Pilot GWAS of caries in African-Americans shows genetic heterogeneity.

BACKGROUND: Dental caries is the most common chronic disease in the US and disproportionately affects racial/ethnic minorities. Caries is heritable, and though genetic heterogeneity exists between ancestries for a substantial portion of loci associated with complex disease, a genome-wide association study (GWAS) of caries specifically in African Americans has not been performed previously. METHODS: We performed exploratory GWAS of dental caries in 109 African American adults (age > 18) and 96 children (age 3-12) from the Center for Oral Health Research in Appalachia (COHRA1 cohort). Caries phenotypes (DMFS, DMFT, dft, and dfs indices) assessed by dental exams were tested for association with 5 million genotyped or imputed single nucleotide polymorphisms (SNPs), separately in the two age groups. The GWAS was performed using linear regression with adjustment for age, sex, and two principal components of ancestry. A maximum of 1 million adaptive permutations were run to determine empirical significance. RESULTS: No loci met the threshold for genome-wide significance, though some of the strongest signals were near genes previously implicated in caries such as antimicrobial peptide DEFB1 (rs2515501; p = 4.54 × 10- 6) and TUFT1 (rs11805632; p = 5.15 × 10- 6). Effect estimates of lead SNPs at suggestive loci were compared between African Americans and Caucasians (adults N = 918; children N = 983). Significant (p < 5 × 10- 8) genetic heterogeneity for caries risk was found between racial groups for 50% of the suggestive loci in children, and 12-18% of the suggestive loci in adults. CONCLUSIONS: The genetic heterogeneity results suggest that there may be differences in the contributions of genetic variants to caries across racial groups, and highlight the critical need for the inclusion of minorities in subsequent and larger genetic studies of caries in order to meet the goals of precision medicine and to reduce oral health disparities.

Nutritional depletion of total mixed rations by European starlings: Projected effects on dairy cow performance and potential intervention strategies to mitigate damage.

European starlings are an invasive bird species in North America that are known to cause damage to commercial dairies through the consumption of total mixed rations (TMR) destined for dairy cows. We hypothesized that large foraging flocks of starlings alter the physical composition of TMR, and that this change may be significant enough to affect milk production. To better determine if production losses could potentially occur in commercial dairies as a consequence of feed consumption by foraging flocks of starlings, we conducted controlled feeding experiments using a TMR sourced from a commercial dairy that is chronically plagued with seasonal starling damage. European starlings selected the high-energy fraction of the TMR and reduced starch and crude fat availability. Using the dairy National Research Council production model equations, the nutritional changes measured in the controlled feeding experiments could potentially reduce the productivity of dairies. Model output suggests that for Holsteins producing 32 kg of milk/d, total required net energy intake (NEI) was 31.5 Mcal/d. Within the reference TMR, NEI supplied was 29.3 Mcal/d, whereas within the starling-consumed TMR NEI supplied was 27.7 Mcal/d. Following our nutrition experiments, we assessed the efficacy of pelleted feed as a deterrent strategy for bird damage management in commercial dairies. Six different pelleted feed treatments of differing diameter were offered to starlings. All pellets of 0.95 cm diameter or larger inhibited starling consumption by ≥79%.

Validation of the Vectra H1 portable three-dimensional photogrammetry system for facial imaging.

Three-dimensional (3D) surface imaging using stereophotogrammetry has become increasingly popular in clinical settings, offering advantages for surgical planning and outcome evaluation. The handheld Vectra H1 is a low-cost, highly portable system that offers several advantages over larger stationary cameras, but independent technical validation is currently lacking. In this study, 3D facial images of 26 adult participants were captured with the Vectra H1 system and the previously validated 3dMDface system. Using error magnitude statistics, 136 linear distances were compared between cameras. In addition, 3D facial surfaces from each system were registered, heat maps generated, and global root mean square (RMS) error calculated. The 136 distances were highly comparable across the two cameras, with an average technical error of measurement (TEM) value of 0.84mm (range 0.19-1.54mm). The average RMS value of the 26 surface-to-surface comparisons was 0.43mm (range 0.33-0.59mm). In each case, the vast majority of the facial surface differences were within a ±1mm threshold. Areas exceeding ±1mm were generally limited to facial regions containing hair or subject to facial microexpressions. These results indicate that 3D facial surface images acquired with the Vectra H1 system are sufficiently accurate for most clinical applications.

Producer survey of bird-livestock interactions in commercial dairies.

