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Background Prosthetic joint infection (PJI) of the knee carries significant morbidity, mortality, and economic cost. We hypothesize that obese and poor medical hosts will have a significant and additive increase in failure rate undergoing 2-stage revision total knee arthroplasty for PJI. Methods All 2-stage revision total knee arthroplasty procedures for PJI performed at one institution were identified between 2005 and 2020. In total, 144 patients were included and defined as success or failure based on published criteria regarding infection eradication, further intervention, or mortality. The American Society of Anesthesiologists score and the Charlson Comorbidity Index were utilized to assess host grade. Patient, surgical, and microbiologic variables were investigated with univariable and multivariable analysis to explore association with risk of failure. Results In the cohort, 32.4% of patients failed with mean follow-up of 5.1 years. In multivariable analysis, the number of major operations requiring arthrotomy and implantation of new material between the primary and first stage, host grade, and elevated body mass index were the major contributors to failure. Combining these factors, with body mass index >30 and 2 or more major operations, the failure rate increased to 76.5% and 71.4% respectively for American Society of Anesthesiologists score 3 (P ≤ .001) and Charlson Comorbidity Index ≥2 (P ≤ .001). Conclusion In this cohort, multiple major operations between the primary and first stage, host grade, and obesity were the major contributors to failure. When combining these factors, patients had an additive increase in failure rate. Treatments such as amputation or less invasive options and suppression should be discussed and considered in these patients.

Summary The broad-spectrum antibiotic d-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analogue of d-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and d-Ala:d-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5’-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes ‘off-target’ two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5–3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.

Background: The aim of this study is to update the 10-year follow-up survivorship and metal ions levels of a cohort of metal-on-metal (MoM) hip resurfacing (HR) and large-diameter-head (LDH) total hip arthroplasty (THA). Methods: The study is a retrospective analysis of prospectively collected data that compared the outcomes of 24 MoM HR (21 patients) and 15 (11 patients) modular LHD MoM THA at >10 years follow-up. Baseline characteristics as well as intraoperative and postoperative information were collected, including complications, revisions, clinical and radiographic outcomes, and serum metal ions level (Cobalt, Chromium). Metal ion levels were compared using a two-tailed unpaired t-test and Wilcoxon signed-rank test (jamovi v2.3.3.0, Sydney, NSW, AU). Results: No significant differences were detected in gender, BMI, and ASA score between the two groups. Patients in the modular THA group were significantly older (57 years vs. 46 years; p < 0.05). The HR overall survivorship was 91.7% (22 of 24 hips) with survivorship from implant failure and/or aseptic loosening and/or metal debris related 100% of problems. The modular THA overall survivorship was 86.7% (13 of 15 hips) with survivorship from implant aseptic loosening and metal ions complications of 93.4% (14 of 15 hips). No significant difference was noted when comparing clinical outcomes. Metal ions were significantly lower in the HR group (Co 25.8 nmol/L vs. 89 nmol/L; p < 0.001–Cr 33.5 nmol/L vs. 55.2 nmol/L; p = 0.026). Conclusion: Both implants reported excellent and comparable clinical outcomes at >10 years follow-up. The Adept HR reported remarkable survivorship, in line with the registry data, proving once again its reliability in young active males. The modular LDH THA, despite being discontinued, presented higher reliability and a lower failure rate when compared with similar withdrawn MoM implants. Trunnionosis did not appear to be a significant problem in this particular modular design.

Mycobacterium tuberculosis resides in the lungs in various lesion types with unique microenvironmental conditions. This diversity is in line with heterogeneous disease progression and divergent drug efficiency. Fluorescent reporter strains can be used to decipher the micromilieu and to guide future treatment regimens. Current reporters using replicating plasmids, however, are not suitable for long-term mouse infections or studies in non-human primates. Using a combination of recombinant DNA and protein optimization techniques, we have developed reporter strains based on integrative plasmids, which exhibit stimulus-response characteristics and fluorescence intensities comparable to those based on replicating plasmids. We successfully applied the concepts by constructing a multi-color reporter strain able to detect simultaneous changes in environmental pH, Mg2+ concentrations, and protein expression levels.

