ABSTRACT
Purpose: To determine the repeatability and reproducibility of anterior segment optical coherence tomography (AS-OCT) and Scheimpflug photography for several measurements of corneal scars, including scar size, scar depth, and corneal thickness. Methods: A series of patients treated for fungal keratitis at a tertiary eye care center in South India were recalled two years after successful treatment. Eyes with corneal scars had a slit lamp examination performed by two ophthalmologists masked to the other's examination. For AS-OCT and Scheimpflug photography, each eye had two scans taken by one technician and a third scan taken by a separate technician. Scar measurements were subsequently assessed from AS-OCT images by three graders masked to each other's results. Repeatability and reproducibility were assessed by calculating the intra-class correlation coefficient (ICC) from mixed effects linear regression models. Results: Fifty eyes had all measurements taken. The corneal scar size, measured as the geometric mean of the two longest perpendicular meridians, ranged from 0.8 to 5.4 (mean 2.8 mm, 95%CI 2.6 to 3.1). Scar size measurements taken by two separate individuals were most reproducible when the border of the scar was traced from the OCT (ICC 0.90, 95%CI 0.86 to 0.94), and least repeatable when assessed from slit lamp examination (ICC 0.80, 95%CI 0.70 to 0.90). Conclusions: AS-OCT and Scheimpflug imaging of corneal scars produced measurements with acceptable reproducibility that could be useful as cornea-specific outcomes for clinical trials.
Subject(s)
Cicatrix/diagnosis , Cornea/pathology , Corneal Pachymetry/methods , Corneal Ulcer/complications , Eye Infections, Fungal/complications , Slit Lamp Microscopy/methods , Tomography, Optical Coherence/methods , Adult , Antifungal Agents/therapeutic use , Cicatrix/etiology , Corneal Ulcer/diagnosis , Corneal Ulcer/drug therapy , Eye Infections, Fungal/diagnosis , Female , Humans , Male , Middle Aged , Reproducibility of ResultsABSTRACT
Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair/physiology , Nucleosomes/metabolism , Chromatin/chemistry , Chromatin/metabolism , Chromatin/radiation effects , DNA/chemistry , DNA/metabolism , DNA/radiation effects , DNA Damage , DNA Glycosylases/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Humans , Models, Molecular , Nucleic Acid Conformation , Oxidation-ReductionABSTRACT
Coiled-coil protein structural motifs have proven amenable to the design of structurally well-defined biomaterials. Mesoscale structural properties can be fairly well predicted based on rules governing the chemical interactions between the helices that define this structural motif. We explore the role of the hydrophobic core residues on the self-assembly of a coiled-coil polymer through a mutational analysis coupled with a salting-out procedure. Because the resultant polymers remain in solution, a thermodynamic approach is applied to characterize the polymer assembly using conventional equations from polymer theory to extract nucleation and elongation parameters. The stabilities and lengths of the polymers are measured using circular dichroism spectropolarimetry, sizing methods including dynamic light scattering and analytical ultracentrifugation, and atomic force microscopy to assess mesoscale morphology. Upon mutating isoleucines at two core positions to serines, we find that polymer stability is decreased while the degree of polymerization is about the same. Differences in results from circular dichroism and dynamic light scattering experiments suggest the presence of a stable intermediate state, and a scheme is proposed for how this intermediate might relate to the monomer and polymer states.
