Machine Learning Models Identify Inhibitors of New Delhi Metallo-ß-lactamase.

The worldwide spread of the metallo-ß-lactamases (MBL), especially New Delhi metallo-ß-lactamase-1 (NDM-1), is threatening the efficacy of ß-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

A comparison of revision rates for stemmed and stemless primary anatomic shoulder arthroplasty with all-polyethylene glenoid components: analysis from the Australian Orthopaedic Association National Joint Replacement Registry.

BACKGROUND: We compared the rate of all-cause revision of 2 classes of primary anatomic shoulder arthroplasty, stemmed (stTSA) and stemless (slTSA), undertaken with cemented all-polyethylene glenoid components. METHODS: A large national arthroplasty registry identified 2 cohort groups for comparison, stTSA and l undertaken for all diagnoses between January 1, 2011, and December 31, 2021. A subanalysis from January 1, 2017, allowed capturing of additional patient demographics including American Society of Anesthesiologists score, body mass index, and glenoid morphology. The cumulative percent revision (CPR) was determined using Kaplan-Meier estimates of survivorship and hazard ratios (HR) from Cox proportional hazard models adjusted for age and gender. RESULTS: Of the 7995 stTSA procedures, the CPR at 9 years was 5.6% (95% confidence interval [CI]: 5.0, 6.4), and for 3156 slTSA procedures, the CPR was 4.4% (95% CI: 3.6, 5.5). There was no significant difference in the rate of revision between the study groups (HR = 0.76 [95% CI: 0.51, 1.14], P = .189, adjusted for age, gender, humeral head size, humeral fixation, bearing surface, glenoid design, and mean surgeon volume [MSV]). There was an increased rate of revision for stTSA and slTSA undertaken with humeral head sizes <44 mm (stTSA <44 mm vs. 44-50 mm, HR = 1.56 [CI: 1.18, 2.08], P = .001; slTSA <44 mm vs. 44-50 mm, HR = 2.08 [CI: 1.32, 3.33], P = .001). MSV as a continuous predictor was not a revision risk to stTSA vs. slTSA, but categorically, a low MSV (<10 stTSA + slTSA cases per annum) was associated with a higher revision rate for stTSA (10-20 cases/yr vs. <10 cases/yr, HR = 0.72 [CI: 0.55, 0.95], P = .019) but was not in slTSA. Revision rates were increased for stTSA with non-crosslinked polyethylene (XLPE) glenoids vs. XPLE after 2 years (HR = 2.20 [CI: 1.57, 3.08], P < .001) but did not significantly differ for slTSA. Metal/XPLE (humeral/glenoid) bearing surface of stTSA rate of revision was not different from each combination of slTSA bearing surface. Instability/dislocation was a revision risk for slTSA vs. stTSA (HR = 1.93 [CI: 1.28, 2.91], P = .001), but from 2017, neither of American Society of Anesthesiologists score, body mass index, and glenoid morphology changed the rate of revision. CONCLUSIONS: Revision rates of stTSA and slTSA did not significantly differ and were associated with humeral head size but not patient characteristics. Surgeon inexperience of anatomic shoulder arthroplasty and non-XLPE glenoids were risk factors for stTSA revision but not slTSA. The metal/XLPE stTSA rate of revision was not found to differ significantly from slTSA regardless of polyethylene or humeral head bearing type. Revision for instability/dislocation was more common for slTSA.

Distal humeral hemiarthroplasty compared to total elbow replacement for distal humeral fractures: a registry analysis of 906 procedures.

