From genes to protein mechanics on a chip.

Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

[Cross-sectoral quality assurance in ambulatory care]. / Sektorenübergreifende Qualitätssicherung in der ambulanten Versorgung.

Overcoming rigid sectoral segmentation in healthcare has also become a health policy target in quality assurance. With the Act to Enhance Competition in Statutory Health Insurance (GKV-WSG) coming into effect, quality assurance measures are to be designed in a cross-sectoral fashion for in- and outpatient sectors equally. An independent institution is currently mandated to develop specific quality indicators for eleven indications. For three of these operating tests have already been commissioned by the Federal Joint Committee. This article depicts the major results of a feasibility study, including a compliance cost estimate, for the aforementioned indications of cross-sectoral quality assurance (cQA). In conclusion, a number of both practical and conceptual basic challenges are still to be resolved prior to the full implementation of cQA, such as a sufficient specification to activate documentation requirements and an inspection system capable of separating actual quality problems from documentary deficits. So far, a comprehensive cost-utility analysis of cQA has not been provided, in particular with comparison to existing QA systems. In order to optimise cost and utility of cQA an evidence-based approach is required for both the extension of cQA areas and for QA provisions.

Protein-DNA force assay in a microfluidic format.

The detailed study of protein-DNA interactions is a core effort to elucidate physiological processes, including gene regulation, DNA repair and the immune response. The molecular force assay (MFA) is an established method to study DNA-binding proteins. In particular, high-affinity binder dissociation is made possible by the application of force. Microfluidic lab-on-a-chip approaches have proven helpful for parallelization, small sample volumes, reproducibility, and low cost. We report the successful combination of these two principles, forming a microfluidic molecular force assay and representing a novel use for the established MITOMI chip design. We present, characterize, validate and apply this integrated method. An alternative confocal fluorescence microscopy readout and analysis method is introduced and validated. In a multiplexing application, EcoRI binding is detected and characterized. This method paves the way for quantitative on-chip force measurements. It is suited for integration with DNA micro-spotting and in vitro expression of transcription factors to form a high-throughput chip for detailed DNA-protein interaction studies.

Local motion analysis reveals impact of the dynamic cytoskeleton on intracellular subdiffusion.

Intracellular transport is a complex interplay of ballistic transport along filaments and of diffusive motion, reliably delivering material and allowing for cell differentiation, migration, and proliferation. The diffusive regime, including subdiffusive, Brownian, and superdiffusive motion, is of particular interest for inferring information about the dynamics of the cytoskeleton morphology during intracellular transport. The influence of dynamic cytoskeletal states on intracellular transport are investigated in Dictyostelium discoideum cells by single particle tracking of fluorescent nanoparticles, to relate quantitative motion parameters and intracellular processes before and after cytoskeletal disruption. A local mean-square displacement (MSD) analysis separates ballistic motion phases, which we exclude here, from diffusive nanoparticle motion. In this study, we focus on intracellular subdiffusion and elucidate lag-time dependence, with particular focus on the impact of cytoskeleton compartments like microtubules and actin filaments. This method proves useful for binary motion state distributions. Experimental results are compared to simulations of a data-driven Langevin model with finite velocity correlations that captures essential statistical features of the local MSD algorithm. Specifically, the values of the mean MSD exponent and effective diffusion coefficients can be traced back to negative correlations of the motion's increments. We clearly identify both microtubules and actin filaments as the cause for intracellular subdiffusion and show that actin-microtubule cross talk exerts viscosifying effects at timescales larger than 0.2 s. Our findings might give insights into material transport and information exchange in living cells, which might facilitate gaining control over cell functions.