Molecular interplay of an assembly machinery for nitrous oxide reductase.

Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N2O) from soils and industrial processes have increased considerably over the last decades1-3. As the final step of bacterial denitrification, N2O is reduced to chemically inert N2 (refs. 1,4) in a reaction that is catalysed by the copper-dependent nitrous oxide reductase (N2OR) (ref. 5). The assembly of its unique [4Cu:2S] active site cluster CuZ requires both the ATP-binding-cassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL (refs. 4,6). Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY and its complexes with NosL and N2OR, respectively. We find that the periplasmic NosD protein contains a binding site for a Cu+ ion and interacts specifically with NosL in its nucleotide-free state, whereas its binding to N2OR requires a conformational change that is triggered by ATP binding. Mutually exclusive structures of NosDFY in complex with NosL and with N2OR reveal a sequential metal-trafficking and assembly pathway for a highly complex copper site. Within this pathway, NosDFY acts as a mechanical energy transducer rather than as a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme N2OR.

Wear and damage in retrieved humeral inlays of reverse total shoulder arthroplasty-where, how much, and why?

BACKGROUND: Polyethylene (PE) wear and material degradation have been reported as complications in reverse total shoulder replacements (rTSAs). In this regard, scapular notching is associated with more clinical complications. Therefore, the purposes of the study were to quantify the linear and volumetric wear, as a measure for the amount of removed material, and to qualitatively assess the PE damage modes to describe the material degradation in retrieved rTSA humeral PE inlays that contribute to failure of shoulder replacements. Furthermore, this study aimed to evaluate the effect of scapular notching on PE wear and rim damage of the humeral components. METHODS: The total study population of 39 humeral inlays contains 2 cohorts that were used for the damage mode analysis and for the wear analysis, respectively. The extent and presence of wear damage modes in 5 defined zones were assessed by a grading system for all PE joint replacements. For quantitative wear analysis the most frequent design (n = 17) was chosen. Using a coordinate-measuring machine and postprocessing software, volumetric wear measurements for the retrieved humeral PE inlays were undertaken. Furthermore, prerevision radiographs were analyzed for scapular notching. Finally, retrieval findings were correlated with clinical and radiographic data to consider the effect of notching and to identify risk of failures for these prostheses. RESULTS: Damage on the rim of the humeral PE inlays was more frequent and severe than on the intended articulation surface. Irrespective of the damage mode, the inferior rim zone sustained the greatest amount of wear damage followed by the posterior zone. Burnishing, scratching, pitting, and embedded particles are most likely to occur in the articular surface area, whereas surface deformation, abrasion, delamination and gross material degradation are predominantly present in the inferior and posterior rim zones. The retrieved inlays exhibited a mean volumetric wear rate of 296.9 mm³/yr ± 87.0 mm³/yr. However, if the notched and non-notched components were compared, a significant higher volumetric wear rate (296.5 ± 106.1 mm³/yr) was found for the notched components compared to the non-notched group (65.7 ± 7.4 mm³/yr). Generally, there was a significantly greater incidence of damage and greater amount of wear if scapular notching occurred. CONCLUSION: The notched components showed a 5-fold increase in PE wear rate. Therefore, scapular notching has a strong effect on PE damage and wear. If scapular notching can be clinically avoided, the PE wear performance is in a similar magnitude as found for hip and knee replacements.

Long-term (35 years) cryopreservation of Echinococcus multilocularis metacestodes.

The metacestode of Echinococcus multilocularis is the etiological agent of alveolar echinococcosis. The metacestode stage used for research is maintained in rodents by serial passages. In order to determine whether cryopreservation of E. multilocularis metacestodes would be suitable for long-term maintenance and replace serial passages, isolates of different geographic origin were cryopreserved in 1984-1986. The aim of the current study was to test the viability of cryopreserved isolates following long-term cryopreservation (up to 35 years) and to determine the phylogenetic clades these isolates belonged to. Cryopreserved isolates were tested for viability in vitro and in vivo in gerbils. In vitro results of 5 isolates indicated protoscolex survival in 13 of 17 experiments (76%) and metacestode survival in 5 of 12 (42%) in vivo experiments. In vivo results showed 'abortive lesions' in 13 of the 36 animals, 15 were negative and 8 harboured proliferating metacestode tissue containing protoscoleces. Genetic analysis confirmed the isolates belonged to European, Asian and North-American clades. In conclusion, the results of the current study indicate that metacestodes of E. multilocularis are able to survive long-term cryopreservation. Therefore, cryopreservation is a suitable method for long-term storage of E. multilocularis metacestode isolates and reduces the number of experimental animals.

