The Impact of Different Rose Bengal Formulations on Corneal Thickness and the Efficacy of Rose Bengal/Green Light Corneal Cross-linking in the Rabbit Eye.

PURPOSE: To examine central corneal thickness (CCT) changes during in vivo rose bengal-green light corneal cross-linking (RG-CXL) and compare the CXL efficacy of different rose bengal formulations. METHODS: After epithelium removal, the right eyes of rabbits were immersed in rose bengal solution prepared by different solvents (water, phosphate buffered saline, dextran, and hydroxypropyl methylcellulos [HPMC]) for 2 or 20 minutes, then the rose bengal distribution in the corneal stroma was analyzed by confocal fluorescence detection. During the RG-CXL process, the CCT was measured at seven time points. The left eyes served as the untreated control group. Corneal enzymatic resistance and corneal biomechanics were tested to compare the RG-CXL efficacy. RESULTS: The rose bengal infiltration depths were 120 and 200 µm for the 2- and 20-minute groups, respectively. CCT increased significantly after infiltration, then decreased significantly in the first 200 seconds of irradiation and decreased slowly for the next 400 seconds. The CCT of the 20-minute groups was significantly thicker than that of the 2-minute groups (P < .0001). All RG-CXL treatments improved the corneal enzymatic resistance and corneal biomechanics, with the effects being greater in the 20-minute groups. The inclusion of 1.1% HPMC in the rose bengal formulation helped to maintain CCT during irradiation while not affecting either the infiltration of rose bengal or the efficacy of RG-CXL. CONCLUSIONS: Within the range studied, RG-CXL efficacy increased with infiltration time. The incorporation of a 20-minute infiltration of 0.1% rose bengal-1.1% HPMC into the RG-CXL procedure may further improve the safety of the treatment and its prospects for clinical use. [J Refract Surg. 2022;38(7):450-458.].

Psychometric Assessment of the Chinese Version of the Indian Vision Functioning Questionnaire Based on the Method of Successive Dichotomizations.

Purpose: The purpose of this study was to assess whether a Chinese translated version of the 33-item Indian Vision Function Questionnaire (IND-VFQ-33) forms a valid measurement scale and to evaluate its psychometric properties based on the method of successive dichotomizations (MSD). Methods: The English version of the IND-VFQ-33 was translated, back translated, and cross-culturally adapted for use in China. It was interviewer administered to patients with cataracts. MSD, a polytomous Rasch model that estimates ordered thresholds, was used to assess and optimize psychometric properties of the overall scale and three subscales separately. Results: One hundred and seventy-nine patients provided complete responses. After the removal of 2 misfitting items, a revised 31-item overall scale demonstrated adequate precision (person reliability [PR] = 0.92) and no misfitting items. The general functioning subscale fit the MSD model well after removing two misfitting items. The psychosocial impact subscale and the visual symptoms subscale were not considered further due to poor measurement precision. After addressing psychometric deficiencies, a 31-item overall scale (IND-VFQ-31-CN) and a 19-item general functioning subscale (IND-VFQ-GF-19-CN) were developed. Conclusions: The original IND-VFQ-33 required re-engineering to form valid measures for use in China. The revised overall scale and general functioning subscale demonstrated adequate MSD based psychometric properties. Translational Relevance: The revised IND-VFQ-33 is a valid patient-reported outcome assessment for Chinese patients with cataract based on MSD analysis.

Simulating structural and thermodynamic properties of carcinogen-damaged DNA.

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.

Role of base sequence context in conformational equilibria and nucleotide excision repair of benzo[a]pyrene diol epoxide-adenine adducts.

We investigate the influence of base sequence context on the conformations of the 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts through molecular dynamics (MD) simulations with free energy calculations, and relate the structural findings to results of nucleotide excision repair (NER) assays in human cell extracts. In previous studies, these adducts were studied in the CA*A sequence context, and here we report results for the CA*C sequence. Our simulations indicate that the base sequence context affects the syn-anti conformational equilibrium in the 10S (+) adduct by modulating the barrier heights between these states on the energy surface, with a higher barrier in the CA*C case. Our nucleotide excision repair assay finds greater NER susceptibilities in the 10S (+) adduct for the CA*C sequence context. A structural rationale ties together these results. A sequence specific hydrogen bond, accompanied by a significantly increased roll and consequent bending in the 10S (+) adduct, has been found in our simulations for the CA*C sequence, which could account for the enhanced nucleotide excision repair as well as the syn-anti equilibrium difference we observe in this isomer and sequence. Such sequence specific differential repair could contribute to the existence of mutational hotspots and thereby contribute to the complexity of cancer initiation.

Analysis of protein sequence/structure similarity relationships.

Current analyses of protein sequence/structure relationships have focused on expected similarity relationships for structurally similar proteins. To survey and explore the basis of these relationships, we present a general sequence/structure map that covers all combinations of similarity/dissimilarity relationships and provide novel energetic analyses of these relationships. To aid our analysis, we divide protein relationships into four categories: expected/unexpected similarity (S and S(?)) and expected/unexpected dissimilarity (D and D(?)) relationships. In the expected similarity region S, we show that trends in the sequence/structure relation can be derived based on the requirement of protein stability and the energetics of sequence and structural changes. Specifically, we derive a formula relating sequence and structural deviations to a parameter characterizing protein stiffness; the formula fits the data reasonably well. We suggest that the absence of data in region S(?) (high structural but low sequence similarity) is due to unfavorable energetics. In contrast to region S, region D(?) (high sequence but low structural similarity) is well-represented by proteins that can accommodate large structural changes. Our analyses indicate that there are several categories of similarity relationships and that protein energetics provide a basis for understanding these relationships.

Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence. A slipped frameshift intermediate model for dinucleotide deletion.

In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 ()-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery.

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability.

A key issue in the nucleotide excision repair (NER) of bulky carcinogen-DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene and the corresponding (-)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (-)-trans-anti-B[c]Ph-N6-dA adducts. Duplexes containing the 1R (+) or 1S (-) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (-)-trans-anti-B[a]P-N6-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (-)-trans-anti-B[c]Ph-N6-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson- Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson-Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.

Conformational analysis of a 4-hydroxyequilenin Guanine adduct using density functional theory.

Equilenin, a component of the drug Premarin (Wyeth), can be metabolized to a quinonoid, 4-hydroxyequilenin (4-OHEN). 4-OHEN can react with 2'-deoxynucleosides to form unusual cyclic adducts, among which 4-hydroxyequilenin-2'-deoxyguanosine (4-OHEN-dG) is the major product under physiological conditions. The structure and stereochemistry of one stereoisomer, 4-OHEN-dG1, has been obtained previously using electrospray mass spectrometry and NMR methods [Shen et al. (1997) J. Am. Chem. Soc. 119, 11126-11127]; however, details of the conformations around the linkage site have not yet been investigated. The objective of this paper was to determine the conformation at the five-membered ring linkage site for this adduct. We have carried out a computational investigation involving high level quantum mechanical geometry optimization using density functional theory (DFT) for the 4-hydroxyequilenin-guanine adduct (4-OHEN-G1). Our results reveal that there are three conformational families which differ in the puckering of the five-membered ring at the linkage site and in the cyclohexene-type A ring conformation. The overall structures of all three families are "V"-shaped; however, two are quite compact while the third is more open. The lowest energy structure contains a half chair-type cyclohexene A ring, while two structures whose energies are approximately 3-4 kcal/mol higher are boat-type. Since the Watson-Crick hydrogen bonding edge of the modified guanine is obstructed by the formation of this bulky nonplanar adduct, it likely would reside in a groove of the DNA double helix.