BACKGROUND: Total elbow replacement (TER) is an accepted treatment for complex intra-articular distal humerus fractures in elderly patients. Distal humeral hemiarthroplasty (HA) is also a potential surgical option for unreconstructable fractures and avoids the concerns regarding mechanical wear and functional restrictions associated with TER. In the current literature, there are limited data available to compare the revision rates of HA and TER for the treatment of fracture. We used data from a large national arthroplasty registry to compare the outcome of HA and TER undertaken for fracture/dislocation and to assess the impact of demographics and implant choice on revision rates. METHODS: Data obtained from the Australian Orthopaedic Association National Joint Replacement Registry from May 2, 2005, to December 31, 2021, included all procedures for primary elbow replacement with primary diagnosis of fracture or dislocation. The analyses were performed using Kaplan-Meier estimates of survivorship and hazard ratios (HRs) from Cox proportional hazards models. RESULTS: There were 293 primary HA and 631 primary TER procedures included. The cumulative percentage revision (CPR) rate at 9 years was 9.7% for HA (95% confidence interval [CI] 6.0, 15.7), and 11.9% (95% CI 8.5, 16.6) for TER. When adjusted for age and gender, there was a significantly higher risk of revision after 3 months for TER compared to HA (HR 2.47, 95% CI 1.22, 5.03, P = .012). There was no difference in the rate of revision for patients aged <55 years or ≥75 years when HA and TER procedures were compared. In primary TER procedures, loosening was the most common cause of revision (3.6% of primary TER procedures), and the most common type of revision in primary TER involved revision of the humeral component only (2.6% of TER procedures). TER has a higher rate of first revision for loosening compared to HA (HR 4.21, 95% CI 1.29, 13.73; P = .017). In HA procedures, instability (1.7%) was the most common cause for revision. The addition of an ulna component was the most common type of revision (2.4% of all HA procedures). CONCLUSION: For the treatment of distal humerus fractures, HA had a lower revision rate than TER after 3 months when adjusted for age and gender. Age <55 or ≥75 years was not a risk factor for revision when HA was compared to TER. Loosening leading to revision is more prevalent in TER and increases with time. In HA, the most common type of revision involved addition of an ulna component with preservation of the humeral component.

Comparable low revision rates of stemmed and stemless total anatomic shoulder arthroplasties after exclusion of metal backed glenoid components: a collaboration between the Australian and Danish national shoulder arthroplasty registries.

BACKGROUND: The stemmed anatomical total shoulder arthroplasty is the gold standard in the treatment of glenohumeral osteoarthritis. However, the use of stemless total shoulder arthroplasties has increased in recent years. The number of revision procedures are relatively low and therefore it has been recommended that national joint replacement registries should collaborate when comparing revision rates. Therefore, we aimed to compare the revision rates of stemmed and stemless TSA used for the diagnosis of glenohumeral osteoarthritis using data from both the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) and the Danish Shoulder Arthroplasty Registry (DSR). METHODS: We included all patients who were registered in the AOANJRR and the DSR from January 1 2012 to December 2021 with an anatomical total shoulder arthroplasty used for osteoarthritis. Revision for any reason was used as the primary outcome. We used the Kaplan-Meier method to illustrate the cumulative revision rates and a multivariate cox regression model to calculate the hazard ratios. All analyses were performed separately for data from AOANJRR and DSR, and the results were then reported using a qualitative approach. RESULTS: 13066 arthroplasties from AOANJRR and 2882 arthroplasties from DSR were included. The hazard ratio for revision of stemmed TSA with stemless TSA as reference, adjusted for age and gender, was 1.67 (95% CI 1.34-2.09, p<0.001) in AOANJRR and 0.57 (95% CI 0.36-0.89, p=0.014) in DSR. When including glenoid type and fixation, surface bearing (only in AOANJRR) and hospital volume in the cox regression model the hazard ratio for revision of stemmed TSA compared to stemless TSA was 1.22 (95% CI 0.85-1.75, p=0.286) in AOANJRR and 1.50 (95% CI 0.91-2.45, p=0.109) in DSR. The adjusted hazard ratio for revision of total shoulder arthroplasties with metal backed glenoid components compared to all-polyethylene glenoid components was 2.54 (95% CI 1.70-3.79, p < 0.001) in AOANJRR and 4.1 (95% CI 1.92-8.58, p<0.001) in DSR. CONCLUSION: Based on data from two national shoulder arthroplasty registries, we found no significant difference in risk of revision between stemmed and stemless total shoulder arthroplasties after adjusting for the type of glenoid component. We advocate that metal-backed glenoid components should be used with caution and not on a routine basis.