Pulsatile Lavage Improves Tibial Cement Penetration and Implant Stability in Medial Unicompartmental Arthroplasty: A Cadaveric Study.

Cemented unicompartmental knee arthroplasty (UKA) shows good survivorship and function. However, implant failure, causing the need for revision, can occur. Aseptic loosening is still among the most common reasons for revision. The purpose of this study was to assess the influence of preimplantation lavage technique on tibial cement penetration depth, tibial cement volume, and load to fracture in the tibial component of mobile-bearing UKA. In 10 pairs of fresh frozen human tibiae, cemented UKA was implanted by an experienced surgeon. Tibial components were then implanted, left and right tibiae were randomly allocated to group A or B. Prior to implantation, irrigation was performed with either syringe lavage or pulsatile jet lavage in a standardized manner. Cement surface was 4170.2 mm2 (3271.6-5497.8 mm2) in the syringe lavage group, whereas the jet lavage group showed 4499.3 mm2 (3354.3-5809.1 mm2); cement volume was significantly higher as well (4143.4 mm3 (2956.6-6198.6 mm3) compared with 5936.9 mm3 (3077.5-8183.1 mm3)). Cement penetration depth was 2.5 mm (1.7-3.2 mm) for the jet lavage, and 1.8 mm (1.2-2.4 mm) for the syringe lavage. The mean fracture load was 4680 N in the jet lavage group and 3800 N in the syringe lavage group (p = 0.001). Subsidence was significantly higher for syringe lavage. This study suggests a correlation of cement penetration depth and cement volume to implant failure in the tibial component of a UKA using a cadaveric model. The type of bone lavage most likely influences these two key parameters.

The primary stability of the femoral component in cemented single and twin peg Oxford unicompartmental knee arthroplasty under adverse conditions.

AIMS: The cemented Oxford unicompartmental knee arthroplasty (OUKA) features two variants: single and twin peg OUKA. The purpose of this study was to assess the stability of both variants in a worst-case scenario of bone defects and suboptimal cementation. METHODS: Single and twin pegs were implanted randomly allocated in 12 pairs of human fresh-frozen femora. We generated 5° bone defects at the posterior condyle. Relative movement was simulated using a servohydraulic pulser, and analyzed at 70°/115° knee flexion. Relative movement was surveyed at seven points of measurement on implant and bone, using an optic system. RESULTS: At the main fixation zone, the twin peg shows less relative movement at 70°/115°. At the transition zone, relative movements are smaller for the single peg for both angles. The single peg shows higher compression at 70° flexion, whereas the twin peg design shows higher compression at 115°. X-displacement is significantly higher for the single peg at 115°. CONCLUSION: Bony defects should be avoided in OUKA. The twin peg shows high resilience against push-out force and should be preferred over the single peg. Cite this article: Bone Joint Res 2022;11(2):82-90.

An optotracer-based antibiotic susceptibility test specifically targeting the biofilm lifestyle of Salmonella.

Antimicrobial resistance is a medical threat of global dimensions. Proper antimicrobial susceptibility testing (AST) for drug development, patient diagnosis and treatment is crucial to counteract ineffective drug use and resistance development. Despite the important role of bacterial biofilms in chronic and device-associated infections, the efficacy of antibiotics is determined using planktonic cultures. To address the need for antibiotics targeting bacteria in the biofilm lifestyle, we here present an optotracing-based biofilm-AST using Salmonella as model. Our non-disruptive method enables real-time recording of the extracellular matrix (ECM) components, providing specific detection of the biofilm lifestyle. Biofilm formation prior to antibiotic challenge can thus be confirmed and pre-treatment data collected. By introducing Kirby-Bauer discs, we performed a broad screen of the effects of antibiotics representing multiple classes, and identified compounds with ECM inhibitory as well as promoting effects. These compounds were further tested in agar-based dose-response biofilm-AST assays. By quantifying the ECM based on the amount of curli, and by visualizing the biofilm size and morphology, we achieved new information directly reflecting the treated biofilm. This verified the efficacy of several antibiotics that were effective in eradicating pre-formed biofilms, and it uncovered intriguing possible resistance mechanisms initiated in response to treatments. By providing deeper insights into the resistances and susceptibilities of microbes, expanded use of the biofilm-AST will contribute to more effective treatments of infections and reduced resistance development.

A semi high-throughput method for real-time monitoring of curli producing Salmonella biofilms on air-solid interfaces.