The objective of this producer survey was to identify and estimate damage caused by bird-livestock interactions in commercial dairies. The interactions between birds and livestock have previously been implicated in causing economic damage while contributing to the environmental dissemination of microorganisms pathogenic to livestock and humans. Very little research exists to help producers understand what bird species use dairies, why they use dairies, or the scope and nature of damage created as a result of bird-livestock interactions. To better characterize these interactions, we surveyed dairy operators within Pennsylvania, New York, and Wisconsin. Survey results suggest that the most common and destructive bird species found on commercial dairies are invasive to North America, and their use of dairies is associated with the loss of cattle feed, increased operating costs, and an increase in dairies self-reporting Salmonella spp. and Mycobacterium avium ssp. paratuberculosis. Cattle feed loss estimates generated from this survey were used to parameterize an input-output (IO) economic model using data from 10 counties in the state of Pennsylvania (Bedford, Berks, Blair, Bradford, Chester, Cumberland, Franklin, Lancaster, Lebanon, and Somerset). This IO model allowed us to estimate direct, indirect, and induced economic effects of feed loss from bird damage to dairies within these counties. The IO model output suggests that feed loss costs Pennsylvania between $4.11 and $12.08 million (mean $10.6 million) in total economic damage, with approximately 43 to 128 jobs (mean 112) forgone statewide in 2009.

Expression of prostaglandin G/H synthase (PGHS) and heat shock protein-70 (HSP-70) in the corpus luteum (CL) of prostaglandin F2 alpha-treated immature superovulated rats.

In this study we examined the mechanism of corpus luteum (CL) regression by measuring changes in expression of prostaglandin G/H synthase-1 (PGHS-1) and -2 (PGHS-2) in day 4 CL and inducible heat shock protein 70 (HSP-70) in day 4 and day 9 CL of immature superovulated rats. The rats were superovulated and treated with 500 microg of prostaglandin F2alpha (PGF2alpha) on day 4 or day 9 after CL formation. Ovaries and serial blood samples were removed during the 24-hour period following treatment. Plasma progesterone was determined by radioimmunoassay while mRNA abundance and protein expression were assessed by semiquantitative RT-PCR and immunoblot analysis, respectively. One hour after PGF2alpha, both day 4 and day 9 rats exhibited a significant decrease in progesterone secretion; however, there was a greater decrease in day 9 rats. In ovarian samples removed on day 4, there was a significant increase in mRNA for PGHS-2 at 1 hour after PGF2alpha. PGHS-1 mRNA content remained unchanged. Immunoblot analyses showed an increase in PGHS-2 protein expression only at 8 h. There were no changes in PGHS-1 protein expression. In day 9 rats, ovarian HSP-70 protein levels increased by 50% after PGF2alpha injection; however, on day 4 there was no change in expression of this protein over the sampling period. These results suggest that expression of PGHS-2 may be involved in inhibiting progesterone production and that expression of HSP-70 may be required for complete CL regression in the rat.

Treatment with prostaglandin F2alpha increases expression of prostaglandin synthase-2 in the rat corpus luteum.

Recent studies indicate that the corpus luteum (CL) may be a source of prostaglandin F2alpha (PGF2alpha) for regression. We investigated expression of mRNA and protein for prostaglandin G/H synthase (PGHS) in the CL of immature superovulated rats following administration of PGF2alpha. We observed an increase in mRNA for PGHS-2, the induced isoform, at 1 h and protein at 8 and 24 h after treatment. One hour after PGF2alpha, there was also a progressive decrease in plasma progesterone concentration. There were no changes, however, in expression of PGHS-1, the constitutive isoform, over the 24 h sampling period. These results indicate that PGHS-2 increases following PGF2alpha treatment and that expression of this enzyme in the rat CL may contribute to the luteolytic mechanism.

Phenanthrenequinone disrupts progesterone production in rat luteal cells.

The ability of the environmental contaminant phenanthrene (PH) and its photooxidized product phenanthrenequinone (PHQ) to disrupt progesterone secretion was examined in a model system of in vitro suspensions of luteal cells from the rat. Treatment with PHQ dramatically inhibited luteinizing hormone (LH) stimulated progesterone secretion. PHQ also generated a significant increase in reactive oxygen species (ROS). In the absence of LH, however, PHQ stimulated a small increase in basal progesterone secretion. The parent compound, PH, did not alter progesterone or ROS release. Since there is evidence that PHQ lowers the activity of nitric oxide synthase (NOS) and that nitric oxide (NO) affects progesterone production, we examined the response to the NOS inhibitors N-monomethyl-L-arginine, Zn protoporphyrin-9, and aminoguanidine in luteal cells. However, there was no effect of these agents on LH stimulated progesterone secretion. These results indicated that PHQ is a potent disrupter of progesterone secretion and should perhaps be considered in assessing the risk of PH to humans.