Post-Activation Performance Enhancement of Punch Force and Neuromuscular Performance in Amateur Boxing: Toward a More Individualized and "Real-World" Approach.

ABSTRACT: Finlay, MJ, Greig, M, Bridge, CA, and Page, RM. Post-activation performance enhancement of punch force and neuromuscular performance in amateur boxing: Toward a more individualized and "real-world" approach. J Strength Cond Res 38(6): 1063-1071, 2024-Previous research on post-activation performance enhancement has been applied in several sporting settings, although this is lacking in a boxing setting. This study explored the effectiveness of 2 upper-body conditioning activities in improving punch-specific performance during an amateur boxing-simulated bout. On 3 separate occasions, 10 male senior elite amateur boxers performed the following conditioning activities before a boxing-specific simulation protocol: isometric (ISO) punch, elastic resistance (ER) punch, and a control trial. Boxers performed maximal punches against a vertically mounted force plate, and countermovement jumps (CMJ) at baseline, before round 1, after each round, and 4 minutes after the simulation. Both conditioning activities, but not the control trial, produced small worthwhile increases (effect size ≥ 0.20; equal to or greater than the smallest worthwhile change) in punch force, although worthwhile increases in rate of force development were limited to the cross during the ISO trial. No group-based improvements in CMJ performance were observed. Individual analysis revealed that 6 boxers improved punch-specific performance to the greatest extent in the ISO trial; in contrast, only 1 boxer did so in the ER trial. Three boxers exhibited similar performance increases across trials. In conclusion, both conditioning activities may be applied to an amateur boxer's warm-up to acutely enhance punch-specific performance. The ISO conditioning activity seems most effective; however, the interindividual variability suggests a need for protocols to be individualized to each athlete. The conditioning activities in the present study may be applied to sparring, competitive bouts, or to other combat sports.

Emergency presentations for farm-related injuries in older adults residing in south-western Victoria, Australia.

INTRODUCTION: Farm workers are at high risk for injuries, and epidemiological data are needed to plan resource allocation. OBJECTIVE: This study identified regions with high farm-related injury rates in the Barwon South West region of Victoria, Australia, for residents aged ≥50 yr. DESIGN: Retrospective synthesis using electronic medical records of emergency presentations occurring during 2017-2019 inclusive for Local Government Areas (LGA) in the study region. For each LGA, age-standardised incidence rates (per 1000 population/year) were calculated. FINDINGS: For men and women combined, there were 31 218 emergency presentations for any injury, and 1150 (3.68%) of these were farm-related. The overall age-standardised rate for farm-related injury presentations was 2.6 (95% CI 2.4-2.7); men had a higher rate than women (4.1, 95% CI 3.9-4.4 versus 1.2, 95% CI 1.0-1.3, respectively). For individual LGAs, the highest rates of farm-related emergency presentations occurred in Moyne and Southern Grampians, both rural LGAs. Approximately two-thirds of farm-related injuries occurred during work activities (65.0%), and most individuals arrived at the hospital by transport classified as "other" (including private car, 83.3%). There were also several common injury causes identified: "other animal related injury" (20.2%), "cutting, piercing object" (19.5%), "fall ⟨1 m" (13.1%), and "struck by or collision with object" (12.5%). Few injuries were caused by machinery (1.7%) and these occurred mainly in the LGA of Moyne (65%). DISCUSSION AND CONCLUSION: This study provides data to inform future research and resource allocation for the prevention of farm-related injuries.

The directed evolution of NDM-1.

