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Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of plateletderived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation., Introduction Untoward thrombus formation is associated with multiple major clinical disorders and is a leading cause of death and disability worldwide (1). Excessive platelet activation and aggregation at sites of [...]

Current high‐speed atomic force microscopy (HS‐AFM) setups reach imaging speeds of several images per second but often have limited options for imaging live cells because of a small scan range, a lack of environmental control, or a missing combination with optical phase‐contrast or fluorescence microscopy. A HS‐AFM setup is therefore developed with a large scan range optimized for imaging live cells. The setup is equipped with temperature and CO2 control and is mounted on an inverted optical microscope providing high‐quality phase‐contrast and fluorescence microscopy. To demonstrate the capabilities of the setup, fast force mapping on live human platelets is performed. Further, HS‐AFM images and optical phase‐contrast and actin fluorescence images of live cancer cells are simultaneously recorded, and two state‐of‐the‐art AFM modes for imaging viscoelastic sample properties, force clamp force mapping and resonance compensating chirp mode, are compared. The setup is then applied to the investigation of viscoelastic material properties of cells in different cell cycle states. Using a melanoma cell line with a fluorescent cell cycle sensor, it is found that during the cell cycle not only cell volume and morphology, but also viscoelastic material properties significantly change, with increasing stiffness and decreasing fluidity from the G1 through the G1/S to the S/G2/M phases. [ABSTRACT FROM AUTHOR]

Abstract Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates. Results Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. Conclusions The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains.

Abstract Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein. Results Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these. Conclusions Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface.

Scanning ion conductance microscopy (SICM) is an emerging tool for the noncontact investigation of biological samples such as live cells. It uses an ion current through the opening of a tapered nanopipette filled with an electrolyte for topography measurements. Despite its successful application to numerous systems no systematic investigation of the image formation process has yet been performed. Here, we use finite element modeling to investigate how the scanning ion conductance microscope images small particles on a planar surface, providing a fundamental characterization of the imaging process. We find that a small particle appears with a height that is only a fraction of its actual height. This has significant consequences for the quantitative interpretation of SICM images. Furthermore, small and low particles are imaged as rings in certain cases. This can cause small, closely spaced particles to appear with a lateral orientation that is rotated by 90°. Considering both real space and spatial frequency space we find that a reasonable and useful definition of lateral resolution of SICM is the smallest distance at which two small particles can clearly be resolved from each other in an image. We find that this resolution is approximately equal to three times the inner radius of the pipette tip opening. [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) andscanning ion conductance microscopy(SICM) are excellent and commonly used techniques for imaging thetopography of living cells with high resolution. We present a directcomparison of AFM and SICM for imaging microvilli, which are smallfeatures on the surface of living cells, and for imaging the shapeof whole cells. The imaging quality on microvilli increased significantlyafter cell fixation for AFM, whereas for SICM it remained constant.The apparent shape of whole cells in the case of AFM depended on theimaging force, which deformed the cell. In the case of SICM, celldeformations were avoided, owing to the contact-free imaging mechanism.We estimated that the lateral resolution on living cells is limitedby the cell’s elastic modulus for AFM, while it is not forSICM. By long-term, time-lapse imaging of microvilli dynamics, weshowed that the imaging quality decreased with time for AFM, whileit remained constant for SICM. [ABSTRACT FROM AUTHOR]