High-fructose feeding of streptozotocin-diabetic rats is associated with increased cataract formation and increased oxidative stress in the kidney.

We examined the effects of high-fructose (FR) feeding on the development of diabetic complications in the lens and the kidney of streptozotocin (STZ)-diabetic rats. Male Wistar Furth rats were treated with one of two doses of STZ (HIGH STZ, 55 mg/kg body weight; MOD STZ, 35 mg/kg body weight) or vehicle alone (SHAM) and were then assigned to a control (CNTL) or 400 g FR/kg diet for 12 weeks. At the end of the study, body weight, plasma glucose and insulin concentrations differed among STZ groups (HIGH v. MOD v. SHAM, P < 0.001) but did not differ due to diet. Plasma FR concentrations were significantly higher in FR-fed v. CNTL-fed groups (P < 0.0001) and in HIGH-STZ groups v. MOD-STZ and SHAM groups (P < 0.0004 and P < 0.0001 respectively). Focal length variability of the lens, a quantitative measure of cataract formation, was increased in the HIGH STZ, FR group compared with the HIGH STZ, CNTL group (P < 0.01). The concentration of H2O2 in kidney microsomes was significantly higher in HIGH STZ, FR rats v. HIGH STZ, CNTL rats (P < 0.01). Micro-albuminuria was not observed in any of the groups examined, and there was no evidence of extensive histological damage in the kidney from any rats. Under conditions of severe hyperglycaemia, high FR intake promotes the development of cataracts in the lens of the eye, and results in increased concentrations of substances indicative of oxidative stress in the kidney. Although FR has been suggested as a carbohydrate source for diabetics, a high FR diet coupled with hyperglycaemia produces effects that may promote some of the complications associated with diabetes.

Oxidative stress in hypopituitary dwarf mice and in transgenic mice overexpressing human and bovine GH.

Growth hormone (GH) stimulates metabolic activity. The purpose of this study was to examine whether it is involved in the aging process by increasing oxidative stress. Inorganic peroxides and lipid peroxides were measured in kidney and liver samples in dwarf mice that are deficient in GH, prolactin and thyrotropin and in transgenic mice that produce high levels of GH. In normal male mice, there was an increase in inorganic peroxides in the kidney with age. Levels were lower in old male dwarfs when compared with normal male mice of similar age. Unexpectedly, concentrations of inorganic peroxides were frequently lower in transgenic male and female mice expressing extra copies of GH than in normal controls. Lipid peroxide concentrations were more variable. Transgenic animals expressing bovine GH had the highest levels of lipid peroxides. In dwarfs, kidney levels were similar to those of normal mice but concentrations in the liver were more variable. This study does not indicate that the decrease in life span in transgenic mice producing high levels of GH is due to an increase of oxidative stress. Rather, it suggests that expression of extra copies of the GH gene may lead to a compensatory increase in antioxidant protection.

A consideration of some notable aging theories.

Aging is characterized as a breakdown process and the relevant events occur after reproduction, when the force of natural selection declines. Studies on the life histories of species reveal that there is an association between resource allocation and longevity and that the aging process is retarded when animals are protected from the deleterious consequences of excess metabolic activity. Although the extent to which aging is caused by environmental or genetic factors is unresolved, our understanding of the field has been enriched by the rapid development of the tools of molecular biology. In his pioneering work, Alex Comfort has postulated a hierarchical clock system as a descriptive paradigm of the aging process, and investigations at the molecular level are bringing to light evidence of a genetic link to life span that seems consistent with Comfort's model. It appears however, that the appropriate context for these mechanistic observations of functional decline is in the postreproductive period.

Luteotropic and luteolytic mechanisms in the bovine corpus luteum.

The function of the corpus luteum (CL) is a key element in many reproductive processes including ovulation, length of the estrous cycle, recognition of pregnancy and embryo survival in all mammalian species. The main function of the CL is to produce progesterone which acts on its tissues to prepare them for successful pregnancy. The CL is controlled by numerous biological compounds which provide luteotropic support during the estrous cycle and pregnancy and for inducing luteolysis at the end of the cycle The purpose of this paper is to review the mechansims responsible for controlling the endocrine function of this tissue in the bovine ovary.

Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum.

