The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and x-ray diffraction at room temperature.

The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures.

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.

Sliding hip screws versus cancellous screws for femoral neck fractures: a systematic review and meta-analysis.

PURPOSE: Both sliding hip screws (SHS) and cancellous screws are used in the surgical management of intracapsular femoral neck fracture. However, there is paucity of information as to which is the superior treatment modality. We performed this systematic review and meta-analysis study to compare the clinical outcomes of SHS and cancellous screws for the treatment of femoral neck fractures in adult patients. METHODS: We searched PubMed, Scopus, Web of Science, and Cochrane CENTRAL, up to December 2017. Randomized controlled trials (RCTs) directly comparing the clinical outcomes of SHS and cancellous screws for femoral neck fractures were retrieved with no language or publication year restrictions. Data retrieved included operative details, nonunion rate, avascular necrosis, reoperation, infection and mortality, hip pain, functional hip scores, and medical complications. These were pooled as risk ratio or mean difference (MD) with their corresponding 95% confidence interval (CI). Heterogeneity was assessed by Chi-square test. RESULTS: Ten RCTs involving 1934 patients were included in the final analysis. The pooled estimate showed that the SHS group was associated with more intraoperative blood loss (MD = 110.01 ml, 95% CI [52.42, 167.60], p = 0.00002) than the cancellous screws. There was no significant difference in terms of operative time, postoperative hip function, nonunion, avascular necrosis, reoperation rate, infection, fracture healing, hip pain, medical complications, and mortality rate. CONCLUSION: Based on our study, the cancellous screws group was associated with less intraoperative blood loss in comparison with the SHS group. No other significant differences were found between the two interventions.

Covalent Modification and Regulation of the Nuclear Receptor Nurr1 by a Dopamine Metabolite.

Nurr1, a nuclear receptor essential for the development, maintenance, and survival of midbrain dopaminergic neurons, is a potential therapeutic target for Parkinson's disease, a neurological disorder characterized by the degeneration of these same neurons. Efforts to identify Nurr1 agonists have been hampered by the recognition that it lacks several classic regulatory elements of nuclear receptor function, including the canonical ligand-binding pocket. Here we report that the dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to and modulates the activity of Nurr1. Using biophysical assays and X-ray crystallography, we show that DHI binds to the ligand-binding domain within a non-canonical pocket, forming a covalent adduct with Cys566. In cultured cells and zebrafish, DHI stimulates Nurr1 activity, including the transcription of target genes underlying dopamine homeostasis. These findings suggest avenues for developing synthetic Nurr1 ligands to ameliorate the symptoms and progression of Parkinson's disease.

Examination under Anesthesia in Patients with Recurrent Patellar Dislocation: Prognostic Study.

Surgical management of patellar instability is a matter of wide debate. While some authors believe that isolated medial patellofemoral ligament (MPFL) reconstruction is sufficient, others advocate procedures to treat the underlying pathology, namely, patella alta and trochlear dysplasia. Radiological studies have been traditionally used as a determinant of these pathologies and to determine the need for an additional procedure. The value of examination under anesthesia (EUA) in formulating a treatment plan remains unknown. Our aim is to assess the prognostic value of EUA in assessing patients presenting for surgical treatment of recurrent patellar dislocations. We retrospectively reviewed the outcome after surgical treatment of 23 patients who had undergone isolated MPFL reconstruction for recurrent patella dislocation by a single surgeon in our tertiary center. All data were completed at the time of index surgery. Primary outcome was recurrent instability requiring revision surgery. The mean age of the 23 patients undergoing MPFL reconstruction was 17.4 years of age. The median trochlea tuberosity-trochlear groove (TT-TG) distance was 15 mm with a median Caton-Deschamps ratio of 1.3. Of the 23 patients who underwent MPFL reconstruction, 9 failed (39%). Median time to failure was 10.5 months. All patients who failed MPFL reconstruction had a dislocating patella on EUA at >30 degrees of flexion. Radiological assessment of the knee to assess its morphology is essential for preoperative surgical planning for patients with recurrent patellar instability. Cutoff values to determine the need for surgical procedures require a consistent method of imaging and are prone to flaws in measurement. However, we demonstrated that EUA provides valuable additional information toward an appropriate surgical approach.Based on our small cohort, we suggest that patients in whom patella remains dislocated past 30 degrees of knee flexion on EUA are unlikely to benefit from isolated MPFL reconstruction.

A Novel Suture Button Construct for Acute Ankle Syndesmotic Injuries; A Prospective Clinical and Radiological Analysis.

