Enhancing the efficacy of AREDS antioxidants in light-induced retinal degeneration.

PURPOSE: Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage. METHODS: Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation. RESULTS: Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions. CONCLUSIONS: Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD.

Objective measurement of erythema in psoriasis using digital color photography with color calibration.

BACKGROUND: Traditional metrics for evaluating the severity of psoriasis are subjective, which complicates efforts to measure effective treatments in clinical trials. METHODS: We collected images of psoriasis plaques and calibrated the coloration of the images according to an included color card. Features were extracted from the images and used to train a linear discriminant analysis classifier with cross-validation to automatically classify the degree of erythema. The results were tested against numerical scores obtained by a panel of dermatologists using a standard rating system. RESULTS: Quantitative measures of erythema based on the digital color images showed good agreement with subjective assessment of erythema severity (κ = 0.4203). The color calibration process improved the agreement from κ = 0.2364 to κ = 0.4203. CONCLUSION: We propose a method for the objective measurement of the psoriasis severity parameter of erythema and show that the calibration process improved the results.

Full-length hdmX transcripts decrease following genotoxic stress.

Previous studies have suggested that the mdmX gene is constitutively transcribed, and that MdmX protein activity is instead controlled by cellular localization and DNA damage induced Mdm2-mediated ubiquitination leading to proteasomal degradation. In these studies, we report that the human mdmX (hdmX) mRNA is reproducibly decreased in various human cell lines following treatment with various DNA-damaging agents. Repression of hdmX transcripts is observed in DNA-damaged HCT116 colon cancer cells and in isogenic p53(-/-) cells, suggesting that this effect is p53-independent. Reduction in the amount of hdmX transcript occurs in both human tumor cell lines and primary human diploid fibroblasts, and results in a significant reduction of HdmX protein. Examination of hdmX promoter activity suggests that damage-induced repression of hdmX mRNA is not significantly impacted by transcription initiation. In contrast, changes in hdmX mRNA splicing appear to partly explain the reduction in full-length hdmX mRNA levels in tumor cell lines with the destabilization of full-length hdmX transcripts, potentially through microRNA miR-34a regulation, also impacting transcript levels. Taken together, this study uncovers previously unrecognized cellular mechanisms by which hdmX mRNA levels are kept low following genotoxic stress.

Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function.

Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of cis-elements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB. Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.

The reliability of measuring physical characteristics of spiculated masses on mammography.

The goal of this study was to assess the reliability of measurements of the physical characteristics of spiculated masses on mammography. The images used in this study were obtained from the Digital Database for Screening Mammography. Two experienced radiologists measured the properties of 21 images of spiculated masses. The length and width of all spicules and the major axis of the mass were measured. In addition, the observers counted the total number of spicules. Interobserver and intraobserver variability were evaluated using a hypothesis test for equivalence, the intraclass correlation coefficient (ICC) and Bland-Altman statistics. For an equivalence level of 30% of the mean of the senior radiologist's measurement, equivalence was achieved for the measurements of average spicule length (p<0.01), average spicule width (p = 0.03), the length of the major axis (p<0.01) and for the count of the number of spicules (p<0.01). Similarly, with the ICC analysis technique "excellent" inter-rater agreement was observed for the measurements of average spicule length (ICC = 0.770), the length of the major axis (ICC = 0.801) and for the count of the number of spicules (ICC = 0.780). "Fair to good" agreement was observed for the average spicule width (ICC = 0.561). Equivalence was also demonstrated for intraobserver measurements. Physical properties of spiculated masses can be measured reliably on mammography. The interobserver and intraobserver variability for this task is comparable with that reported for other measurements made on medical images.

Bayesian networks of BI-RADStrade mark descriptors for breast lesion classification.

We investigated Bayesian network structure learning and probability estimation from mammographic feature data in order to classify breast lesions into different pathological categories. We compared the learned networks to naive Bayes classifiers, which are similar to the expert systems previously investigated for breast lesion classification. The learned network structures reflect the difference in the classification of biopsy outcome and the invasiveness of malignant lesions for breast masses and microcalcifications. The difference between masses and microcalcifications should be taken into consideration when interpreting systems for automatic pathological classification of breast lesions. The difference may also affect use of these systems for tasks such as estimating the sampling error of biopsy.

A neural network approach to breast cancer diagnosis as a constraint satisfaction problem.