ß-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward ß-lactam antibiotics is the production of ß-lactamases. These enzymes are capable of hydrolyzing the ß-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- ß-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- ß-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 ß-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1-TRAF6-transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

Forecasting the future burden of primary total shoulder replacement in Australia.

OBJECTIVE: To forecast the number of primary total shoulder replacements (TSR) in Australia to the year 2035, and associated costs. METHODS: De-identified TSR data for 2009-2019 were obtained from the Australian Orthopaedic Association National Joint Replacement Registry. Population data, including population projections to 2035, were obtained from the Australian Bureau of Statistics. Three forecasting scenarios were used: constant TSR rates from 2019 onwards (Scenario 1, conservative); continued growth in TSR rates using negative binomial regression (Scenario 2, exponential); and continued growth using negative binomial regression with monotone B-splines (Scenario 3, moderate). Healthcare costs were estimated using TSR projections and average procedure costs, inflated to 2035 Australian dollars. RESULTS: The use of TSR increased by 242% in Australia from 2009 to 2019 (from 1983 to 6789 procedures for people ≥40 years). Under Scenario 1, the incidence of TSR is conservatively projected to rise to 9676 procedures by 2035 (43% increase from 2019), at a cost of $AUD 312.6 million to the health system. Under Scenario 2, TSR incidence would increase to 45,295 procedures by 2035 (567% increase), costing $AUD 1.46 billion. Under Scenario 3, 28,257 TSR procedures are forecast in 2035 (316% increase) at a cost of $913 million. CONCLUSIONS: Recent growth in TSR likely relates to prosthesis improvements, greater surgeon proficiency, and expanded clinical indications. Under moderate and exponential scenarios that consider rising TSR rates and population projections, Australia would face three- to five-fold growth in procedures by 2035. This would have profound implications for the healthcare budget, clinical workforce, and infrastructure.

Real-time bio-layer interferometry ubiquitination assays as alternatives to western blotting.

Ubiquitination is a crucial cellular pathway enabling normal cellular functions. Abnormalities in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases. Efforts to screen and develop small molecule inhibitors targeting portions of the ubiquitination cascade require rapid and robust methods for detecting ubiquitination. Enormous efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, NMR, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming and difficult to use when seeking fine-grained time course experiments. Here we present the use of bio-layer interferometry to rapidly assay ubiquitination in real-time. An E3 ligase auto-ubiquitination system and a substrate ubiquitination assay have been applied as tests for the newly developed assay. The developed BLI ubiquitination assay provides one-second time resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously, and reagent concentrations are identical to those used by traditional western blot-based ubiquitination assays. The developed BLI ubiquitination assay is a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner.

Outcomes after Genicular Artery Embolization Vary According to the Radiographic Severity of Osteoarthritis: Results from a Prospective Single-Center Study.

This prospective single-center study compared outcomes after genicular artery embolization (GAE) in people with mild radiographic knee osteoarthritis (OA) (n = 11) versus moderate to severe radiographic knee OA (n = 22). The mild OA group was older and had experienced symptoms for less time (P < .05). All participants underwent complete embolization of neovessels from genicular arteries. The primary outcome was the proportion of responders at 6 months according to predetermined improvements in pain, function, and/or global change. Results indicated that a higher proportion of participants (n = 9, 81.8%) with mild OA fulfilled responder criteria after treatment compared with people with moderate to severe OA (n = 8, 36.4%) (P = .014). Secondary outcomes for pain, quality of life, and global change were also better in the mild OA group (P < .05). No serious adverse events occurred, including the absence of osteonecrosis on magnetic resonance imaging. The study demonstrated that outcomes after GAE varied according to the severity of baseline radiographic OA.

Polymer modification of SARS-CoV-2 spike protein impacts its ability to bind key receptor.