Tuberculosis (TB) is one of the primary causes of deaths due to infectious diseases. The current TB regimen is long and complex, failing of which leads to relapse and/or the emergence of drug resistance. There is a critical need to understand the mechanisms of resistance development. With increasing drug pressure, Mycobacterium tuberculosis (Mtb) activates various pathways to counter drug-related toxicity. Signaling modules steer the evolution of Mtb to a variant that can survive, persist, adapt, and emerge as a form that is resistant to one or more drugs. Recent studies reveal that about 1/3rd of the annotated Mtb proteome is modified post-translationally, with a large number of these proteins being essential for mycobacterial survival. Post-translational modifications (PTMs) such as phosphorylation, acetylation, and pupylation play a salient role in mycobacterial virulence, pathogenesis, and metabolism. The role of many other PTMs is still emerging. Understanding the signaling pathways and PTMs may assist clinical strategies and drug development for Mtb. In this review, we explore the contribution of PTMs to mycobacterial physiology, describe the related cellular processes, and discuss how these processes are linked to drug resistance. A significant number of drug targets, InhA, RpoB, EmbR, and KatG, are modified at multiple residues via PTMs. A better understanding of drug-resistance regulons and associated PTMs will aid in developing effective drugs against TB.

Mycobacterium tuberculosis is a facultative anaerobe and its characteristic pathological hallmark, the granuloma, exhibits hypoxia in humans and in most experimental models. Thus the host and bacillary adaptation to hypoxia is of central importance in understanding pathogenesis and thereby to derive new drug treatments and vaccines.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations., D-cycloserine (DCS) has been used for decades to treat Mycobacterium tuberculosis (Mtb) but resistance is rarely observed in clinical isolates. Here, the authors report ultra-low rate of emergence of resistance mutations as the underlying mechanism of DCS resistance evasion in Mtb.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated whyMycobacterium tuberculosisfails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysisin vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms.One Sentence SummaryWe show that the lack of D-cycloserine resistance inMycobacterium tuberculosisis due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

In July 2008, the Western Australia (WA) Department of Health embarked on a landmark 5-year project to implement a sustainable comprehensive health-system-wide Patient Blood Management Program. Fundamentally, it was a quality and safety initiative, which also had profound resource and economic implications. Unsustainable escalating direct and indirect costs of blood, potentially severe blood shortages due to changing population dynamics, donor deferrals, loss of altruism, wide variations in transfusion practice and growing knowledge of transfusion limitations and adverse outcomes necessitate a paradigm shift in the management of anemia and blood loss. The concept of patient-focused blood management is proving to be an effective force for change. This approach has now evolved to embrace comprehensive hospital-wide Patient Blood Management Programs. These programs show significant reductions in blood utilisation, and costs while achieving similar or improved patient outcomes. The WA Program is achieving these outcomes across a health jurisdiction in a sustained manner.

Case: We present a case of systemic cobalt toxicity secondary to third-body wear of a cobalt-chromium (CoCr) femoral head following revision of a fractured ceramic bearing. Removal of the CoCr head was followed by resolution of much of the systemic symptoms. Conclusion: This case supports previous reports of cobalt toxicity secondary to catastrophic third-body wear of a CoCr femoral head following a fractured ceramic bearing. It also demonstrates the potential reversibility of many systemic sequelae associated with cobalt toxicity. To our knowledge, this case represents the highest documented blood cobalt level (45,840 nmol/L).

Redirective, periacetabular osteotomies (PAO) represent a group of surgical procedures for treatment of developmental dysplasia of the hip (DDH) in skeletally mature and immature patients. The ultimate goal of all procedures is to reduce symptoms, improve function and delay or prevent progression of osteoarthritis. During the last two decades, the understanding of the underlying pathomechanisms has continuously evolved. This is mainly attributable to the development of the femoroacetabular impingement concept that has increased the awareness of the underlying three-dimensional complexity associated with DDH. With increasing knowledge about the pathobiomechanics of dysplastic hips, diagnostic tools have improved allowing for sophisticated preoperative analyses of the morphological and pathobiomechanical features, and early recognition of degenerative changes, which may alter the long-term outcome. As redirective, PAO are technically demanding procedures, preoperative planning is crucial to avoid intraoperative obstacles and to sufficiently address the patient-specific deformity. Although conventional radiography has been used for decades, it has not lost its primary role in the diagnostic work-up of patients with DDH. Furthermore, an increasing number of modern imaging techniques exists allowing for assessment of early cartilage degeneration (biochemical magnetic resonance imaging) as well as 3D planning and computer-based virtual treatment simulation of PAO. This article reviews the literature with regard to the current concepts of imaging of DDH, preoperative planning and treatment recommendations for redirective, PAO.

