, by 10.3 points (95% CI, 1.6-20.2) between the second and third groups, and by 18.4 points (95% CI, 7.7-28.0) between the third and worst groups. Conclusions and Relevance: This study demonstrates that during early recovery, patients with orthopedic trauma can be classified into risk and protective clusters that account for a substantial amount of the variance in 12-month functional and health outcomes. Early screening and classification may allow a personalized approach to postsurgical care that conserves resources and targets appropriate levels of care to more patients.

Fragment-based approaches to TB drugs.

Tuberculosis is an infectious disease associated with significant mortality and morbidity worldwide, particularly in developing countries. The rise of antibiotic resistance in Mycobacterium tuberculosis (Mtb) urgently demands the development of new drug leads to tackle resistant strains. Fragment-based methods have recently emerged at the forefront of pharmaceutical development as a means to generate more effective lead structures, via the identification of fragment molecules that form weak but high quality interactions with the target biomolecule and subsequent fragment optimization. This review highlights a number of novel inhibitors of Mtb targets that have been developed through fragment-based approaches in recent years.

Effect of DMSO on Protein Structure and Interactions Assessed by Collision-Induced Dissociation and Unfolding.

Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization-ion mobility-mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.

Does a Staged Posterior Approach Have a Negative Effect on OTA 43C Fracture Outcomes?

OBJECTIVE: To determine whether multiple approaches pose an increased risk to fracture healing when compared with a standard single approach in the treatment of pilon (OTA 43C) fractures. DESIGN: Retrospective review of a prospective database. SETTING: Level I academic trauma center and level II community trauma center. METHODS: From January 1, 2005 to December 31, 2011, all records of patients treated for OTA 43C fractures of the distal tibia were reviewed. Patients were grouped according to multiple (posterior-anterior) and single (anterior-alone) approaches. Medical charts and surgical documentation were reviewed and postoperative computed tomography (CT) scans were examined for residual articular displacement and quantified. Ultimate union rate was correlated with approach strategy. Articular reduction was subdivided into 3 groups (<1, 1-2, and >2 mm). RESULTS: A total of 116 patients were identified as having had 43C fractures treated surgically with postoperative CT scans completed. Twenty-six fractures presented as an open injury. Of these 116 patients, 35 underwent staged fixation of the posterior malleolar component at an average of 2 days postinjury, followed by delayed anterior fixation at an average of 14 days postinjury. The remaining 81 patients underwent anterior fixation alone, on average 17 days postinjury. Twenty-one patients were lost to follow-up before 12 months. Of the 95 patients with sufficient follow-up (≥12 months), there were 24 nonunions. There was a statistically significant association of nonunion with staged posterior approach (40% vs. 19%, P = 0.015). CT reduction for staged posterior versus anterior-alone approach was not significantly different for any of the 3 categories (63% vs. 57% <1 mm, 31% vs. 26% 1-2 mm, and 6% vs. 17% >2 mm). CONCLUSIONS: In this series, there was no statistically proven benefit to combined surgical approaches to tibial pilon fractures with regard to the quality of articular reduction. It appears from this investigation that there may be a significantly higher risk of nonunion associated with the addition of the staged posterior approach. Although articular reduction is of paramount importance, multiple approaches for direct reduction and fixation of all fragments may lead to further complications. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors.

The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 µM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.

Suprapatellar Versus Infrapatellar Tibial Nail Insertion: A Prospective Randomized Control Pilot Study.