The global spread of SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has caused the loss of many human lives and severe economic losses. SARS-CoV-2 mediates its infection in humans via the spike glycoprotein. The receptor binding domain of the SARS-CoV-2 spike protein binds to its cognate receptor, angiotensin converting enzyme-2 (ACE2) to initiate viral entry. In this study, we examine how polymer modification of the spike protein receptor binding domain impacts binding to ACE2. The horseradish peroxidase conjugated receptor binding domain was modified with a range of polymers including hydrophilic N,N-dimethylacrylamide, hydrophobic N-isopropylacrylamide, cationic 3-(N,N-dimethylamino)propylacrylamide, and anionic 2-acrylamido-2-methylpropane sulfonic acid polymers. The effect of polymer chain length was observed using N,N-dimethylacrylamide polymers with degrees of polymerization of 5, 10 and 25. Polymer conjugation of the receptor binding domain significantly reduced the interaction with ACE2 protein, as determined by an enzyme-linked immunosorbent assay. Stability analysis showed that these conjugates remained highly stable even after seven days incubation at physiological temperature. Hence, this study provides a detailed view of the effect specific type of modification using a library of polymers with different functionalities in interrupting RBD-ACE2 interaction.

Survivorship of shoulder arthroplasty in young patients with osteoarthritis: an analysis of the Australian Orthopaedic Association National Joint Replacement Registry.

BACKGROUND: The treatment of shoulder osteoarthritis in the young patient remains challenging. The higher functional demands and higher expectations of the young patient cohort are often coupled with increased failure and revision rates. Consequently, shoulder surgeons are faced with a unique challenge with implant selection. The aim of this study was to compare the survivorship and reasons for revision of 5 classes of shoulder arthroplasty in patients aged <55 years with a primary diagnosis of osteoarthritis by use of data from a large national arthroplasty registry. METHODS: The study population included all primary shoulder arthroplasty procedures undertaken for osteoarthritis in patients aged <55 years and reported to the registry between September 1999 and December 2021. Procedures were grouped into the following classes: total shoulder arthroplasty (TSA), hemiarthroplasty resurfacing (HRA), hemiarthroplasty stemmed metallic head (HSMH), hemiarthroplasty stemmed pyrocarbon head (HSPH), and reverse total shoulder arthroplasty (RTSA). The outcome measure was the cumulative percent revision, which was defined using Kaplan-Meier estimates of survivorship to describe the time to the first revision. Hazard ratios (HRs) were calculated from Cox proportional hazards models, adjusting for age and sex, to compare revision rates among groups. RESULTS: There were 1564 shoulder arthroplasty procedures in patients aged <55 years, of which 361 (23.1%) were HRA, 70 (4.5%) were HSMH, 159 (10.2%) were HSPH, 714 (45.7%) were TSA, and 260 (16.6%) were RTSA. HRA had a higher rate of revision than RTSA after 1 year (HRA = 2.51 (95% CI 1.30, 4.83), P = .005), with no difference prior to that time. In addition, HSMH had a higher rate of revision than RTSA for the entire period (HR, 2.69 [95% confidence interval, 1.28-5.63], P = .008). There was no significant difference in the rate of revision for HSPH and TSA when they were compared with RTSA. Glenoid erosion was the most common cause of revision for HRA (28.6% of revisions) and HSMH (50%). Instability/dislocation was the leading cause of revision for RTSA (41.7%) and HSPH (28.6%), and for TSA, the majority of revisions were for either instability/dislocation (20.6%) or loosening (18.6%). CONCLUSION: These results should be interpreted within the context of the lack of availability of long-term data on RTSA and HSPH stems. RTSA outperforms all implants regarding revision rates at mid-term follow-up. The high early dislocation rate associated with RTSA, as well as the lack of revision options available to address this, indicates that careful selection of patients and a greater appreciation of anatomic risk factors are needed in the future.

Obesity is associated with an increased risk of undergoing shoulder arthroplasty in Australia.