Directed enzyme evolution is now a routine approach to improve desirable biocatalytic properties. When only a low-throughput screen is available to detect improved variants from a mutant gene library, it is imperative that cloning efficiency be maximized during library synthesis to avoid wasting effort screening empty plasmids. To achieve this we developed pUCXKT, a gain-of-function positive selection expression vector. Insertion of genes amplified using a specialized downstream PCR primer restores key regulatory and genetic elements necessary for co-expression of a kanamycin resistance marker adjacent to the pUCXKT cloning region. We show that pUCXKT enables 100 % cloning efficiency as well as high-level expression of inserted genes. Unlike previous positive selection expression plasmids, the strategy we used to design pUCXKT is readily adaptable to different vector backbones, antibiotic marker genes, and multiple cloning regions.

Recent technological advances in accurate mass spectrometry and data analysis have revolutionized metabolomics experimentation. Activity-based and global metabolomic profiling methods allow simultaneous and rapid screening of hundreds of metabolites from a variety of chemical classes, making them useful tools for the discovery of novel enzymatic activities and metabolic pathways. By using the metabolome of the relevant organism or close species, these methods capitalize on biological relevance, avoiding the assignment of artificial and non-physiological functions. This review discusses state-of-the-art metabolomic approaches and highlights recent examples of their use for enzyme annotation, discovery of new metabolic pathways, and gene assignment of orphan metabolic activities across diverse biological sources.

Intravenous Tranexamic acid has been shown to decrease blood transfusion requirements in sur- gery. Little evidence exists regarding the much cheaper oral form. The objective of this cohort study was to evaluate whether oral tranexamic acid administration in patients undergoing elec- tive hip and knee replacement surgery resulted in decreased transfusion requirements. Methods: We assessed the transfusion requirements of 332 patients following unilateral total hip or knee arthroplasty, with the first 140 receiving no tranexamic acid and the next 192 given 1 g oral tranexamic acid one hour prior to and a further 1 g 4 hours post joint arthroplasty. Haemoglobin before and after surgery, the number of units transfused post-operatively and the incidence of deep vein thrombosis or pulmonary embolism were recorded. Results: In the first group, there were 22 instances of transfusion (15.7%) and a mean haemoglobin drop of 32.2 g/L, while the second (tranexamic acid) group had just 12 patients transfused (6.3%) and a mean haemoglobin drop of 24.6 g/L (both significantly less, p < 0.01). There was no significant difference in deep vein thrombosis or pulmonary embolism rates between groups. Conclusions: This is the first prospective study to analyze the outcome of oral tranexamic acid administration in hip and knee replacement. We conclude that oral tranexamic acid administration is a safe and effective means to decrease transfusion requirements in joint arthroplasty and is a much cheaper alternative to intravenous preparations of tranexamic acid.

Background: There is much interest regarding metal-on-metal implants in medical and general media. Much of this has been regarding failure of specific implant systems and metal ion toxicity. We present our early mid-term experience of the ADEPT metal-on-metal system which has both modular and non-modular hip options. Methods: Functional assessment, blood metal ion quantification, and radiographic analysis were performed for the modular and non-modular ADEPT variants. Fifty implants were implanted with a mean follow up time of 28 months. Unpaired t-tests were used to compare modular and resurfacing groups, standardised hip scores were used to compare function to conventional total hip arthroplasty. Results: Metal ion levels were significantly higher in modular prostheses compared to resurfacing implants, but not at “harmful” levels (as determined by a previous metal-on-metal implant recall). Functional outcomes were excellent and revision rates were lower than expected. Conclusions: At our institution we have good outcomes with the ADEPT hip prosthesis. Though patient selection and implant position are crucial, poor performance of metal-on-metal hip replacements is implant specific.