In this study we examined the prospect that the superoxide radical (SOR) is involved in the mechanism by which LH stimulates progesterone secretion in the rat corpus luteum (CL). Treatment of dispersed CL cells with low doses of LH or a SOR-generating system (xanthine-xanthine oxidase) resulted in a significant increase in progesterone release and SOR production. High doses of each treatment were inhibitory. SOR generation also decreased hCG binding. To determine whether SOR may be required for progesterone secretion, dispersed cells were electroporated with antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] and treated with either low (50 ng) stimulatory or high (20 micrograms) inhibitory doses of LH. At 50 ng LH, insertion of SOD or CAT dose-dependently inhibited progesterone secretion. However, at high doses of LH (20 micrograms), which are associated with high levels of SOR, electroporation of SOD or CAT produced the opposite response. This stimulatory response of SOD or CAT on progesterone release was also dose related. These results indicate that SOR may be involved in the mechanisms that stimulate as well as those that inhibit progesterone release. The effect on progesterone secretion appears to be dose related, with small increases associated with stimulation and high levels involved in inhibition of secretion.

Generation of free radicals and messenger function.

Free radicals are toxic agents that are produced as by-products of metabolic activity. A number of antioxidant mechanisms work to protect cells from damage. Recent evidence indicates, however, that free radicals and related oxidants such as hydrogen peroxide may also have a beneficial role, working as messengers to control cell function. These agents are generated in response to agonists, production is regulated by intracellular signal pathways, and they appear to be used to control particular cellular processes. Free radicals may perform these functions in a number of cell types. Also, they are produced in muscles and there is evidence that they may work as messengers in smooth muscle cells.

Stimulation of progesterone secretion in dispersed cells of rat corpora lutea by antioxidants.

Antioxidants were used to investigate the role of free radicals in control of luteal steroidogenesis. Corpora lutea from pseudopregnant rats were enzymatically dispersed, the cells were incubated with antioxidants, and progesterone production was measured. Addition of the antioxidants nordihydroguaiaretic acid (NDGA), butylated hydroxytoluene (BHT), and the gonadotropin luteinizing hormone (LH) resulted in a dose-dependent increase in progesterone secretion. However, the response pattern to these treatments differed with the age of the corpora lutea, and unlike LH neither NDGA nor BHT treatment resulted in an increase in the intracellular second messenger cAMP. Nevertheless, LH and antioxidant-induced progesterone stimulation could be blocked by the addition of either aminoglutethimide or ketoconazole, cytochrome P450 side-chain cleavage (cytochrome P450 SCC) enzyme inhibitors, which prevent the conversion of cholesterol to pregnenolone and thus block steroid hormone synthesis. Also, unlike exposure to LH, exposure to antioxidants resulted in an additional increase in progesterone production in luteal tissue saturated with 25 hydroxycholesterol, a soluble cholesterol analog which serves as a substrate for cytochrome P450 SCC. This study suggests that the site of antioxidant action in affecting progesterone secretion may be at the cytochrome P450 SCC enzyme. Based on these results and on studies in other steroid hormone-producing cells, it appears that free radicals may be involved in regulating synthesis by modulating activity of cytochrome P450 SCC enzyme in rat luteal tissue.

Studies on the mechanism controlling generation of superoxide radical in luteinized rat ovaries during regression.

The mechanism regulating the luteolytic release of superoxide radical (SOR) was examined in prostaglandin F2 alpha (PGF2 alpha)-treated rats. Tail vein injection of PGF2 alpha caused a rapid increase in SOR in mitochondria and plasma membrane samples prepared from luteinized rat ovaries. The peak in the mitochondria preceded that in the plasma membrane, and both occurred before progesterone concentrations decreased in the blood. The amount of SOR produced was greater when samples from the plasma membrane, mitochondria, and cytosol were combined. In plasma membrane samples, SOR generation was lowered by inhibitors of intracellular signaling pathways, but not by cyanide, which blocks electron transport in respiratory enzymes. In mitochondria samples, however, SOR was blocked by cyanide, but not by inhibitors of intracellular signaling enzymes. The addition of phospholipase-A2, phorbol myristate acetate (protein kinase-C activator), or arachidonic acid stimulated SOR production in plasma membrane samples from ovaries of control rats, and phorbol myristate acetate and arachidonic acid inhibited LH-stimulated progesterone secretion in dispersed rat luteal cells. Also, when mitochondria prepared from ovaries of PGF2 alpha-treated rats were added to dispersed corpus luteum cells, there was an increase in SOR generation and an inhibition of LH-stimulated cAMP formation and progesterone secretion. These results indicate that SOR production in the corpus luteum after PGF2 alpha treatment is generated by several subcellular components. Formation in the plasma membrane may be initiated by SOR generation from the mitochondria and regulated by intracellular signaling pathways. Our results indicate that formation of SOR may lead to the disruption of LH stimulation of progesterone secretion.

