New perspectives on keratoconus as revealed by corneal confocal microscopy: Invited Review

Confocal microscopy (CM) of keratoconus is reviewed. In the Manchester Keratoconus Study (MKS), slit scanning CM was used to evaluate 29 keratoconic patients and light microscopy (LM) was performed on two of the keratoconic corneas post-keratoplasty. The findings of the MKS are compared with other CM studies. Consideration of the differences between studies of cell counts is confounded by the use of different experimental controls. A consensus exists among studies with respect to qualitative observations. The epithelium appears more abnormal with increasing severity of keratoconus. In severe disease, the superficial epithelial cells are elongated and spindle shaped, epithelial wing cell nuclei are larger and more irregularly spaced and basal epithelial cells are flattened. Bowman's layer is disrupted and split in the region of the cone and intermixed with epithelial cells and stromal keratocytes. Stromal haze and hyper-reflectivity observed with CM correspond with apical scarring seen with the slitlamp biomicroscope (SLB). Hyper-reflective keratocyte nuclei are thought to indicate the presence of fibroblastic cells. Increased haze detected with CM is found with LM to be due to fibroblastic accumulation and irregular collagen fibres. Dark stromal bands observed with CM correlate with the appearance of Vogt's striae with SLB. Desçemet's membrane appears normal with both CM and LM. Some evidence of endothelial cell elongation is observed with CM. The application of CM to ophthalmic practice has facilitated a greater understanding of medical and surgical approaches that are used to treat keratoconus. This review offers new perspectives on keratoconus and provides a framework, against which tissue changes in this visually debilitating condition can be studied in a clinical context in vivo using CM

Assessment of Local Friction in Protein Folding Dynamics Using a Helix Cross-Linker

Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measureable via folding kinetic studies. Specifically, we show that when a helix cross-linker, m-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C), but has little effect on protein stability. Thus, these results, in conjunction with those obtained with another cross-linked trp-cage and two uncross-linked variants, demonstrate the feasibility of using a non-perturbing cross-linker to help quantify the effect of internal friction. In addition, we estimate that an m-xylene cross-linker could lead to an increase in the roughness of the folding energy landscape by as much as 0.4-1.0k(B)T