Stable isotope-mass spectrometry (MS)-based metabolomic profiling is a powerful technique for following changes in specific metabolite pool sizes and metabolic flux under various experimental conditions in a test organism or cell type. Here, we use a metabolomics approach to interrogate the mechanism of antibiotic action of d-cycloserine (DCS), a second line antibiotic used in the treatment of multidrug resistant Mycobacterium tuberculosis infections. We use doubly labeled 13C α-carbon-2H l-alanine to allow tracking of both alanine racemase and d-alanine:d-alanine ligase activity in M. tuberculosis challenged with DCS and reveal that d-alanine:d-alanine ligase is more strongly inhibited than alanine racemase at equivalent DCS concentrations. We also shed light on mechanisms surrounding d-Ala-mediated antagonism of DCS growth inhibition and provide evidence for a postantibiotic effect for this drug. Our results illustrate the potential of metabolomics in cellular drug-target engagement studies and consequently have broad implications in future drug development and target validation ventures.

d-Cycloserine is a second-line drug approved for use in the treatment of patients infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The unique mechanism of action of d-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show that d-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the M. tuberculosis enzyme orthologue. Furthermore, evidence is presented that indicates d-cycloserine binds exclusively to the C-terminal d-alanine binding site, even in the absence of bound d-alanine at the N-terminal binding site. Together, these results led us to propose a new model of d-alanine:d-alanine ligase inhibition by d-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target.

The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis , where it has so far been underexploited. d -Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d -alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis . During our studies on alternative inhibitors of the d -alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro– d -alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d -alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.

Following the recognition of femoroacetabular impingement (FAI) as a clinical entity, diagnostic tools have continuously evolved. While the diagnosis of FAI is primarily made based on the patients' history and clinical examination, imaging of FAI is indispensable. Routine diagnostic work-up consists of a set of plain radiographs, magnetic resonance imaging (MRI) and MR-arthrography. Recent advances in MRI technology include biochemically sensitive sequences bearing the potential to detect degenerative changes of the hip joint at an early stage prior to their appearance on conventional imaging modalities. Computed tomography may serve as an adjunct. Advantages of CT include superior bone to soft tissue contrast, making CT applicable for image-guiding software tools that allow evaluation of the underlying dynamic mechanisms causing FAI. This article provides a summary of current concepts of imaging in FAI and a review of the literature on recent advances, and their application to clinical practice.

Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the nfsB gene from Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (k(cat)/K(m)) and when over-expressed in an E. coli nfsA(-)/nfsB(-) gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.

Background and purpose The outcome of modern resurfacing remains to be determined. The Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) started collection of data on hip resurfacing at a time when modern resurfacing was started in Australia. The rate of resurfacing has been higher in Australia than in many other countries. As a result, the AOANJRR has one of the largest series of resurfacing procedures. This study was undertaken to determine the results of this series and the risk factors associated with revision.Patients and methods Data from the AOANJRR were used to analyze the survivorship of 12,093 primary resurfacing hip replacements reported to the Joint Replacement Registry between September 1999 and December 2008. This was compared to the results of primary conventional total hip replacement reported during the same period. The Kaplan-Meier method and proportional hazards models were used to determine risk factors such as age, sex, femoral component size, primary di...

Enzymes fuel the biochemical activities of all cells. Their substrates and products thus offer a potential window into the physiologic state of a cell. Metabolomics focuses on the global, or systems-level, study of small molecules in a given biological system and thus provided an experimental tool with which to study cellular physiology on a global biochemical scale. While metabolomic studies of Mycobacterium tuberculosis are still in their infancy, recent studies have begun to deliver unique insights into the composition, organization, activity, and regulation of M. tuberculosis' physiologic network. Here, we outline practical methods for the culture, collection, and analysis of metabolomic samples from Mycobacterium tuberculosis that emphasize minimal sample perturbation, broad and native metabolite recovery, and sensitive, biologically agnostic metabolite detection.

Enzymes fuel the biochemical activities of all cells. Their substrates and products thus represent a potential window into the physiologic state of a cell. Metabolomics focuses on the global, or systems-level, study of small molecules in a given biological system and has thus provided an experimental tool with which to study cellular physiology, including the biochemistry within pathogenic microorganisms. While metabolomic studies of Mycobacterium tuberculosis are still in their infancy, recent studies have begun to deliver unique insights into the composition, organization, activity, and regulation of the bacterium's physiologic network not accessible by other approaches. Here, we outline practical methods for the culture, collection, and analysis of metabolomic samples from M. tuberculosis that emphasize minimally perturbing sample handling, broad and native metabolite recovery, and sensitive, biologically agnostic metabolite detection.

