The retina is the light-sensitive tissue of the eye that facilitates vision. Mutations within genes affecting eye development and retinal function cause a host of degenerative visual diseases, including retinitis pigmentosa and anophthalmia/microphthalmia. The characin fish Astyanax mexicanus includes both eyed (surface fish) and eyeless (cavefish) morphs that initially develop eyes with normal retina; however, early in development, the eyes of cavefish degenerate. Since both surface and cave morphs are members of the same species, they serve as excellent evolutionary mutant models with which to identify genes causing retinal degeneration. In this study, we crossed the eyed and eyeless forms of A. mexicanus and quantified the thickness of individual retinal layers among 115 F2 hybrid progeny. We used next generation sequencing (RAD-seq) and microsatellite mapping to construct a dense genetic map of the Astyanax genome, scan for quantitative trait loci (QTL) affecting retinal thickness, and identify candidate genes within these QTL regions. The map we constructed for Astyanax includes nearly 700 markers assembled into 25 linkage groups. Based on our scans with this map, we identified four QTL, one each associated with the thickness of the ganglion, inner nuclear, outer plexiform, and outer nuclear layers of the retina. For all but one QTL, cavefish alleles resulted in a clear reduction in the thickness of the affected layer. Comparative mapping of genetic markers within each QTL revealed that each QTL corresponds to an approximately 35 Mb region of the zebrafish genome. Within each region, we identified several candidate genes associated with the function of each affected retinal layer. Our study is the first to examine Astyanax retinal degeneration in the context of QTL mapping. The regions we identify serve as a starting point for future studies on the genetics of retinal degeneration and eye disease using the evolutionary mutant model Astyanax.

The sclera is the tough outer covering of the eye that provides structural support and helps maintain intraocular pressure. In some fishes, reptiles, and birds, the sclera is reinforced with an additional ring of hyaline cartilage or bone that forms from scleral ossicles. Currently, the evolutionary and genetic basis of scleral ossification is poorly understood, especially in teleost fishes. We assessed scleral ossification among several groups of the Mexican tetra (Astyanax mexicanus), which exhibit both an eyed and eyeless morph. Although eyed Astyanax surface fish have bony sclera similar to other teleosts, the ossicles of blind Astyanax cavefish generally do not form. We first sampled cavefish from multiple independent populations and used ancestral character state reconstructions to determine how many times scleral ossification has been lost. We then confirmed these results by assessing complementation of scleral ossification among the F1 hybrid progeny of two cavefish populations. Finally, we quantified the number of scleral ossicles present among the F2 hybrid progeny of a cross between surface fish and cavefish, and used this information to identify quantitative trait loci (QTL) responsible for this trait. Our results indicate that the loss of scleral ossification is common–but not ubiquitous–among Astyanax cavefish, and that this trait has been convergently lost at least three times. The presence of wild-type, ossified sclera among the F1 hybrid progeny of a cross between different cavefish populations confirms the convergent evolution of this trait. However, a strongly skewed distribution of scleral ossicles found among surface fish x cavefish F2 hybrids suggests that scleral ossification is a threshold trait with a complex genetic basis. Quantitative genetic mapping identified a single QTL for scleral ossification on Astyanax linkage group 1. We estimate that the threshold for this trait is likely determined by at least three genetic factors which may control the severity and onset of lens degeneration in cavefishes. We conclude that complex evolutionary and genetic patterns underlie the loss of scleral ossification in Astyanax cavefish.

Grape byproducts are a rich source of phenolics having immense medicinal properties, but usually wasted from juice/wine processing industries. The present study investigates the phenolic antioxidants and the insulinotropic effect of extracts prepared from seed, skin and stems of two red wine grape cultivars: Pusa Navarang and Merlot. Pusa Navarang cultivar has shown high amounts of total phenolics (95.8 mg/ml), flavonoids (30.5 mg/ml) and flavan-3-ols (21.8 mg/ml) in seed extract and total anthocyanin (4.9 mg/ml) in its skin extract as compared to Merlot cultivar. As determined using HPLC, higher amounts of catechin hydrate (14909 mg/l) and epicatechin (9299 mg/l) were observed in its seed extract, while quercetin hydrate (5849 mg/l) was abundant in its skin extract. Similarly, ferric reducing antioxidant power (FRAP) and ABTS + . [2,2′-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid] and DPPH. (1,1-diphenyl-2-picrylhy-drazyl) radicals scavenging, were higher in its seed extract, respectively being 134.8 mg/ml of Quercetin equivalent (QE), 18.7 mM of trolox equivalent (TE) and 33.5 mM of TE. Strong correlation was obtained between FRAP and total phenolics, flavonoids and flavan-3-ols contents with correlation coefficients (r 2 ) of 0.915, 0.738 and 0.838 respectively. Interestingly, there was a 2-8 fold increase in insulin secretion by isolated mice pancreatic islets at 5.5 mM and 16.5 mM glucose concentration in presence of various extracts. Overall, the seed, skin and stem byproducts of both cultivars are rich sources of phenolics and antioxidants and represent a source of new insulin secretagogues.

The phenolic composition and antioxidant potential were determined in grape berries, berry stem, leaves, leaf petiole and shoots of commercially popular variety Kishmish Chornyi (Sharad Seedless) at various stages of maturation. The berries and berry stems at the initial stage of maturation showed high amounts of total phenolics, flavonoids, flavonols, flavan-3-ols and antioxidant activity in terms of ferric reducing antioxidant power (FRAP). The effect of maturation was profound in grapes with a drastic reduction in various phenolic components and the FRAP value. The berries obtained at the last two harvests showed high anthocyanins and reducing sugars, but the least amount of all phenolic components and thus the antioxidant activity; however, berry stems, leaves, petioles and shoots, respectively, showed 7.6-, 2.9-, 2.3-and 1.5-fold higher FRAP values than berries. Thus, besides the berries, different grapevine parts could also become a potential source of phenolics for pharmaceuticals or functional foods.

Apoptosis, a form of cell death ("programmed" cell death) in which the nucleus and cytoplasm shrink and often fragment, serves to eliminate excessive or unwanted cells during remodeling of embryonic tissues, during organ involution, and in tumor regression. In acute pathological states, such as ischemia, the cells tend to swell and lyse--a process called necrosis. We hypothesize that the delayed neural death clinically associated with hypoxia may, in part, represent apoptosis. A tissue culture model of 24 hours of hypoxia was employed using sympathetic neurons. Pretreatment with an endonuclease inhibitor (aurintricarboxylic acid) decreased cell death by 53%, depolarizing conditions (55 mM potassium chloride) decreased cell death by 33%, and an RNA synthesis inhibitor (actinomycin D) by 26% (all have been shown to prevent apoptosis). Pretreatment with antisense c-myc had no effect. Fluorescent staining with propidium iodide (a DNA marker) demonstrated chromatin condensation and agarose gel electrophoresis demonstrated a DNA "ladder." These data suggest that apoptosis may play a role in hypoxic cell death and that in this paradigm, expression of c-myc is unnecessary. This would suggest a new approach to our understanding of hypoxia and open new strategies to lessen neuronal damage secondary to this process.