Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 μm thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell-cell interaction, pores with 0.5 to 5.0 μm diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures was demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest this new biomaterial system offers important potential benefits for corneal tissue regeneration.

The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.

Osteoblasts (OSTs) are derived from mesenchymal stem cells (MSCs) and coexist in close proximity with MSCs in bone during development and remodelling. Interactions between these two cell types remain obscure. Through a well-defined co-culture model, the present work demonstrated that OSTs regulate MSCs through the WNT and cadherin pathways. The regulation mechanism depends on the cell-cell contact mode (indirect or direct) between the two cell types. When physically separated (indirect contact), OSTs express WNTs and stimulate the osteogenic differentiation of MSCs through the activation of the WNT pathway and suppression of the cadherin pathway. This mechanism is evidenced by: (a) the elevation of cytoplasmic and nuclear unphosphorylated beta-catenin protein levels; (b) the suppression of beta-catenin degradation; (c) the increase in WNT-related transcription factor TCF1/LEF1; and (d) the loss of major bone-related cadherins (N-CAD and CAD11). Addition of DKK1 antagonizes the WNT pathway and diminishes the stimulatory effect of OSTs on MSCs. When in direct cell-cell contact, OSTs still secrete WNTs, whose binding still stabilizes the beta-catenin in MSCs. However, direct cell-cell contact induces the upregulation of cadherin pathway in MSCs, which suppresses the WNT pathway by containing cytoplasmic beta-catenin protein at a low level; consequently, the stimulatory effect of OSTs is negated. Regulation of cytoplasmic beta-catenin protein levels through concerted action or crosstalk between the WNT and cadherin pathways is the key to the signalling transduction in these cellular communication networks.

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