We prospectively followed 112 hips, undergoing THA with a Spectron EF stem. At mean follow-up of 11.2years, 21 patients had died. We obtained radiological follow-up in 99% and clinical follow-up in 100% of the surviving 91 hips. Fifty-four percent demonstrated osteolysis in at least one Gruen zone. Twenty-two hips required revision for all causes, with a further five stems radiologically loose. With endpoint being stem revision for aseptic loosening or radiological failure, survivorship at 11years was 0.783. We believe the addition of a rougher surface finish has contributed to the high levels of osteolysis and stem failure seen with the Spectron EF.

Two potentially complementary approaches to improve the anti-cancer strategy gene-directed enzyme prodrug therapy (GDEPT) are discovery of more efficient prodrug-activating enzymes, and development of more effective prodrugs. Here we demonstrate the utility of a flexible screening system based on the Escherichia coli SOS response to evaluate novel nitroreductase enzymes and prodrugs in concert. To achieve this, a library of 47 candidate genes representing 11 different oxidoreductase families was created and screened to identify the most efficient activators of two different nitroaromatic prodrugs, CB1954 and PR-104A. The most catalytically efficient nitroreductases were found in the NfsA and NfsB enzyme families, with NfsA homologues generally more active than NfsB. Some members of the AzoR, NemA and MdaB families also exhibited low-level activity with one or both prodrugs. The results of SOS screening in our optimised E. coli reporter strain SOS-R2 were generally predictive of the ability of nitroreductase candidates to sensitise E. coli to CB1954, and of the kcat/Km for each prodrug substrate at a purified protein level. However, we also found that not all nitroreductases express stably in human (HCT-116 colon carcinoma) cells, and that activity at a purified protein level did not necessarily predict activity in stably transfected HCT-116. These results highlight a need for all enzyme-prodrug partners for GDEPT to be assessed in the specific context of the vector and cell line that they are intended to target. Nonetheless, our oxidoreductase library and optimised screens provide valuable tools to identify preferred nitroreductase-prodrug combinations to advance to preclinical evaluation.

D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents.

The painful subluxed or dislocated hip in adults with cerebral palsy presents a challenging problem. Prosthetic dislocation and heterotopic ossification are particular concerns. We present the first reported series of 19 such patients (20 hips) treated with hip resurfacing and proximal femoral osteotomy. The pre-operative Gross Motor Function Classification System (GMFCS) was level V in 13 (68%) patients, level IV in three (16%), level III in one (5%) and level II in two (11%). The mean age at operation was 37 years (13 to 57). The mean follow-up was 8.0 years (2.7 to 11.6), and 16 of the 18 (89%) contactable patients or their carers felt that the surgery had been worthwhile. Pain was relieved in 16 of the 18 surviving hips (89%) at the last follow-up, and the GMFCS level had improved in seven (37%) patients. There were two (10%) early dislocations; three hips (15%) required revision of femoral fixation, and two hips (10%) required revision, for late traumatic fracture of the femoral neck and extra-articular impingement, respectively. Hence there were significant surgical complications in a total of seven hips (35%). No hips required revision for instability, and there were no cases of heterotopic ossification. We recommend hip resurfacing with proximal femoral osteotomy for the treatment of the painful subluxed or dislocated hip in patients with cerebral palsy.

CB1954 is an anti-cancer prodrug that can be reduced at either of two nitro groups to form cytotoxic metabolites. We describe here two efficient and previously uncharacterized nitroreductases, YfkO from Bacillus subtilis which reduces CB1954 exclusively at the 4-NO(2) position, and NfsA from Klebsiella pneumoniae which preferentially reduces the 2-NO(2) group. Utilizing these novel enzymes, together with three previously characterized nitroreductases, we demonstrate that the Escherichia coli SOS-chromotest assay can differentially detect the 4-nitro versus 2-nitro reduction products of CB1954 following deletion of the nucleotide excision repair gene uvrB, but not mismatch repair (mutS) or methyltransferase (ada/ogt) genes. These findings may hold significance for identification and selection of nitroreductases for CB1954-mediated gene therapy, particularly when targeting tumors that are deficient in nucleotide excision repair. Moreover, we demonstrate that comparative SOS chromotest analysis in wild type and uvrB mutant strains can be used to determine whether or not nucleotide excision repair plays a significant role in processing DNA damage resulting from activation of different nitroaromatic prodrugs.