A constraint satisfaction neural network (CSNN) approach is proposed for breast cancer diagnosis using mammographic and patient history findings. Initially, the diagnostic decision to biopsy was formulated as a constraint satisfaction problem. Then, an associative memory type neural network was applied to solve the problem. The proposed network has a flexible, nonhierarchical architecture that allows it to operate not only as a predictive tool but also as an analysis tool for knowledge discovery of association rules. The CSNN was developed and evaluated using a database of 500 nonpalpable breast lesions with definitive histopathological diagnosis. The CSNN diagnostic performance was evaluated using receiver operating characteristic analysis (ROC). The results of the study showed that the CSNN ROC area index was 0.84+/-0.02. The CSNN predictive performance is competitive with that achieved by experienced radiologists and backpropagation artificial neural networks (BP-ANNs) presented before. Furthermore, the study illustrates how CSNN can be used as a knowledge discovery tool overcoming some of the well-known limitations of BP-ANNs.

Increased thermohaline stratification as a possible cause for an ocean anoxic event in the Cretaceous period.

Ocean anoxic events were periods of high carbon burial that led to drawdown of atmospheric carbon dioxide, lowering of bottom-water oxygen concentrations and, in many cases, significant biological extinction. Most ocean anoxic events are thought to be caused by high productivity and export of carbon from surface waters which is then preserved in organic-rich sediments, known as black shales. But the factors that triggered some of these events remain uncertain. Here we present stable isotope data from a mid-Cretaceous ocean anoxic event that occurred 112 Myr ago, and that point to increased thermohaline stratification as the probable cause. Ocean anoxic event 1b is associated with an increase in surface-water temperatures and runoff that led to decreased bottom-water formation and elevated carbon burial in the restricted basins of the western Tethys and North Atlantic. This event is in many ways similar to that which led to the more recent Plio-Pleistocene Mediterranean sapropels, but the greater geographical extent and longer duration (approximately 46 kyr) of ocean anoxic event 1b suggest that processes leading to such ocean anoxic events in the North Atlantic and western Tethys were able to act over a much larger region, and sequester far more carbon, than any of the Quaternary sapropels.

Application of the mutual information criterion for feature selection in computer-aided diagnosis.

The purpose of this study was to investigate an information theoretic approach to feature selection for computer-aided diagnosis (CAD). The approach is based on the mutual information (MI) concept. MI measures the general dependence of random variables without making any assumptions about the nature of their underlying relationships. Consequently, MI can potentially offer some advantages over feature selection techniques that focus only on the linear relationships of variables. This study was based on a database of statistical texture features extracted from perfusion lung scans. The ultimate goal was to select the optimal subset of features for the computer-aided diagnosis of acute pulmonary embolism (PE). Initially, the study addressed issues regarding the approximation of MI in a limited dataset as it is often the case in CAD applications. The MI selected features were compared to those features selected using stepwise linear discriminant analysis and genetic algorithms for the same PE database. Linear and nonlinear decision models were implemented to merge the selected features into a final diagnosis. Results showed that the MI is an effective feature selection criterion for nonlinear CAD models overcoming some of the well-known limitations and computational complexities of other popular feature selection techniques in the field.

Fatal carbon monoxide poisoning after the detonation of explosives in an underground mine: a case report.

An unusual death caused by carbon monoxide poisoning after the detonation of explosives in an underground mine was investigated by the Office of the Medical Investigator of the State of New Mexico. The 50-year-old miner had 18 years of mining experience but no documented safety training. He collapsed approximately 20 minutes after entering the mine and working at the bottom of the single vertical shaft. The tight confines of the mine shaft hindered rescue personnel from reaching him, and the body was not recovered until 2 days later. The autopsy showed severe coronary artery atherosclerosis with remote and resolving myocardial microinfarcts, as well as the characteristic pink lividity of carbon monoxide poisoning, which was confirmed by laboratory analysis. Detailed investigation of the scene revealed no sources of carbon monoxide other than the explosives. The case represents an uncommon cause of death in mining that may have been avoided through the use of proper safety procedures, and illustrates the importance of recognizing the many sources of carbon monoxide.

Toward objective selection of representative microscope images.

Scientists wishing to communicate the essential characteristics of a pattern (such as an immunofluorescence distribution) currently must make a subjective choice of one or two images to publish. We therefore developed methods for objectively choosing a typical image from a set, with emphasis on images from cell biology. The methods involve calculation of numerical features to describe each image, calculation of similarity between images as a distance in feature space, and ranking of images by distance from the center of the feature distribution. Two types of features were explored, image texture measures and Zernike polynomial moments, and various distance measures were utilized. Criteria for evaluating methods for assigning typicality were proposed and applied to sets of images containing more than one pattern. The results indicate the importance of using distance measures that are insensitive to the presence of outliers. For collections of images of the distributions of a lysosomal protein, a Golgi protein, and nuclear DNA, the images chosen as most typical were in good agreement with the conventional understanding of organelle morphologies. The methods described here have been implemented in a web server (http://murphylab.web.cmu.edu/services/TyplC).

Automated recognition of patterns characteristic of subcellular structures in fluorescence microscopy images.