BACKGROUND: The importance of the syndesmosis in ankle stability is well recognized. Numerous means of fixation have been described for syndesmotic injuries including the suture button technique. Significant cost limits the use the commercially available options. We, therefore, designed a cheap and readily available alternative construct. We aim to assess the results of using a novel suture-button construct in treatment of syndesmotic ankle injuries. METHODS: Fifty-two patients (34 males and 18 females) fulfilled our inclusion/exclusion criteria. Five patients were lost to follow-up. The remaining 47 patients were successfully followed up for a minimum of 24 months. The pre and post-surgery American Orthopedic Foot and Ankle Society scores (AOFAS) together with reported complications and post-operative radiological analysis were assessed. In this innovative construct, we utilized polyester braided surgical sutures jointly with double mini two- holed plates, a No.2 polygalactin 910 suture, a 4 mm drill bit, together with a 15 cm long suture needle with slotted end. This technique was supported with the use of the image intensifier. RESULTS: The AOFAS score improved significantly from a mean of 32.4 to 94.2 (P<0.004). Radiologically, the medial clear space (MCS), tibio-fibular clear space (TFCS) (P=0.05) and tibio-fibular overlap (TFO) measurements showed a significant improvement postoperatively (P=0.02). Patients reported good satisfaction rates with a 96% success rate (95% CI: 94.0% to 99.3%). CONCLUSION: We have observed that this low cost suture button construct is a simple, safe and cost effective treatment option for acute syndesmotic injuries.

A systematic review of the concept and clinical applications of bone marrow aspirate concentrate in tendon pathology.

Tendon pathologies are a group of musculoskeletal conditions frequently seen in clinical practice. They can be broadly classified into traumatic, degenerative and overuse-related tendinopathies. Rotator cuff tears, Achilles tendinopathy and tennis elbow are common examples of these conditions. Conventional treatments have shown inconsistent outcomes and might fail to provide satisfactory clinical improvement. With the growing trend towards the use of mesenchymal stem cells (MSCs) in other branches of medicine, there is an increasing interest in treating tendon pathologies using the bone marrow MSC. In this article, we provide a systematic literature review documenting the current status of the use of bone marrow aspirate concentrate (BMAC) for the treatment of tendon pathologies. We also asked the question on the safety of BMAC and whether there are potential complications associated with BMAC therapy. Our hypothesis is that the use of BMAC provides safe clinical benefit when used for the treatment of tendinopathy or as a biological augmentation of tendon repair. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist while preparing this systematic review. A literature search was carried out including the online databases of PubMed, EMBASE, ClinicalTrial.gov and the Cochrane Library from 1960 to the end of May 2015. Relevant studies were selected and critically appraised. Data from eligible studies were extracted and classified per type of tendon pathology. We included 37 articles discussing the application and use of BMAC for the treatment of tendon pathologies. The Critical Appraisal Skills Program (CASP) appraisal confirmed a satisfactory standard of 37 studies. Studies were sub-categorised into: techniques of extraction, processing and microscopic examination of BMAC (n = 18), where five studies looked at the evaluation of aspiration techniques (n = 5), augmentation of rotator cuff tears (n = 5), augmentation of tendo-achilles tendon (n = 1), treatment of gluteal tendon injuries (n = 1), management of elbow epicondylitis (n = 2), management of patellar tendinopathy (n = 1) and complications related to BMAC (n = 5). Multiple experimental studies investigated the use of BMAC for tendon repair; nonetheless, there are only limited clinical studies available in this field. Unfortunately, due to the scarcity of studies, which were mainly case series, the current level of evidence is weak. We strongly recommend further future randomised controlled studies in this field to allow scientists and clinicians make evidence-based conclusions.

A systematic review of the clinical applications and complications of bone marrow aspirate concentrate in management of bone defects and nonunions.