PURPOSE: The purpose of this OTA-approved pilot study was to compare the clinical and functional outcomes of the knee joint after infrapatellar (IP) versus suprapatellar (SP) tibial nail insertion. DESIGN: Prospective, randomized. SETTING: Level I trauma center. METHODS: After institutional review board approval, skeletally mature patients with OTA 42 tibial shaft fractures were randomized into either an IP or SP nail insertion group after informed consent was obtained. The SP also underwent prenail and postnail insertion patella-femoral (PF) joint arthroscopy. Patients underwent follow-up (6 weeks, 3, 6, and 12 months) with standard radiographs, as well as visual analog score and pain diagram documentation. At the 6-month and 12-month visits, knee function questionnaires (Lysholm knee scale and SF-36) were completed. Magnetic resonance imaging/image (MRI) of the affected knee was obtained at 12 months. Ten patients in each group were required for a power analysis for the anticipated larger randomized control trial, but enrollment in each arm was not limited because of known problems with patient follow-up over a 12-month period. RESULTS: A total of 41 patients/fractures were enrolled in this study. Of those, only 25 patients/fractures (14 IP, 11 SP) fully complied with and completed 12 months of follow-up. Six of 11 SP presented with articular changes (chondromalacia) in the PF joint during the preinsertion arthroscopy. Three patients displayed a change in the articular cartilage based on postnail insertion arthroscopy. At 12 months, all fractures in both groups had proceeded to union. There were no differences between the affected and unaffected knee with respect to range of motion. Functional visual analog score and Lysholm knee scores showed no significant differences between groups (P > 0.05). The SF-36v2 comparison also revealed no significant differences in the overall score, all 4 mental components, and 3/4 physical components (P > 0.05). The bodily pain component score was superior in the SP group (45 vs. 36, P = 0.035). All 11 SP patients obtained MRIs at 1 year. Five of these patients had evidence of chondromalacia on MRI. These findings did not correlate with either the prenail or postnail insertion arthroscopy. Importantly, no patient in the SP group with postnail insertion arthroscopic changes had PF joint pain at 1 year. CONCLUSIONS: Overall, there seemed to be no significant differences in pain, disability, or knee range of motion between these 2 tibial intramedullary nail insertion techniques after 12 months of follow-up. Based on this pilot study data, larger prospective trial with long-term follow-up is warranted. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Synthesis, biological evaluation and structure-activity relationship studies of isoflavene based Mannich bases with potent anti-cancer activity.

Phenoxodiol, an analogue of the isoflavone natural product daidzein, is a potent anti-cancer agent that has been investigated for the treatment of hormone dependent cancers. This molecular scaffold was reacted with different primary amines and secondary amines under different Mannich conditions to yield either benzoxazine or aminomethyl substituted analogues. These processes enabled the generation of a diverse range of analogues that were required for structure-activity relationship (SAR) studies. The resulting Mannich bases exhibited prominent anti-proliferative effects against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines. Further cytotoxicity studies against MRC-5 normal lung fibroblast cells showed that the isoflavene analogues were selective towards cancer cells.

Effect of distal interlocking screw number and position after intramedullary nailing of distal tibial fractures: a biomechanical study simulating immediate weight-bearing.

OBJECTIVE: To quantify the changes in biomechanical stability conferred by the addition of a single medial blocking screw or a single bicortical interlocking screw to 2 existing distal points of screw fixation in a distal tibial fracture model repaired with intramedullary nailing. METHODS: After simulation of a distal tibial metaphyseal fracture, 21 synthetic tibiae were repaired with an intramedullary nail and: (1) two bicortical locking screws placed in the 2 most distal screw holes (IM-L2); (2) three distal bicortical locking screws (IM-L3); and 2 distal locking screws and a single blocking screw positioned in the sagittal plane on the medial aspect of the nail (IM-L2B). The specimens were tested under combined cyclic axial and torsional loading for up to 16k cycles. The former was stepwise increasing, whereas the latter was with constant amplitude in internal rotation. RESULTS: All constructs survived 12k cycles without hardware deformation or failure. IM-L3 constructs displayed the highest baseline axial stiffness at the beginning of the test (1130.9 ± 246.9 N/mm), which was significant compared with the IM-L2 construct (701.8 ± 189.57 N/mm, P = 0.004). No significant difference in baseline axial stiffness was identified between the IM-L3 and IM-L2B constructs (881.1 ± 182.4 N/mm, P = 0.125). Relative varus interfragmentary deformation at baseline was smaller in the IM-L3 treatment group (1.3 ± 0.3 degrees) relative to the IM-L2 group (2.4 ± 0.7 degrees, P = 0.012). No differences in torsional rigidity or relative interfragmentary torsional deformation were identified between groups (P > 0.168). Failure was breakage or backout of the distal bicortical screws, fracture of the distal fragment, or proximal screw breakage. There was no significant difference in number of cycles to failure between instrumentation groups (IM-L2: 14,345 ± 1438 cycles; IM-L3: 15,634 ± 626 cycles; and IM-L2B: 14,862 ± 1511 cycles, P = 0.184). CONCLUSION: Results suggest that each of the constructs tested here may be a biomechanically viable option allowing for immediate weight-bearing after fixation of fractures of the distal third of the tibia. The addition of a single bicortical interlocking screw to create 3 points of distal fixation improves construct stiffness while reducing interfragmentary motion relative to 2 interlocking points of screw fixation with or without a blocking screw.