Biofilms enable bacteria to colonize numerous ecological niches. Bacteria within a biofilm are protected by the extracellular matrix (ECM), of which the fibril-forming amyloid protein curli and polysaccharide cellulose are major components in members of Salmonella, Eschericha and Mycobacterium genus. A shortage of real-time detection methods has limited our understanding of how ECM production contributes to biofilm formation and pathogenicity. Here we present optotracing as a new semi-high throughput method for dynamic monitoring of Salmonella biofilm growth on air-solid interfaces. We show how an optotracer with binding-induced fluorescence acts as a dynamic fluorescent reporter of curli expression during biofilm formation on agar. Using spectrophotometry and microscopic imaging of fluorescence, we analyse in real-time the development of the curli architecture in relation to bacterial cells. With exceptional spatial and temporal precision, this revealed a well-structured, non-uniform distribution of curli organised in distally projecting radial channel patterns. Dynamic monitoring of the biofilm also showed defined regions undergoing different growth phases. ECM structures were found to assemble in regions of late exponential growth phase, suggesting that ECM forms on site after bacteria colonize the surface. As the optotracer biofilm method expedites screening of curli production, providing exceptional spatial-temporal understanding of the surface-associated biofilm lifestyle, this method adds a new technique to further our understanding of bacterial biofilms.

Bearing Thickness Is Not a Predictive Factor for Damage and Penetration in Oxford Unicompartmental Knee Arthroplasty-A Retrieval Analysis.

The medial Oxford unicompartmental knee arthroplasty (OUKA) shows good survivorship, as well as clinical results. Aseptic loosening, however, remains one of the main reasons for revision and polyethylene debris is known to cause aseptic loosening. The role of bearing thickness in total as well as unicondylar knee arthroplasty has been the subject of controversial discussions, especially the longevity of lower thickness bearings in total knee arthroplasty was questioned. The purpose of this study was to assess the influence of bearing thickness on time to revision, damage pattern, penetration, and volumetric material loss. A cohort of 47 consecutively retrieved medial OUKA bearings was analyzed with conventional direct light microscopy applying the Hood damage analysis, as well as measuring the penetration depth. In this retrieval cohort, a difference on survival time, damage, penetration, as well as volumetric material loss could not be seen. We conclude that low as well as high thickness bearings can safely be used in OUKA without any relevant differences in terms of wear and damage.

Minimally invasive lateral unicompartmental knee replacement: Early results from an independent center using the Oxford fixed lateral prosthesis.

PURPOSE: The aim of the present study was to assess clinical outcome as well as short-term survivorship of fixed-bearing lateral unicompartmental knee replacement (UKR) from a non-designer centre using the Oxford Fixed Lateral prosthesis. METHODS: This single-centre retrospective cohort study reports the short-term results of 51 consecutive patients (52 knees) after fixed-bearing lateral UKR with a minimum follow-up of one year. Survivorship analysis was performed with different endpoints and clinical outcome was measured using the Oxford Knee Score (OKS), objective American Knee Society Score (AKSS-O), range-of-motion (ROM), visual analog scale for pain (VAS), Tegner activity score and UCLA score. RESULTS: There was no revision surgery, defined as exchange of at least one of the components resulting in a survival rate of 100% at two years. Three patients required further surgical treatment resulting in a survival rate of 94.2% (95% confidence interval (CI): 83.2-98.1%) with the endpoint 'any reoperation'. Outcome scores, VAS and ROM showed a statistically significant improvement at final follow-up (P < .001). The OKS improved from 26.4 ±â€¯6.9 (12-41) preoperatively to 39.7 ±â€¯8.4 (15-48), the AKSS-O from 54.3 ±â€¯15.3 (18-90) to 82.2 ±â€¯15.6 (40-100), the American Knee Society Functional Score from 56.4 ±â€¯21.3 (10-100) to 83.1 ±â€¯20.2 (five to 100) and the ROM from 123.5 ±â€¯13.5 (90-140) to 134 ±â€¯10.3 (95-150). CONCLUSION: Early results of fixed-bearing lateral UKR using the Oxford-Fixed-Lateral prosthesis were encouraging with a significant improvement in pain and knee function as well as an excellent survivorship of 100% at a mean follow-up of two years. Further follow-up is necessary to evaluate the long-term effectiveness of this device and the surgical technique. LEVEL OF EVIDENCE: Level IV, retrospective cohort study.

Electrocautery Damage Can Reduce Implant Fatigue Strength: Cases and in Vitro Investigation.