PURPOSE: Fracture healing encompasses a succession of dynamic multifactorial metabolic events, which ultimately re-establishes the integrity of the biomechanical properties of the bone. Up to 10% of the fractures occurring annually will need additional surgical procedures because of impaired healing. The aim of this article is to review the current literature regarding the use of bone marrow aspirate concentrate (BMAC) and its effectiveness in the management of bone defects. METHODS: We have included all published clinical literature investigating the development, techniques and applications of BMAC. Language, design and risk of bias did not deter the initial inclusion of any study. Our search was exclusively limited to studies involving human subjects. A PRISMA compliant search was carried out as published in 2009. This included the online databases: PubMed, EMBASE, clinical trial.gov and the Cochrane library from 1960 to the end of May 2015. MeSH terms used included: "Bone" AND "Marrow" AND "Aspirate" AND "Concentrate" AND "Bone Defects" AND "NONUNION". Eligible studies were independently appraised by two authors using the Critical Appraisal Skills Program checklist. For the purpose of narrative review, relevant studies were included irrespective of methodology or level of evidence. RESULTS: Thirty-four of the 103 (48 PubMed and 55 EMBASE) results yielded by the preliminary search were included. Exclusions included three duplicate records, six letters, 17 non-orthopaedics related studies and four records irrelevant to our search topic. The CASP appraisal confirmed a satisfactory standard of 31 studies. They all had clearly defined objectives, were well designed and conducted appropriately to meet them. The published studies reported the use of BMAC in non-union and fracture healing (15 studies), bone defects (nine studies), spine fusion (two studies), distraction osteogensis (two studies) and complications related to the use of BMAC (seven studies). CONCLUSIONS: Stem cells found in BMAC have the potential to self-renew, undertake clonal expansion and differentiate into different musculoskeletal tissues. The commercial processing of BMAC needs to be optimized in order to achieve a consistent end product, which will provide predicable and translatable results. The future potential of cell characterization in order to determine the optimum cell for repair/regeneration of bone also needs to be explored. LEVEL OF EVIDENCE: Systematic Review of minimum level IV studies.

The computed tomography-based fractal analysis of trabecular bone structure may help in detecting decreased quality of bone before urgent spinal procedures.

BACKGROUND CONTEXT: To date, no reliable method is available to determine the parameters of bone density based on the routine spinal computed tomography (CT) in the emergency setup. We propose the use of fractal analysis to detect patients with poor quality of bone before urgent or semi-urgent spinal procedures. PURPOSE: This study aimed to validate the hypothesis that the CT-based fractal analysis of the trabecular bone structure may help in detecting patients with poor quality of bone before urgent spinal procedures. STUDY DESIGN: This is a retrospective analysis of prospectively collected data. METHODS: Patients in whom the dual-energy x-ray absorptiometry (DEXA) scan and lumbar spine CT were performed at an interval of no more than 3 months were randomly selected from a prospectively collected database. Diagnostic axial CT scans of L2, L3, and L4 vertebrae were processed to determine the fractal dimension (FD) of the trabecular structure of each spinal level. Box-count method and ImageJ 1.49 software were used. The FD was compared with the results of the DEXA scan: bone mineral density (BMD) and T-score by mean of correlation coefficients. Receiver operating characteristic curve analysis was later performed to determine the cutoff value of FD. RESULTS: A total of 102 vertebral levels obtained from 35 patients (mean age 60±18 years; 29 female) were analyzed. The FD was significantly higher in the group of patients with decreased bone density (DBD) (T-score<-1.0) (1.67 vs. 1.43; p<.0001) and negatively correlated with BMD (R Spearman, -0.53; p<.0001) and T-score (-0.49; p<.0001). Receiver operating characteristic curve analysis revealed that a cutoff value of FD>1.53 indicates DBD (p<.0001; area under the ROC curve [AUC], 0.84; 95% confidence interval [CI], 0.76-0.91). CONCLUSIONS: This study shows that fractal analysis of the lumbar spine CT images may be used to determine bone density before spinal instrumentation (eg, metastatic or traumatic cord compression). Further prospective studies comparing results of the fractal analysis of CT scans with quantitative CT (qCT) are warranted.

A systematic review of the concept and clinical applications of Bone Marrow Aspirate Concentrate in Orthopaedics.

INTRODUCTION: Mesenchymal stem cells (MSC's) are believed to have multipotent plasticity with the capability to differentiate along multiple cell lineages such as cartilage, bone, tendon, muscle, and nerve. Such multipotency has the potential to play an important role in the repair and reconstruction of multiple tissues across a number of orthopaedic specialties. Bone marrow and fat are the most abundant and accessible source of MSC's with bone marrow aspirate the most commonly being reported to stimulate healing. METHODS: This review examines the current reported 20 Q2 clinical applications of bone marrow aspirate concentrate and its effectiveness. RESULTS: The published studies reported techniques of collection and preparation of BMAC in addition to its applications in a number of orthopaedic sub-specialities. Studies could be sub-categorised into: techniques of extraction, processing and microscopic examination of BMAC (31), reconstruction of osseous defects/non-union (20), treatment of avascular necrosis (9), repair of cartilage defects (8), treatment of sports injuries and tendon injury/repair (9), injection in regenerative therapy (4), treatment of spine conditions (4) including enhancing postoperative fusion and degenerative disc pathology and orthopaedic oncology (4). A few published studies combined the use of platelet-rich plasma (PRP) with BMAC (4) or compared them in different applications (5). CONCLUSIONS: BMAC has been used in bone, cartilage and tendon injuries with encouraging results.