Alterations in free radical activity in aging Drosophila.

Drosophila were examined to see if there is an increase in damage associated with free radical activity in older flies. The levels of superoxide radical and of lipid peroxides were higher in membrane samples from older flies. Also, in older Drosophila there was a significant decrease in membrane fluidity, as determined by fluorescence polarization, and an increase in ATP-dependent calcium uptake. In homogenates from whole flies, the concentration of inorganic peroxides and activity of the proteolytic enzyme, cathepsin B, increased with age. During their lifetime, vestigial wing Drosophila displayed a greater level of free radical activity than wild-type flies and a significantly shorter life span (26.7 +/- 0.7 days vs. 34.4 +/- 1.0, p < 0.01). These results indicate that the level of oxidative stress is closely coupled to cellular damage and to life span, and they indicate that free radicals may play a central role in the aging process in Drosophila.

Plasma membrane changes in the rat corpus luteum induced by oxygen radical generation.

The luteolytic mechanism was investigated in rat corpora lutea (CL). This study focused on the changes that occur in the plasma membrane. Previous experiments with rat luteal cells indicated that in vitro generation of superoxide radicals by xanthine oxidase disrupted LH-stimulated cAMP production and progesterone secretion similar to the effect of prostaglandin F2 alpha, the luteolytic hormone. In the present study, we observed that xanthine oxidase treatment of plasma membrane samples from CL caused a large decrease in fluidity, which also occurs during prostaglandin F2 alpha-induced luteolysis. This fluidity change was blocked by catalase, bromophenacyl bromide, an inhibitor of phospholipase-A activity, indomethacin, and free radical scavengers, and it was reversed by removal of FFA from the membrane. In addition, xanthine oxidase treatment caused phospholipid breakdown, formation of neutral lipids, a burst of inorganic peroxides, and a sustained rise in the level of lipid peroxides. These results indicate that free radical generation causes several changes that disrupt the plasma membrane of CL cells, and they raise the possibility that phospholipid breakdown could be involved in the mechanism that inhibits LH stimulation of steroidogenesis during luteolysis.

Changes in superoxide radical formation, lipid peroxidation, membrane fluidity and cathepsin B activity in aging and spawning male Chinook salmon (Oncorhynchus tschawytscha).

Superoxide radical (SOR) formation in the brain and the liver of male Chinook salmon (Oncorhynchus tschawytscha) increased in mitochondrial and plasma membrane samples as they aged. In 2-year-old salmon, spawning also lead to a significant elevation in SOR formation in mitochondrial and plasma membrane samples. The rise in this free radical was associated with an increase in lipid peroxidation, a decrease in plasma membrane fluidity, and an elevation in cathepsin B activity in the brain and liver. In 2-year-old spawning salmon, the changes in these parameters was greater than in 2-year-old non-spawning salmon. These observations suggest that free radical levels increase with aging and during spawning and indicate that these changes may be involved in cellular degeneration. In addition, these results support the suggestion that cellular degeneration accelerates during the spawning process.

Isolation and characterization of deteriosomes from rat liver.

Deteriosomes, a new class of microvesicles, have been isolated from rat liver tissue. These microvesicles are similar to those isolated previously from plant tissue [Yao et al., Proc Natl Acad Sci USA 88:2269-2273, 1991] in that they are nonsedimentable and enriched in membrane catabolites, particularly products of phospholipid degradation. Liver deteriosomes range in size from 0.05 microns to 0.11 microns in radius. They are also much more permeable than microsomal membrane vesicles indicating that the deteriosome bilayer is perturbed. The data are consistent with the proposal that deteriosomes are formed from membranes by microvesiculation and that they represent an intermediate stage of membrane deterioration. Furthermore, liver deteriosomes were found to contain phospholipase A2 activity. This suggests that they not only serve as a means of moving destabilizing macromolecular catabolites out of membranes into the cytosol but also possess enzymatic activity. The fact that the specific activity of phospholipase A2 is higher in deteriosomes than in deteriosome-free cytosol suggests that some of the enzymatic activity traditionally assumed to be cytosolic may in fact be associated with deteriosomes.