BACKGROUND: The aim of this study was to examine the incidence of overweight and obesity in patients undergoing primary total shoulder arthroplasty (TSA) for osteoarthritis (OA) in Australia compared to the incidence of obesity in the general population. MATERIALS AND METHODS: A 2017/18 cohort consisting of 2997 patients from the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) who underwent TSA were compared with matched controls from the Australian Bureau of Statistics (ABS) National Health Survey from the same time period. The 2 groups were analyzed according to body mass index (BMI) category, sex, and age. RESULTS: According to the 2017/18 National Health Survey, 35.6% of Australian adults were overweight and 31.3% were obese. Of the primary TSA cases performed, 34.9% were overweight and 50.1% were obese. The relative risk of requiring TSA for OA increased with increasing BMI category. Class III obese females, aged 55-64, were 8.6 times more likely to receive a TSA compared to their normal weight counterparts. Males in the same age and BMI category were 2.5 times more likely. Class III obese patients underwent TSA 4 years (female) and 6 years (male) younger than their normal weight counterparts. CONCLUSION: Obesity significantly increases the risk of requiring TSA. The association appears to be particularly strong for younger females.

The influence of fixture congestion on physical performance response to U23 soccer match-play.

This study sought to examine differences in measures of intense periods of physical performance during competitive match-play, between one-match (1 match weeks) and two-match microcycles (2 match week). 1, 3 and 5 min "peak" and mean averages for total distance (TD), high-speed running (HSR) and metabolic power (MP) were analysed for each 15 min period of match-play. Linear mixed models were employed to examine the differences in dependent variables for each method of measurement between the 1 and 2 game microcycles. No differences were reported for "peak" values for all epoch lengths, however, results revealed significantly reduced "average" values, during periods of fixture congestion, for 1, 3 and 5 min epochs for average TD, and 3 min epochs for average HSR towards the end of the match (75-90 min split). There was, however, a trend for the opposite response to occur in the 60-75-min period. The current data suggests that players potentially display altered pacing strategies during periods of fixture congestion, with these observed responses being dependent on sampling method and epoch length.

Different Conformations Revealed by NMR Underlie Resistance to Ceftazidime/Avibactam and Susceptibility to Meropenem and Imipenem among D179Y Variants of KPC ß-Lactamase.

ß-Lactamase-mediated resistance to ceftazidime-avibactam (CZA) is a serious limitation in the treatment of Gram-negative bacteria harboring Klebsiella pneumoniae carbapenemase (KPC). Herein, the basis of susceptibility to carbapenems and resistance to ceftazidime (CAZ) and CZA of the D179Y variant of KPC-2 and -3 was explored. First, we determined that resistance to CZA in a laboratory strain of Escherichia coli DH10B was not due to increased expression levels of the variant enzymes, as demonstrated by reverse transcription PCR (RT-PCR). Using timed mass spectrometry, the D179Y variant formed prolonged acyl-enzyme complexes with imipenem (IMI) and meropenem (MEM) in KPC-2 and KPC-3, which could be detected up to 24 h, suggesting that IMI and MEM act as covalent ß-lactamase inhibitors more than as substrates for D179Y KPC-2 and -3. This prolonged acyl-enzyme complex of IMI and MEM by D179Y variants was not observed with wild-type (WT) KPCs. CAZ was studied and the D179Y variants also formed acyl-enzyme complexes (1 to 2 h). Thermal denaturation and differential scanning fluorimetry showed that the tyrosine substitution at position 179 destabilized the KPC ß-lactamases (KPC-2/3 melting temperature [Tm] of 54 to 55°C versus D179Y Tm of 47.5 to 51°C), and the D179Y protein was 3% disordered compared to KPC-2 at 318 K. Heteronuclear 1H/15N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy also revealed that the D179Y variant, compared to KPC-2, is partially disordered. Based upon these observations, we discuss the impact of disordering of the Ω loop as a consequence of the D179Y substitution. These conformational changes and disorder in the overall structure as a result of D179Y contribute to this unanticipated phenotype.

Fluorescence dequenching assay for the activity of TEV protease.