Methods for numerical description and subsequent classification of cellular protein localization patterns are described. Images representing the localization patterns of 4 proteins and DNA were obtained using fluorescence microscopy and divided into distinct training and test sets. The images were processed to remove out-of-focus and background fluorescence and 2 sets of numeric features were generated: Zernike moments and Haralick texture features. These feature sets were used as inputs to either a classification tree or a neural network. Classifier performance (the average percent of each type of image correctly classified) on previously unseen images ranged from 63% for a classification tree using Zernike moments to 88% for a backpropagation neural network using a combination of features from the 2 feature sets. These results demonstrate the feasibility of applying pattern recognition methods to subcellular localization patterns, enabling sets of previously unseen images from a single class to be classified with an expected accuracy greater than 99%. This will provide not only a new automated way to describe proteins, based on localization rather than sequence, but also has potential application in the automation of microscope functions and in the field of gene discovery.

Conformational changes of an Hsp70 molecular chaperone induced by nucleotides, polypeptides, and N-ethylmaleimide.

Hsp70 molecular chaperones are highly conserved ATPases that guide the folding and assembly of proteins in many cellular pathways. They use the energy of ATP binding and hydrolysis to regulate their interactions with hydrophobic regions of unfolded proteins. The activities and the conformations of the N-terminal nucleotide- and C-terminal polypeptide-binding domains of Hsp70s are coupled. We recently reported that the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) inactivates the yeast Hsp70 Ssa1p by reacting with its three cysteine residues which are located in the nucleotide-binding domain. To further characterize conformational changes associated with interdomain coupling and to determine whether NEM alters Ssa1p's conformation, the structures of Ssa1p and NEM-modified Ssa1p (NEM-Ssa1p) were compared using a variety of biophysical techniques. Size exclusion chromatography revealed that NEM-Ssa1p is more oligomeric and more resistant to nucleotide- or polypeptide-dependent depolymerization than Ssa1p. Measurement of the thermal stability indicated that NEM modification has an effect very similar to that of binding of nucleotides to the unmodified protein. Circular dichroism demonstrated small differences in the secondary structure of Ssa1p and NEM-Ssa1p, and in their complexes with nucleotides. NEM modification increased the ANS fluorescence of Ssa1p and exposed numerous trypsin-sensitive sites in its nucleotide-binding domain. The intrinsic fluorescence of Ssa1p's only tryptophan residue, which is located in a C-terminal alpha-helical region adjacent to the polypeptide-binding cleft, was quenched in the presence of ATP, but not ADP. NEM modification altered nucleotide-dependent changes in the intrinsic fluorescence of Ssa1p. Together, these results demonstrate that NEM alters the conformation of Ssa1p and disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidence for a model in which the polypeptide-binding cleft of Hsp70s is covered by an alpha-helical lid that is open in the presence of ATP, but closed in the presence of ADP.

Humoral immune responses to mycetoma organisms: characterization of specific antibodies by the use of enzyme-linked immunosorbent assay and immunoblotting.

Levels of antibodies were determined by enzyme-linked immunosorbent assay (ELISA) in 13 patients with eumycetomas due to Madurella mycetomatis infections. Raised levels of specific IgM were observed in 12 patients, compared with normal human controls. By contrast, low levels of specific IgG were detected in some patients. Specific responses to separated protein antigens were investigated by immunoblotting. Of 10 patients' sera tested, IgM in 2 recognized up to 7 of the blotted antigens between 45 and 84 kDa. Gold-labelled protein A (which predominantly binds to IgG) indicated that sera from 2 patients reacted with at least 6 protein bands with relative molecular masses between 64 and 95. The demonstration of significant IgM levels by ELISA, but few antigenic bands in sera from the same patients by immunoblotting, may point to an antibody response against polysaccharide fungal antigens in mycetoma patients. The use of the ELISA to detect antibodies of different classes and the characterization of their antigenic specificities by immunoblotting may have both diagnostic and prognostic value.

Ultrastructural and immunogenic changes in the formation of mycetoma grains.

The differences in the fine structure and antigenic determinants of mycetoma fungi in the mycelial phase in vitro and in grains in vivo facilitate an interpretation of grain formation. Aggregates of hyphal elements with multiple and thickened walls was a feature of the fungi in vivo. Associated with hyphal wall material, numerous polysaccharide microfibrils were detected in grains of Madurella mycetomatis. These were not seen associated with hyphal elements in vitro and it is suggested that these structures may be concerned with the aggregation of fungal elements in the formation of grains. Antibodies directed against the fungi in vitro were shown, by indirect immunogold labelling, to bind at identical sites in fungal material grown in vitro as in mycetoma grains. However the grain matrix was not labelled, suggesting that part of the structure formed in vivo is composed of modified antigen or is host derived.