BACKGROUND: The risk of femoral stem fracture after total hip replacement is low and can often be associated with a specific implant system or other factors that may reduce the fatigue strength. Additionally, damage to a metal component during revision surgery by an electrocautery device may further affect the fatigue behavior. METHODS: Two clinical cases of stem failure after revision of fractured ceramic components are presented; the retrieved components were analyzed for the cause of failure. In vitro cyclic load-to-failure testing of titanium alloy femoral stems after electrocautery application at 2 different locations (at the base and about midway on the femoral neck) was performed using a stepwise increase in load until implant fracture occurred. In addition, a detailed characterization of the local material structure around the electrocautery marks was performed. RESULTS: Superficial discoloration and melting marks were found on the retrieved components, including at the location of crack initiation in the anterolateral region, which may have reduced the fatigue strength of the material. In addition, elemental analysis indicated material transfer from the electrocautery tip. Damage to the surface by the electrocautery device significantly reduced the in vitro load to failure by up to 47% compared with that of undamaged femoral neck specimens. Material analysis revealed a relevant modification in microstructure, with an extension of approximately 2.7 mm and a depth of 550 µm, which could be divided in 3 structural zones. CONCLUSIONS: Intraoperative electrocautery device contact with the implant during surgical revision of a total hip replacement cannot always be avoided. However, on the basis of our findings, the risk of implant failure is increased due to a change in microstructure and a potential reduction of the implant's fatigue strength. Surgeons and manufacturers of electrocautery devices should be aware of this concern. CLINICAL RELEVANCE: During revision surgery, contact between an electrocautery device and the femoral component should be avoided to reduce the chance of subsequent femoral neck fracture.

Can intraoperative measurement of bone quality help in decision making for cementless unicompartmental knee arthroplasty?

BACKGROUND: In uncemented total hip arthroplasty (THA), low bone mineral density (BMD) is associated with aseptic loosening. BMD is usually assessed via dual-energy X-ray absorptiometry (DXA) or quantitative computed tomography, which takes time and exposes patients to radiation. Due to its low risk profile, intraoperative measurement of the trabecular stability might be a useful alternative to DXA. METHODS: In 24 human femora, BMD was analysed using DXA at the femoral necks and the knees. Performing the standard Oxford Unicompartmental Knee Arthroplasty (OUKA) implantation procedure, a wingblade (DensiProbe) coupled to a torque probe was used to evaluate the trabecular peak torque. The standard procedure was modified: before the completion of the central peg drill hole, the DensiProbe was inserted into the pre-drilled hole and then turned until a loss of resistance was achieved. The obtained data was then correlated with BMD at the femoral neck as well as the knee. RESULTS: In all tested regions, a higher peak torque was observed in correlation with a higher BMD. CONCLUSIONS: As demonstrated, the DensiProbe can be a helpful tool to assess the bone quality intraoperatively in OUKA. It can be a valuable decision guidance when faced with choosing between a cemented and a cementless implant. Due to the fact that the central peg hole of the OUKA can be used for the procedure, no additional risk for the patient exists, while the additional work for the surgeon is minimal.

Haycocknema-like nematodes in muscle fibres of a horse.

A 14-year-old horse (imported to Switzerland from Ireland 8 years earlier) showed signs of chewing muscle atrophy. A severe chronic myositis, caused by numerous immature and mature female nematodes, was diagnosed in muscle samples obtained by biopsy and subsequently at necropsy. Most of the nematodes had invaded muscle fibres of the masseter, root of the tongue and anterior breast, only a few were found in the intermuscular interstitium. Isolated nematodes and parasite sections were clearly different from muscle larvae of Trichinella spp. but showed morphological similarities to Haycocknema perplexum, a nematode species (order Enoplida, family Robertdollfusidae) recently found in the musculature of a human patient in Australia. However, our material did not allow the precise identification of the nematode genus nor the unequivocal differentiation from Halicephalobus gingivalis. This species infects horses and humans and can cause severe granuloma formation in muscles and many other organ systems, but has never been observed to invade individual muscle fibres. Our findings show that nematodes of another genus than Trichinella may invade muscle fibres of the horse and cause myositis. These nematodes are provisionally regarded as Haycocknema-like.

Fretting and Corrosion in Modular Shoulder Arthroplasty: A Retrieval Analysis.

Tribocorrosion in taper junctions of retrieved anatomic shoulder arthroplasty implants was evaluated. A comparison of the tribocorrosion between cobalt-chromium and titanium alloy stems was conducted and the observations were correlated with the individual's clinical data. Adverse effects caused by metal debris and subsequent elevated serum metal ion levels are frequently reported in total hip arthroplasty. In total shoulder arthroplasty, to date only a small number of retrieval analyses are available and even fewer address the issue of tribocorrosion at the taper junctions. A total of 36 retrieved hemiarthroplasties and total shoulder arthroplasties were assessed using the modified Goldberg score. The prevalence of fretting and corrosion was confirmed in this cohort. Titanium stems seem to be more susceptible to damage caused by tribocorrosion than cobalt-chromium stems. Furthermore, stemless designs offered less tribocorrosion at the taper junction than stemmed designs. A weak correlation between time to revision and increased levels of tribocorrosion was seen. Whether or not tribocorrosion can lead to adverse clinical reactions and causes failure of shoulder arthroplasties remains to be examined.

