Susanna M. Früh

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody’s Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.

The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 10⁵ CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.

Saliva offers many advantages for point-of-care (PoC) diagnostic applications due to non-invasive, easy, and cost-effective methods of collection. However, the complex matrix with its non-Newtonian behavior and high viscosity poses handling challenges. Several tedious and long pre-analytic steps, incompatible with PoC use, are required to liquefy and homogenize saliva samples before protein analysis can be performed. We apply magnet-beating to reduce hands-on time and to simplify sample preparation. A magnet in a chamber containing the whole saliva is actuated inside a centrifugal microfluidic cartridge by the interplay of centrifugal and magnetic forces. Rigorous mixing, which homogenizes the saliva sample, is then initiated. Consequently, fewer manual steps are required to introduce the whole saliva into the cartridge. After 4 min of magnet-beating, the processed sample can be used for protein analysis. The viscosity of whole saliva has been reduced from 10.4 to 2.3 mPa s. Immunoassay results after magnet-beating for three salivary periodontal markers (MMP-8, MMP-9, TIMP-1) showed a linear correlation with a slope of 0.99 when compared to results of reference method treated samples. Conclusively, magnet-beating has been shown to be a suitable method for the pre-analytic processing of whole saliva for fully automated PoC protein analysis.