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Objective: Clinical trials for osteoarthritis (OA), the leading cause of global disability, are unable to pinpoint the early, potentially reversible disease with clinical technology. Hence, disease-modifying drug candidates cannot be tested early in the disease. To overcome this obstacle, we asked whether early OApathology detection is possible with current clinical technology. Methods: We determined the relationship between two sensitive early OA markers, atomic force microscopy (AFM)-measured human articular cartilage (AC) surface stiffness, and location-matched superficial zone chondrocyte spatial organizations (SCSOs), asking whether a significant loss of surface stiffness can be detected in early OA SCSO stages. We then tested whether current clinical technology can visualize and accurately diagnose the SCSOs using an approved probe-based confocal laser-endomicroscope and a random forest (RF) model. Results: We demonstrated a correlation between AC surface stiffness and the SCSO (r rm ¼ À0.91; 95% CI: À0.97, À0.73), and an extensive loss of surface stiffness specifically in those ACs with early OA-typical SCSO (95%CIs: string SCSO: 269-173 kPa, double string SCSO: 77-46 kPa). This established the SCSO as a visualizable, functionally relevant surrogate marker of early OA AC surface pathology. Moreover, SCSObased stiffness discrimination worked well in each patient's AC. We then demonstrated feasibility of visualizing the SCSO by clinical laser-endomicroscopy and, importantly, accurate SCSO diagnosis using RF. Conclusion: We present the proof-of-concept of early OA-pathology detection with available clinical technology, introducing a future-oriented, AI-supported, non-destructive quantitative optical biopsy for early disease detection. Operationalizing SCSO recognition, this approach allows testing for correlations between local tissue architectures with other experimental and clinical read-outs, but needs clinical validation and a larger sample size for defining diagnostic thresholds.

SummaryThe NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 Å unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston-like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine-scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spinprobe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement.

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A crucial step of exotoxin action is the attack on the membrane. Many exotoxins show an architecture following the AB model, where a binding subunit translocates an “action” subunit across a cell membrane. Atomic force microscopy is an ideal technique to study these systems because of its ability to provide structural as well as dynamic information at the same time. We report first images of toxins Photorhabdus luminescens TcdA1 and Clostridium difficile TcdB on a supported lipid bilayer. A significant amount of toxin binds to the bilayer at neutral pH in the absence of receptors. Lack of diffusion indicates that toxin particles penetrate the membrane. This observation is supported by fluorescence recovery after photobleaching measurements. We mimic endocytosis by acidification while imaging the particles over time; however, we see no large conformational change. We therefore conclude that the toxin particles we imaged in neutral conditions had already formed a pore and speculate that there is no “pre-pore” state in our imaging conditions (i.e., in the absence of receptor).

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