Human alveolar echinococcosis after fox population increase, Switzerland.

We analyzed databases spanning 50 years, which included retrospective alveolar echinococcosis (AE) case finding studies and databases of the 3 major centers for treatment of AE in Switzerland. A total of 494 cases were recorded. Annual incidence of AE per 100,000 population increased from 0.12-0.15 during 1956-1992 and a mean of 0.10 during 1993-2000 to a mean of 0.26 during 2001-2005. Because the clinical stage of the disease did not change between observation periods, this increase cannot be explained by improved diagnosis. Swiss hunting statistics suggested that the fox population increased 4-fold from 1980 through 1995 and has persisted at these higher levels. Because the period between infection and development of clinical disease is long, the increase in the fox population and high Echinococcus multilocularis prevalence rates in foxes in rural and urban areas may have resulted in an emerging epidemic of AE 10-15 years later.

Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern.

Echinococcosis in humans is a zoonotic infection caused by larval stages (metacestodes) of cestode species of the genus Echinococcus. Cystic echinococcosis (CE) is caused by Echinococcus granulosus, alveolar echinococcosis (AE) is caused by E. multilocularis, and polycystic forms are caused by either E. vogeli or E. oligarthrus. In untreated cases, AE has a high mortality rate. Although control is essentially feasible, CE remains a considerable health problem in many regions of the northern and southern hemispheres. AE is restricted to the northern hemisphere regions of North America and Eurasia. Recent studies have shown that E. multilocularis, the causative agent of AE, is more widely distributed than previously thought. There are also some hints of an increasing significance of polycystic forms of the disease, which are restricted to Central and South America. Various aspects of human echinococcosis are discussed in this review, including data on the infectivity of genetic variants of E. granulosus to humans, the increasing invasion of cities in Europe and Japan by red foxes, the main definitive hosts of E. multilocularis, and the first demonstration of urban cycles of the parasite. Examples of emergence or reemergence of CE are presented, and the question of potential spreading of E. multilocularis is critically assessed. Furthermore, information is presented on new and improved tools for diagnosing the infection in final hosts (dogs, foxes, and cats) by coproantigen or DNA detection and the application of molecular techniques to epidemiological studies. In the clinical field, the available methods for diagnosing human CE and AE are described and the treatment options are summarized. The development of new chemotherapeutic options for all forms of human echinococcosis remains an urgent requirement. A new option for the control of E. granulosus in the intermediate host population (mainly sheep and cattle) is vaccination. Attempts are made to reduce the prevalence of E. multilocualaris in fox populations by regular baiting with an anthelmintic (praziquantel). Recent data have shown that this control option may be used in restricted areas, for example in cities, with the aim of reducing the infection risk for humans.

Immunosurveillance of alveolar echinococcosis by specific humoral and cellular immune tests: long-term analysis of the Swiss chemotherapy trial (1976-2001).

BACKGROUND/AIMS: Long-term chemotherapy with benzimidazoles is beneficial in non-resectable alveolar echinococcosis (AE). Criteria to track early therapeutic efficacy are lacking and the clinical impact of immunosurveillance is unsettled. We aimed to analyze this issue particularly for assessing the putative parasitocidal efficacy of chemotherapy. METHODS: The present study is part of our prospective Swiss trial outlined previously and comprises 57 patients with a median follow-up of 18.5 (3-30) years and with repeated tests of humoral and cell-mediated immunity. The series was subdivided into group A (n=23; curative surgery) and group B (n=34: non-resectable AE). RESULTS: Long-term survival was 87% (group A) and 76% (group B). The profiles of specific antibodies against EmII/3-10 antigen normalized within 3 years in most group A-patients, but remained above the cut-off value in 40% of group B-patients. This lack of normalization was associated with lower bioavailability of mebendazole. AE-recurrence after 'radical' surgery (up to 13 years) was associated with high anti-EmII/3-10 concentrations in 7 of 8 cases. Following abrogation of longterm chemotherapy in group B, no AE-recurrence occurred in 9/18 patients, suggestive of parasitocidal efficacy and documented by a normal EmII/3-10 profile. CONCLUSIONS: The EmII/3-10 profile is of value in monitoring AE after surgery and/or chemotherapy.