Tobacco etch virus (TEV) protease is a widely used protease for fusion tag cleavage. Despite its widespread usage, an assay to quickly and easily quantify its activity in laboratory settings is still lacking. Thus, researchers may encounter inefficient cleavage of the desired fusion proteins due to poor activity of a given TEV protease preparation. Here, we describe the development and implementation of a fluorescence dequenching-based assay to quantify TEV protease activity and assess kinetic parameters. The peptide substrate used in this assay consists of a C-terminal TAMRA fluorophore, an N-terminal fluorescein fluorophore, and the canonical TEV protease recognition sequence. The assay is based on a reduction of fluorescence quenching of fluorescein upon cleavage by TEV protease. The substrate peptide was studied spectroscopically to assess feasibility and to propose a plausible mechanism of the assay. The assay was optimized and applied to obtain rapid assessments of TEV protease activity in purified samples and crude lysate extracts. The kinetic data obtained from improved TEV protease variants were compared with a traditional SDS-PAGE assay. Finally, the assay was applied to determine the optimum pH for TEV protease. Further, the study found that the assay is a rapid and simple approach to quantify TEV protease activity. The findings of the assay on crude lysate extracts, activity assay of TEV protease variants, and assessment of optimum pH for TEV protease reactions demonstrate the robust utility of the assay.

Investigating the Impact of Polymer Length, Attachment Site, and Charge on Enzymatic Activity and Stability of Cellulase.

The thermophilic cellulase Cel5a from Fervidobacterium nodosum (FnCel5a) was conjugated with neutral, cationic, and anionic polymers of increasing molecular weights. The enzymatic activity toward an anionic soluble cellulose derivative, thermal stability, and functional chemical stability of these bioconjugates were investigated. The results suggest that increasing polymer chain length for polymers compatible with the substrate enhances the positive impact of polymer conjugation on enzymatic activity. Activity enhancements of nearly 100% were observed for bioconjugates with N,N-dimethyl acrylamide (DMAm) and N,N-dimethyl acrylamide-2-(N,N-dimethylamino)ethyl methacrylate (DMAm/DMAEMA) due to proposed polymer-substrate compatibility enabled by potential noncovalent interactions. Double conjugation of two functionally distinct polymers to wild-type and mutated FnCel5a using two conjugation methods was achieved. These doubly conjugated bioconjugates exhibited similar thermal stability to the unmodified wild-type enzyme, although enzymatic activity initially gained from conjugation was lost, suggesting that chain length may be a better tool for bioconjugate activity modulation than double conjugation.

Reduced Revision Rates in Total Shoulder Arthroplasty With Crosslinked Polyethylene: Results From the Australian Orthopaedic Association National Joint Replacement Registry.