Background and purpose: Recent years have seen a rapid increase in the use of resurfacing hip arthroplasty despite the lack of literature on the long-term outcome. In particular, there is little evidence regarding the outcome of revisions of primary resurfacing. The purpose of this analysis was to examine the survivorship of primary resurfacing hip arthroplasties that have been revised. Patients and methods: Over 12,000 primary resurfacing hip arthroplasties were recorded by the Australian Orthopaedic Association National Joint Replacement Registry between September 1, 1999 and December 31, 2008. During this time, 397 revisions for reasons other than infection were reported for these primary resurfacings and classified as acetabular, femoral, or both acetabular and femoral revisions. The survivorship of the different types of revisions was estimated using the Kaplan-Meier method and compared using proportional hazard models. Additionally, the outcome of a femoral-only revision was compared to that of primary conventional total hip arthroplasty. Results: Acetabular-only revision had a high risk of re-revision compared to femoral-only and both acetabular and femoral revision (5-year cumulative per cent revision of 20%, 7%, and 5% respectively). Femoral-only revision had a risk of re-revision similar to that of revision of both the acetabular and femoral components. Femoral-only revision had over twice the risk of revision of primary conventional total hip arthroplasty. Interpretation: Revision of a primary resurfacing arthroplasty is associated with a major risk of re-revision. The best outcome is achieved when either the femoral-only or both the acetabular and femoral components are revised. Technically straightforward femoral-only revisions generally have a worse outcome than a primary conventional total hip arthroplasty. Refereed/Peer-reviewed

d-Cycloserine is a second-line drug approved for use in the treatment of patients infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The unique mechanism of action of d-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show that d-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the M. tuberculosis enzyme orthologue. Furthermore, evidence is presented that indicates d-cycloserine binds exclusively to the C-terminal d-alanine binding site, even in the absence of bound d-alanine at the N-terminal binding site. Together, these results led us to propose a new model of d-alanine:d-alanine ligase inhibition by d-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target.

Background Adult Hip Dysplasia (AHD) has been strongly linked with the development of hip osteoarthritis. The complexity and therefore resultant steep learning curve of the Bernese osteotomy for AHD has been well described. The purpose of this study was to analyse the efficacy of a technically less demanding interlocking pelvic triple osteotomy. Methods Pre and postoperative pelvic radiographs of 8 hips in 7 patients who underwent pelvic osteotomy between January 2010 and December 2011 were corrected to a standardised orientation using a validated software package, Hip2NormTM, and this tool was then used to measure hip parameters used for assessing dysplasia. The Lateral Centre Edge Angle (LCEA), the Acetabular Index of the Weight-Bearing Zone (AIWB), and the percentage Acetabular Coverage of the Femoral Head (ACFH) were all calculated and compared. Oxford hip scores, WOMAC hip scores, and UCLA activity scores were clinical outcome measures. Results Average LCEA correction was 23.8 deg, from a mean of 8.8 deg preoperatively to 32.6 deg postoperatively. AIWB was corrected an average of 21.3 deg (mean 22.5 to 1.2 deg postoperatively) and ACFH was increased on average 23.8% (mean 59.0 to 82.8% postoperatively). At a minimum follow-up of 3 months Oxford hip scores improved from an average of 19.6 preoperatively to 39.4, and the mean UCLA activity index was increased from 3.3 to 7.1 postoperatively. There were two technical complications in the studied procedures, which have resulted in no long-term sequelae. Conclusions This study demonstrates the safe and effective use of an interlocking pelvic triple osteotomy to provide correction of radiological parameters and symptomatic improvement of AHD.

