Characterization of synovial fluid from periprosthetic infection in revision total joint arthroplasty by single-molecule microscopy.

Periprosthetic joint infection is among the most common and severe complications in total joint arthroplasty. Today, a combination of different methods is used for diagnosis because no single method with sufficient sensitivity and specificity is available. In this study, we explored the usability of single-molecule microscopy to characterize synovial fluid samples from periprosthetic joint infections. Patients (n = 27) that needed revision arthroplasty underwent the routine diagnostic procedures for periprosthetic joint infection of the University Hospital in Bonn. Additionally, the diffusion rate of two probes, dextran and hyaluronan, was measured in small volumes of periprosthetic synovial fluid samples using single-molecule microscopy. To evaluate the suitability of single-molecule microscopy to detect PJI the AUC for both markers was calculated. The diffusion rate of hyaluronan in periprosthetic synovial fluid from patients with septic loosening was faster than in samples from patients with aseptic loosening. Single-molecule microscopy showed excellent diagnostic performance, with an area under the receiver operating characteristic curve of 0.93, and allowed the detection of periprosthetic joint infection in patients that would be challenging to diagnose with current methods. For the first time, single-molecule microscopy was used to detect periprosthetic joint infection. Our results are encouraging to study the value of single-molecule microscopy in a larger patient cohort. The speed and accuracy of single-molecule microscopy can be used to further characterize synovial fluid, potentially allowing intraoperative diagnosis of periprosthetic joint infections in the future.

The Structure of Murine N1-Acetylspermine Oxidase Reveals Molecular Details of Vertebrate Polyamine Catabolism.

N1-Acetylspermine oxidase (APAO) catalyzes the conversion of N1-acetylspermine or N1-acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N1-acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.

Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis.

Osteoarthritis is a common and progressive joint disorder. Despite its widespread, in clinical practice only late phases of osteoarthritis that are characterized by severe joint damage are routinely detected. Since osteoarthritis cannot be cured but relatively well managed, an early diagnosis and thereby early onset of disease management would lower the burden of osteoarthritis. Here we evaluated if biophysical parameters of small synovial fluid samples extracted by single molecule microscopy can be linked to joint damage. In healthy synovial fluid (ICRS-score < 1) hyaluronan showed a slower diffusion (2.2 µm(2)/s, N = 5) than in samples from patients with joint damage (ICRS-score > 2) (4.5 µm(2)/s, N = 16). More strikingly, the diffusion coefficient of hyaluronan in healthy synovial fluid was on average 30% slower than expected by sample viscosity. This effect was diminished or missing in samples from patients with joint damage. Since single molecule microscopy needs only microliters of synovial fluid to extract the viscosity and the specific diffusion coefficient of hyaluronan this method could be of use as diagnostic tool for osteoarthritis.

Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking.

Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1-3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.

Nuclear trafficking and export of single, native mRNPs in Chironomus tentans salivary gland cells.

Real-time observation of single molecules or biological nanoparticles with high spatial resolution in living cells provides detailed insights into the dynamics of cellular processes. The salivary gland cells of Chironomus tentans are a well-established model system to study the processing of RNA and the formation and fate of messenger ribonucleoprotein particles (mRNPs). For a long time, challenging imaging conditions limited the access to this system for in vivo fluorescence microscopy. Recent technical and methodical advantages now allow observing even single molecules in these cells. We describe here the experimental approach and the optical techniques required to analyze intranuclear trafficking and export of single native mRNPs across the nuclear envelope.

Light-inducible molecular beacons for spatio-temporally highly defined activation.

We have developed a new molecular beacon design that requires an additional UV pulse for fluorescence activation. This improves the signal-to-noise ratio tremendously compared to previous approaches and allows for a precise control of the time point and location of RNA labelling.

Single-molecule imaging of hyaluronan in human synovial fluid.

Human synovial fluid contains a high concentration of hyaluronan, a high molecular weight glycosaminoglycan that provides viscoelasticity and contributes to joint lubrication. In osteoarthritis synovial fluid, the concentration and molecular weight of hyaluronan decrease, thus impairing shock absorption and lubrication. Consistently, substitution of hyaluronan (viscosupplementation) is a widely used treatment for osteoarthritis. So far, the organization and dynamics of hyaluronan in native human synovial fluid and its action mechanism in viscosupplementation are poorly characterized at the molecular level. Here, we introduce highly sensitive single molecule microscopy to analyze the conformation and interactions of fluorescently labeled hyaluronan molecules in native human synovial fluid. Our findings are consistent with a random coil conformation of hyaluronan in human synovial fluid, and point to specific interactions of hyaluronan molecules with the synovial fluid matrix. Furthermore, single molecule microscopy is capable of detecting the breakdown of the synovial fluid matrix in osteoarthritis. Thus, single molecule microscopy is a useful new method to probe the structure of human synovial fluid and its changes in disease states like osteoarthritis.