BACKGROUND: Loss of glenoid fixation is a key factor affecting the survivorship of primary total shoulder arthroplasty (TSA). It is not known whether the lower revision rates associated with crosslinked polyethylene (XLPE) compared with those of non-XLPE identified in hip and knee arthroplasty apply to shoulder arthroplasty. QUESTIONS/PURPOSES: We used data from the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) to compare the revision rates of primary stemmed anatomic TSA using XLPE to procedures using non-XLPE. In patients receiving a primary stemmed anatomic TSA for osteoarthritis, we asked: (1) Does the rate of revision or reason for revision vary between XLPE and non-XLPE all-polyethylene glenoid components? (2) Is there any difference in the revision rate when XLPE is compared with non-XLPE across varying head sizes? (3) Is there any difference in survival among prosthesis combinations with all-polyethylene glenoid components when they are used with XLPE compared with non-XLPE? METHODS: Data were extracted from the AOANJRR from April 16, 2004, to December 31, 2020. The AOANJRR collects data on more than 97% of joint replacements performed in Australia. The study population included all primary, stemmed, anatomic TSA procedures performed for osteoarthritis using all-polyethylene glenoid components. Procedures were grouped into XLPE and non-XLPE bearing surfaces for comparison. Of the 10,102 primary stemmed anatomic TSAs in the analysis, 39% (3942 of 10,102) used XLPE and 61% (6160 of 10,102) used non-XLPE. There were no differences in age, gender, or follow-up between groups. Revision rates were determined using Kaplan-Meier estimates of survivorship to describe the time to the first revision, with censoring at the time of death or closure of the database at the time of analysis. Revision was defined as removal, replacement, or addition of any component of a joint replacement. The unadjusted cumulative percent revision after the primary arthroplasty (with 95% confidence intervals [CIs]) was calculated and compared using Cox proportional hazard models adjusted for age, gender, fixation, and surgeon volume. Further analyses were performed stratifying according to humeral head size, and a prosthesis-specific analysis adjusted for age and gender was also performed. This analysis was restricted to prosthesis combinations that were used at least 150 times, accounted for at least four revisions, had XLPE and non-XLPE options available, and had a minimum of 3 years of follow-up. RESULTS: Non - XLPE had a higher risk of revision than XLPE after 1.5 years (HR 2.3 [95% CI 1.6 to 3.1]; p < 0.001). The cumulative percent revision at 12 years was 5% (95% CI 4% to 6%) for XLPE and 9% (95% CI 8% to 10%) for non-XLPE. There was no difference in the rate of revision for head sizes smaller than 44 mm. Non-XLPE had a higher rate of revision than XLPE for head sizes 44 to 50 mm after 2 years (HR 2.3 [95% CI 1.5 to 3.6]; p < 0.001) and for heads larger than 50 mm for the entire period (HR 2.2 [95% CI 1.4 to 3.6]; p < 0.001). Two prosthesis combinations fulfilled the inclusion criteria for the prosthesis-specific analysis. One had a higher risk of revision when used with non-XLPE compared with XLPE after 1.5 years (HR 3.7 [95% CI 2.2 to 6.3]; p < 0.001). For the second prosthesis combination, no difference was found in the rate of revision between the two groups. CONCLUSION: These AOANJRR data demonstrate that noncrosslinked, all-polyethylene glenoid components have a higher revision rate compared with crosslinked, all-polyethylene glenoid components when used in stemmed anatomic TSA for osteoarthritis. As polyethylene type is likely an important determinant of revision risk, crosslinked polyethylene should be used when available, particularly for head sizes larger than 44 mm. Further studies will need to be undertaken after larger numbers of shoulder arthroplasties have been performed to determine whether this reduction in revision risk associated with XLPE bears true for all TSA designs. LEVEL OF EVIDENCE: Level III, therapeutic study.

The influence of angle-specific torque of the knee flexors and extensors on the angle-specific dynamic control ratio in professional female soccer players.

The purpose of this study was to assess whether dynamic torque ratios (DCR) from isokinetic strength assessments of eccentric knee flexors (eccKF) and concentric knee extensors (conKE) display differences when stratified into specific angle-specific DCR (DCRAST) groups. Fifty-two professional female soccer players (age 21.30 ± 4.44 years; height 166.56 ± 5.17 cm; mass 61.55 ± 5.73 kg) from the English Women's Super League completed strength assessments of both lower limbs on an isokinetic dynamometer at 60°âˆ™s-1. Angle-specific torque (AST) were used to calculate DCRAST to create sub-groups using clustering algorithms. The results identified for the dominant side that the Medium DCRAST group elicited significantly higher conKE AST when compared to Low and High DCRAST groups at increased knee extension (P ≤ 0.05). For the non-dominant side, the High DCRAST group had significantly higher and lower eccKF and conKE AST compared to the Low DCRAST group at increased knee extension (P ≤ 0.05). This study highlights that the inclusion of AST data may subsequently help practitioners to prescribe exercise that promotes strength increases at targeted joint angles. In turn, these approaches can be used to help reduce injury risk, identify rehabilitation responses and help inform return to play.