Tumor-targeting viruses and bacteria hold great promise as anti-cancer agents. They kill cells by entirely different mechanisms to radio- and chemotherapies, and have potential to synergize with these treatments without overlapping toxicities. Furthermore, these agents can be ‘armed’ with genes that encode enzymes that activate prodrugs - compounds that are deactivated in their administered form, but become highly toxic upon metabolic activation. This not only improves killing of infected cells, but also neighboring non-infected cells, as the prodrug metabolites can diffuse locally and exert a bystander effect. A highly efficient activating enzyme in partnership with a prodrug that has a strong bystander effect can address some of the historical limitations of cancer gene therapy including the inability of biological vectors to reach every target cell. Phase I/II trials of the first-generation nitroaromatic prodrug CB1954 in conjunction with the prototype gene therapy nitroreductase, Escherichia coli NfsB, have been conducted in prostate and liver cancer. These trials provided some evidence of anti-tumor activity but, due to dose-limiting hepatotoxicity, the highest achievable plasma concentration of CB1954 was less than 1% of NfsB's Km. As well as highlighting a need for more efficient nitroreductase enzymes, this has fuelled a search for superior nitroaromatic prodrugs. The next-generation dinitrobenzamide mustard prodrug PR-104A is not only 5–50 fold more dose-potent upon activation, but also better tolerated in humans (MTD 1100 mg/m2 vs 24 mg/m2; q3w, iv). However, E. coli NfsB also has relatively poor (millimolar) affinity for this substrate. To discover more efficient nitroreductases we developed screens for genotoxic prodrug activation, based on their ability to induce reporter genes linked to the E. coli SOS (DNA damage repair) response. We used these to screen a large library of candidate enzymes for DNBM activity, and selected E. coli NfsA as a top candidate for further improvement by random and targeted mutagenesis. High throughput screening of large error prone PCR libraries coupled with medium throughput screening of targeted mutagenesis libraries revealed 10 individual mutations that significantly increased NfsA activity. These mutations were then combined in a synthetic “smart” library, from which eight poly-mutant enzymes were selected for kinetic analysis. Relative to wild type the engineered variants display an 18–40 fold improvement in PR-104A Km with respect to E. coli NfsA, and are 860–1700 fold better than NfsB. Importantly they also retain, or are improved in, their ability to co-metabolize preferred 2-nitroimidazole probes with PET-imaging capabilities (see abstract: Patterson et al, “Molecular imaging using bacterial nitroreductase reporter genes by repurposing the clinical stage hypoxia PET probe EF5”). The enhanced prodrug activation and in vivo imaging potential of these enzymes is now being evaluated in human gene therapy models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B88.

Oncolytic viruses and tumor-tropic bacteria offer promise as cancer therapeutics of the future. There is a need to develop technologies to monitor the spatio-temporal distribution of these live vectors in a manner that is predictive of normal tissue toxicity and antitumor efficacy. Positron emission tomography (PET) is the preferred non-invasive imaging modality (biomarker) but suitable advanced reporter gene/PET probe combinations are lacking. A range of 2-nitroimidazole (2-NI) probes are currently in clinical use for the detection of hypoxia (EF5, FMISO, HX4, FAZA) and thus have already attained a high level of clinical validation. We hypothesized that (2-NI) PET agents might be repurposed to monitor the biodistribution of replicating biological agents, thereby leveraging two decades of research efforts to optimise hypoxia PET probe pharmacology. Bacterial nitroreductases (NTRs; type I) are efficient O2-independent enzymes that provide the necessary catalytic flexibility to achieve this goal. Historically E. coli NfsB has been the focus of gene therapy applications. We cloned eleven candidate NTRs from E. coli namely; AzoR, KefF, MdaB, NemA, NfsA, NfsB, WrbA, YcaK, YcdI, YdjA and Yief. Using HCT116 cells engineered to express each NTR, we showed NfsA alone was able to metabolise a range of 2-NI probe molecules, including EF5 (pentafluoroetanidazole), leading to efficient cellular retention. We confirmed catalytic efficiency of EF5 reduction by recombinant NfsA (kcat/Km 97 s−1/mM). In contrast NfsB only weakly metabolised EF5 (kcat/Km 0.24 s−1/mM). We compared EF5 adduct retention in HCT116 cells, detected by mAb using flow cytometry, either under anoxia (< 10ppm O2) or engineered to express NfsA or NfsB. Following exposure to EF5 in monolayer, median fluorescence was >100-fold greater in NfsA expressing cells than anoxic cells, whereas NfsB cells were negative. Mixed Wt : NfsA multicellular layers containing various ratios of cells demonstrated the capacity to detect, with precision, single NfsA-positive cells in mixed cell populations. HCT116 xenografts composed of increasing proportions of NfsA cells (0% to 25%) were established in nude mice and analysed by immunohistochemistry and flow cytometry. Ex-vivo pimonidazole-binding confirmed all NfsA positive cells were detected in vivo following administration of EF5. NfsA, like NfsB, can bioactivate the clinical-stage prodrug PR-104. We determined the relationship between EF5 retention and PR-104 cytotoxicity in HCT116 xenografts harbouring variable proportions of NfsA cells (0%-25%). A correlation was observed between total EF5 retention and global clonogenic cell kill (r2 = 0.828; p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B89.