The Basic Science of Bone Marrow Aspirate Concentrate in Chondral Injuries.

There has been great interest in bone marrow aspirate concentrate (BMAC) as a cost effective method in delivering mesenchymal stem cells (MSCs) to aid in the repair and regeneration of cartilage defects. Alongside MSCs, BMAC contains a range of growth factors and cytokines to support cell growth following injury. However, there is paucity of information relating to the basic science underlying BMAC and its exact biological role in supporting the growth and regeneration of chondrocytes. The focus of this review is the basic science underlying BMAC in relation to chondral damage and regeneration.

Bone Marrow Aspirate in the Treatment of Chondral Injuries.

The ability of mesenchymal stem cells (MSCs) to transdifferentiate into a desired cell lineage has captured the imagination of scientists and clinicians alike. The limited ability for chondrocytes to regenerate in chondral injuries has raised the concept of using MSCs to help regenerate and repair damaged tissue. The expansion of cells in a laboratory setting to be delivered back to the patient is too costly for clinical use in the present tough economic climate. This process is slow with due to the complexity of trying to imitate the natural environment and biological stimulation of chondral cell replication and proliferation. Bone marrow aspirate concentrate (BMAC) has the potential to provide an easily accessible and readily available source of MSCs with key growth factors that can be used in treating chondral injuries. This review summarizes the underlying basic science of MSCs and the therapeutic potential of BMAC.

Cementless Total Hip Replacement for the Management of Severe Developmental Dysplasia of the Hip in the Middle Eastern Population: A Prospective Analysis.

INTRODUCTION: In the Middle East, severe developmental dysplasia of the hip with subsequent high dislocation is often seen. We assessed the efficiency of total hip replacement (THR) with subtrochanteric shortening femoral osteotomy and trochanteric advancement in this population. METHODS: This prospective study assessed 25 female patients with symptomatic and severe (Crowe IV). Pre- and postoperative Harris hip score (HHS) and Oxford hip score (OHS) were performed alongside assessment of leg length discrepancy (LLD) and the ability to sit in a cross-legged position. RESULTS: The mean HHS and OHS improved pre-operatively at 1 and 10 years, respectively (p-value < 0.001). The mean postoperative LLD was 3 mm (0-8 mm). Functionally, 22/25 patients were able to sit cross-legged. None of the 25 hips underwent revision during this period. CONCLUSION: Total hip replacement with subtrochanteric shortening osteotomy in combination with trochanteric advancement is sufficient for the management of Crowe type IV hips in this population.

Bartsocas-Papas Syndrome: A Case Report and Review of the Literature.

Bartsocas-Papas syndrome (BPS) is an autosomal recessively inherited form of the popliteal pterygium syndrome characterized by severe growth retardation, midface hypoplasia, popliteal pterygia, and syndactyly. Almost all affected babies die in utero or infancy. We report the difficulties of reconstruction and ongoing plastic surgical management in an 8-year-old child with BPS. With increasingly sophisticated resuscitation and supportive techniques, it is possible that more patients with BPS will survive beyond the neonatal period. This raises new challenges with reconstruction highlighted by this case with a difficult balance between trying to overcome some of the profound effects of the syndrome versus diminishing quality of life for the child by repeated and often unsuccessful surgical procedures.

Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion.

Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

Peptide crystal simulations reveal hidden dynamics.

Molecular dynamics simulations of biomolecular crystals at atomic resolution have the potential to recover information on dynamics and heterogeneity hidden in X-ray diffraction data. We present here 9.6 µs of dynamics in a small helical peptide crystal with 36 independent copies of the unit cell. The average simulation structure agrees with experiment to within 0.28 Å backbone and 0.42 Å all-atom RMSD; a model refined against the average simulation density agrees with the experimental structure to within 0.20 Å backbone and 0.33 Å all-atom RMSD. The R-factor between the experimental structure factors and those derived from this unrestrained simulation is 23% to 1.0 Å resolution. The B-factors for most heavy atoms agree well with experiment (Pearson correlation of 0.90), but B-factors obtained by refinement against the average simulation density underestimate the coordinate fluctuations in the underlying simulation where the simulation samples alternate conformations. A dynamic flow of water molecules through channels within the crystal lattice is observed, yet the average water density is in remarkable agreement with experiment. A minor population of unit cells is characterized by reduced water content, 310 helical propensity and a gauche(-) side-chain rotamer for one of the valine residues. Careful examination of the experimental data suggests that transitions of the helices are a simulation artifact, although there is indeed evidence for alternate valine conformers and variable water content. This study highlights the potential for crystal simulations to detect dynamics and heterogeneity in experimental diffraction data as well as to validate computational chemistry methods.