Wound complications associated with bone morphogenetic protein-2 in orthopaedic trauma surgery.

OBJECTIVE: To document the incidence of postoperative wound complications associated with the use of rhBMP-2 in a large series of patients for both acute traumatic and reconstructive extremity cases. DESIGN: Retrospective chart and radiographic review. SETTING: Level I trauma center. METHODS: A retrospective chart and x-ray review was performed on cases between 2002 and 2009, in which rhBMP-2 (Infuse) was used in acute trauma or posttraumatic reconstruction. The following data points were collected: age, surgical site, purpose (acute vs. reconstructive), associated wound factors (open fractures, soft tissue injury requiring coverage, or history of infection), signs of infection (seroma, erythema, prolonged drainage, abscess), reoperation rate secondary to wound complication, culture results, and union. These cases were then compared with a matched cohort without the use of bone morphogenetic protein-2 (matched for age, type of case, anatomic site, and open injury) for statistical analysis. RESULTS: Group 1 was comprised a total of 193 patients whose treatment included rhBMP-2 (155 reconstructive and 38 acute open fractures). Group 2 was comprised 181 patients treated without the use of rhBMP-2 (145 reconstructive and 36 acute open fractures). The incidences of documented wound complications were 31% (60/193) in group 1 and 18% (33/181) in group 2 (P = 0.004). Reoperation rates for wound complications were in 3.1% of group 1 and 8.3% of group 2 (P = 0.04). Age, sex, anatomic site, acute trauma, open fracture, and the need for soft tissue reconstruction did not correlate with the need to return to the operating room for presumed or actual wound infection. The rates of union between rhBMP-2 and control groups were 90% versus 74% (P < 0.001); for acute trauma cases, 94% versus 79% (P = 0.220); and for reconstructive cases, 89% versus 73% (P = 0.002). CONCLUSIONS: The use of rhBMP-2 in both acute traumatic and posttraumatic reconstructive extremity surgery may increase the incidence of prolonged postoperative serous wound drainage. However, this does not seem to correlate with an increased incidence of postoperative wound infection or the need for reoperation. The use of rhBMP-2 seems to have a beneficial effect in improving union rates for both acute trauma and posttraumatic reconstruction of the extremities (P = 0.002); however, this and the mechanism for prolonged serous drainage require further study before definitive recommendations can be made. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

The insertion of intramedullary nail locking screws without fluoroscopy: a faster and safer technique.

OBJECTIVE: This study was designed to compare the accuracy, time, and radiation exposure during the insertion of intramedullary nail locking screws using either standard fluoroscopic assistance or an electromagnetic (EM)-based navigational system without fluoroscopy. DESIGN: Prospective. SETTING: Level I academic trauma center. METHODS: Patients were divided into 2 groups: group 1 (fluoroscopic assistance), consisted of standard freehand fluoroscopically assisted insertion of locking screws (OEC 9900; G.E. HealthCare, Waukesha, WI), whereas group 2 (EM), consisted of EM navigationally assisted insertion without fluoroscopy (SureShot; Smith & Nephew, Memphis, TN). Technician arrival time, setup (SU) time, screw insertion (SI) time (seconds), fluoroscopy time (seconds), radiation exposure (mrads), and accuracy (hit or miss) were recorded for each screw. For group 1, the SU time was recorded as the time and radiation required to obtain "perfect circles" before insertion, and for group 2, the SU time was recorded as the time required to set up the navigational EM unit. Data collected regarding SI were then compared using standard analysis of variance. RESULTS: Forty-one locking screws were inserted in group 1, whereas 60 screws were inserted in group 2. Accuracy was 100% for both groups. For group 1, mean technician wait time was 77 seconds plus a mean perfect circle SU time of 105 seconds (9.2 mrads and 10 seconds of fluoroscopy). Mean SU time for group 2 was 94 seconds (no fluoroscopy). Mean insertion time was 342 seconds per screw for group 1 (32.9 mrads and 18 seconds of fluoroscopy) compared with 234 seconds per screw for group 2 (no fluoroscopy). These differences were statistically significant (P = 0.006). CONCLUSIONS: The use of EM navigation (SureShot; Smith & Nephew) for the insertion of intramedullary nail locking screws demonstrated accuracy similar to conventional fluoroscopic-guided insertion. However, EM-guided locking SI resulted in a significantly shorter total procedural time and completely eliminated radiation exposure. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB.

Transcription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity.