Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy.

Fluorescence resonance energy transfer in single enzyme molecules (smFRET, single-molecule measurement) allows the measurement of multicomponent distance distributions in complex biomolecules similar to pulsed electron-electron double resonance (PELDOR, ensemble measurement). Both methods use reporter groups: FRET exploits the distance dependence of the electric interaction between electronic transition dipole moments of the attached fluorophores, whereas PELDOR spectroscopy uses the distance dependence of the interaction between the magnetic dipole moments of attached spin labels. Such labels can be incorporated easily to cysteine residues in the protein. Comparison of distance distributions obtained with both methods was carried out with the H+-ATPase from Escherichia coli (EF0F1). The crystal structure of this enzyme is known. It contains endogenous cysteines, and as an internal reference two additional cysteines were introduced (EF0F1-γT106C-εH56C). These positions were chosen to allow application of both methods under optimal conditions. Both methods yield very similar multicomponent distance distributions. The dominating distance distribution (> 50%) is due to the two cysteines introduced by site-directed mutagenesis and the distance is in agreement with the crystal structure. Two additional distance distributions are detected with smFRET and with PELDOR. These can be assigned by comparison with the structure to labels at endogenous cysteines. One additional distribution is detected only with PELDOR. The comparison indicates that under optimal conditions smFRET and PELDOR result in the same distance distributions. PELDOR has the advantage that different distributions can be obtained with ensemble measurements, whereas FRET requires single-molecule techniques.

Risk of infection in primary, elective total hip arthroplasty with direct anterior approach or lateral transgluteal approach: a prospective cohort study of 1104 hips.

BACKGROUND: The direct anterior approach (DAA) is increasingly popular for hip replacement. However, the small incision and the location near to the groin might increase the risk of periprosthetic joint infection (PJI). We asked the questions (i) whether there is an increased risk of infection for this approach, and (ii) whether the spectrum of microorganisms differs between patients with DAA and those with lateral transgluteal approach (LAT). METHODS: All patients operated between 08/2006 and 12/2013 were followed prospectively in an in house register. The DAA was introduced as routine in 02/2009 at our hospital. Patients with primary elective hip replacement without previous operations were included. Follow-up was scheduled after 6, 12 weeks and 1, 2 years. PJI was defined according to standardized criteria. RESULTS: One thousand one hundred four patients were studied, 700 were operated with DAA and 404 with LAT. No patient was lost to follow-up. PJI was diagnosed in 23/1104 (2.1 %) patients, 16 (2.3 %) in the group with DAA, and 7 (1.7 %) in the group with LAT. Patients with infection had a higher BMI (p < 0.001) and a higher ASA score (p < 0.001). Only patients with the DAA had exogenous PJI caused by gramnegative bacilli (35.7 % vs 0 %, p = 0.26). In the DAA-group, the fraction of patients with polymicrobial infection was somewhat higher than in the LAT-group (50 % vs 33 %, P = 0.64). CONCLUSION: There was no increased risk of infection for the DAA.

Eradication of infection, survival, and radiological results of uncemented revision stems in infected total hip arthroplasties.

Background and purpose - The use of uncemented revision stems is an established option in 2-stage procedures in patients with periprosthetic joint infection (PJI) after total hip arthroplasty (THA). However, in 1-stage procedures, they are still rarely used. There are still no detailed data on radiological outcome after uncemented 1-stage revisions. We assessed (1) the clinical outcome, including reoperation due to persistent infection and any other reoperation, and (2) the radiological outcome after 1- and 2-stage revision, using an uncemented stem. Patients and methods - Between January 1993 and December 2012, an uncemented revision stem was used in 81 THAs revised for PJI. Patients were treated with 1- or 2-stage procedures according to a well-defined algorithm (1-stage: n = 28; 2-stage: n = 53). All hips had a clinical and radiological follow-up. Outcome parameters were eradication of infection, re-revision of the stem, and radiological changes. Survival was calculated using Kaplan-Meier analysis. Radiographs were analyzed for bone restoration and signs of loosening. The mean clinical follow-up time was 7 (2-15) years. Results - The 7-year infection-free survival was 96% (95% CI: 92-100), 100% for 1-stage revision and 94% for 2-stage revision (95% CI: 87-100) (p = 0.2). The 7-year survival for aseptic loosening of the stem was 97% (95% CI: 93-100), 97% for 1-stage revision (95% CI: 90-100) and 97% for 2-stage revision (95% CI: 92-100) (p = 0.3). No further infection or aseptic loosening occurred later than 7 years postoperatively. The radiographic results were similar for 1- and 2-stage procedures. Interpretation - Surgical management of PJI with stratification to 1- or 2-stage exchange according to a well-defined algorithm combined with antibiotic treatment allows the safe use of uncemented revision stems. Eradication of infection can be achieved in most cases, and medium- and long-term results appear to be comparable to those for revisions for aseptic loosening.

Thermodynamics of proton transport coupled ATP synthesis.

The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the ß-subunits in F(1), is c/ß=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase.

One-stage revision of infected hip arthroplasty: outcome of 39 consecutive hips.

PURPOSE: There are various options for treating periprosthetic joint infection (PJI). Two-stage exchange has traditionally been the gold standard. However, if the appropriate surgical intervention is chosen according to a rational algorithm, the outcome is similar when using all types of interventions. In an observational cohort study, the outcome of patients with PJI after hip replacement treated with one-stage revision was analysed. METHODS: All patients fulfilling all criteria for one-stage exchange according to the Infectious Diseases Society of America (IDSA) guidelines and six without preoperative identification of a microorganism were included. Implant removal, debridement and cemented or uncemented reimplantations were performed in a single intervention. If a cemented device was implanted, commercially available gentamicin cement was used in all cases. Antibiotic treatment was administered intravenously for at least 2 weeks, followed by oral therapy for a total duration of 3 months. Patients had standardised clinical and radiological follow-up visits. RESULTS: Between 1996 and 2011, 38 patients (39 hips) were treated with a one-stage procedure and followed for at least 2 years. Coagulase-negative staphylococci were the most frequent pathogens, and polymicrobial infection was observed in five cases. In 25 hips, an uncemented revision stem was implanted, and 37 hips received an acetabular reinforcement ring. The mean follow-up was 6.6 (2.0-15.1) years. No patient had persistent, recurrent or new infection. There were four stem revisions for aseptic loosening. The mean Harris Hip Score was 81 points (26-99) at the final follow-up. CONCLUSIONS: Excellent cure rate and function seen in our study suggest that one-stage exchange is a safe procedure, even without local antibiotic treatment, provided that the patient has no sinus tract or severe soft tissue damage, no major bone grafting is required and the microorganism is susceptible to orally administered agents with high bioavailability.

Single-particle EM reveals plasticity of interactions between the adenovirus penton base and integrin αVß3.

Human adenoviruses are double-stranded DNA viruses responsible for numerous infections, some of which can be fatal. Furthermore, adenoviruses are currently used in clinical trials as vectors for gene therapy applications. Although initial binding of adenoviruses to host attachment receptors has been extensively characterized, the interactions with the entry receptor (integrins) remain poorly understood at the structural level. We characterized the interactions between the adenovirus 9 penton base subunit and αVß3 integrin using fluorescence correlation spectroscopy and single-particle electron microscopy to understand the mechanisms underlying virus internalization and infection. Our results indicate that the penton base subunit can bind integrins with high affinity and in several different orientations. These outcomes correlate with the requirement of the pentameric penton base to simultaneously bind several integrins to enable their clustering and promote virus entry into the host cell.

Recurrent bone fractures due to tenofovir-induced renal phosphate wasting.

A 42-y-old HIV-infected man suffered from several stress fractures due to tenofovir-induced proximal tubular injury. Laboratory examination revealed hypophosphatemia due to renal phosphate wasting. Therefore, more attention has to be paid to the monitoring of serum phosphate and alkaline phosphatase levels, since tenofovir-related nephrotoxicity increases the risk of osteomalacia.

Risk factors for periprosthetic ankle joint infection: a case-control study.

BACKGROUND: Periprosthetic ankle joint infection is a feared complication of total ankle arthroplasty because the implant fails in the majority of cases. However, risk factors for developing these infections are unknown. METHODS: We aimed to determine risk factors for infection in a matched case-control study that included twenty-six patients with periprosthetic ankle joint infection and two control groups, each consisting of fifty-two patients. RESULTS: The prevalence of periprosthetic ankle joint infection within our cohort was 4.7%. Four infections (15%) had a hematogenous origin and twenty-two (85%), an exogenous origin. Staphylococcus aureus was the most common pathogen, followed by coagulase-negative staphylococci. Preoperative predisposing factors associated with infection included prior surgery at the site of infection (odds ratio [OR] = 4.56, 95% confidence interval [CI] = 0.98 to 21.35, and OR = 4.78, 95% CI = 1.53 to 14.91, in comparison with the two control groups) and a low American Orthopaedic Foot & Ankle Society (AOFAS) hindfoot score (35.8 versus 49.8 and 47.6 in the two control groups, p ≤ 0.02). The mean duration of the index surgery was significantly longer in the case group than in both control groups (119 versus eighty-four and ninety-three minutes, p ≤ 0.02). After surgery, persistent wound dehiscence (OR = 15.38, 95% CI = 2.91 to 81.34, p = 0.01, in comparison with both control groups) and secondary wound drainage (OR = 7.00, 95% CI = 1.45 to 33.70, and OR = 5.31, 95% CI = 1.01 to 26.78, in comparison with the two control groups, p ≤ 0.04) were associated with the development of a periprosthetic ankle joint infection. CONCLUSIONS: Patients at risk for periprosthetic ankle joint infection following total ankle arthroplasty include those with a history of surgery on the ankle, a low preoperative AOFAS hindfoot score, and a long operative time. Postoperatively, patients with a prolonged wound dehiscence or a secondary wound-healing problem are also at risk for infection.

Comparison of the H+/ATP ratios of the H+-ATP synthases from yeast and from chloroplast.

F(0)F(1)-ATP synthases use the free energy derived from a transmembrane proton transport to synthesize ATP from ADP and inorganic phosphate. The number of protons translocated per ATP (H(+)/ATP ratio) is an important parameter for the mechanism of the enzyme and for energy transduction in cells. Current models of rotational catalysis predict that the H(+)/ATP ratio is identical to the stoichiometric ratio of c-subunits to ß-subunits. We measured in parallel the H(+)/ATP ratios at equilibrium of purified F(0)F(1)s from yeast mitochondria (c/ß = 3.3) and from spinach chloroplasts (c/ß = 4.7). The isolated enzymes were reconstituted into liposomes and, after energization of the proteoliposomes with acid-base transitions, the initial rates of ATP synthesis and hydrolysis were measured as a function of ΔpH. The equilibrium ΔpH was obtained by interpolation, and from its dependency on the stoichiometric ratio, [ATP]/([ADP]·[P(i)]), finally the thermodynamic H(+)/ATP ratios were obtained: 2.9 ± 0.2 for the mitochondrial enzyme and 3.9 ± 0.3 for the chloroplast enzyme. The data show that the thermodynamic H(+)/ATP ratio depends on the stoichiometry of the c-subunit, although it is not identical to the c/ß ratio.

Prosthetic valve endocarditis caused by a Pasteurella dagmatis-like isolate originating from a patient's cat.

Pasteurella species are part of the oral flora of cats and dogs. In humans, they are frequently found in infected animal bite wounds, but invasive infections are rare. This is the first report of prosthetic-valve endocarditis with a Pasteurella dagmatis-like species, which originated from the patient's cat.

Periprosthetic joint infection following Staphylococcus aureus bacteremia.

OBJECTIVES: The incidence of haematogenous periprosthetic joint infections (PJI) among patients with remote infections has been reported to be less than 1%. This incidence may be much higher in cases after documented Staphylococcus aureus bacteremia (SAB). We evaluated the incidence of haematogenous PJI following SAB in patients with previously uninfected arthroplasties. METHODS: A retrospective analysis of our cohort including patients with SAB and prosthetic joints at the Basel University Medical Clinic Liestal from 1998 to 2008. RESULTS: We identified 31 patients with 45 uninfected prosthetic joints in situ at the time of SAB. In 12 patients (39%) and 13 arthroplasties (29%), SAB caused PJI. In comparison to nosocomial SAB, infections occurred only in cases with community-acquired SAB (p=0.002). PJI was diagnosed within a median time of 2.5 days (IQR 1-3.5) after admission. The comparison between patients with and without PJI revealed no significant difference in gender, age, comorbidities and number of prostheses per patient and age of the prosthesis. CONCLUSIONS: The rate of PJI after SAB is high, ranging from 30% to 40%, and clearly higher than rates reported for bacteremia with other pathogens. PJIs were observed in community-onset bacteremia, in which there is a typically delay from symptoms to antimicrobial treatment.

Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.

H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.

Proton transport coupled ATP synthesis by the purified yeast H+ -ATP synthase in proteoliposomes.

The H(+)/ATP synthase from yeast mitochondria, MF0F1, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF0F1 liposomes were energized by acid-base transitions (DeltapH) and a K(+)/valinomycin diffusion potential (Deltaphi). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120s⁻¹ at a transmembrane pH difference of 3.2 units (at pH(in)=4.8 and pH(out)=8.0) and a Deltaphi of 133mV (Nernst potential). Functional studies showed that the monomeric MF0F1, was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K(M) on pH(out) indicated that the substrate for ATP synthesis is the monoanionic phosphate species H2PO⁻4.

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication.

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Subunit movements in single membrane-bound H+-ATP synthases from chloroplasts during ATP synthesis.

Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

Quantum dots for single-pair fluorescence resonance energy transfer in membrane- integrated EFoF1.

spFRET (single-pair fluorescence resonance energy transfer) with organic fluorophores has been used to demonstrate rotation of the subunits gamma and epsilon in membrane-integrated FoF1 during proton transport-coupled ATP synthesis. Owing to the high light intensities used in single-molecule spectroscopy, organic fluorophores show a high probability for photobleaching. Luminescent CdSe/ZnS nanocrystals with a hydrophilic shell have been covalently bound to FoF1 either to the stator subunit b or to the rotor subunit c. TIRFM (total internal reflection microscopy) shows that covalent binding of the QD (quantum dot) via cysteine to FoF1 leads to a significant decrease in the blinking probability in the microsecond-to-second time range. This effect allows the observation of subunit movements in an extended time range. If the QD is bound to the rotor subunit c, the fluorescence anisotropy shows fluctuations in the presence of ATP, in contrast with the constant anisotropy observed in the absence of ATP.