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%.

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.

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.

Alterations in phospholipase A2 activity during luteal regression in pseudopregnant and pregnant rats.

The activity of phospholipase A2 was measured in microsomes prepared from ovaries of superovulated pseudopregnant rats during spontaneous and prostaglandin F2 alpha-induced regression and during regression in pregnant rats. Microsome samples were incubated at 40 C for 90 min in Tris buffer (pH 8.3) with 1.0 mM CaCl2 added. The substrate, radiolabeled phosphatidylcholine, was incorporated into liposomes. During spontaneous regression, there was a significant 2- to 4-fold increase in phospholipase A2 activity, when compared with levels at mid-pseudopregnancy (days 8-9). This elevation was correlated with a significant decrease in plasma progesterone concentration. On day 6 or 7 of pseudopregnancy, treatment of rats with luteolytic doses of prostaglandin F2 alpha also caused a significant increase in phospholipase A2 activity, which remained elevated throughout the 72-h sampling period. In pregnant rats there was a small but significant rise in phospholipase A2 activity after parturition. These results indicate that the activity of phospholipase A2 increases during luteal regression in pregnant and pseudopregnant rats and that it could be involved in the mechanism that causes the loss in progesterone secretion.

Rapid plasma membrane changes in superoxide radical formation, fluidity, and phospholipase A2 activity in the corpus luteum of the rat during induction of luteolysis.

Early luteolytic changes in the plasma membrane of luteal cells were examined in the rat. Treatment with prostaglandin F2 alpha in vivo caused a rapid transient increase in superoxide radical formation and a decrease in fluidity in plasma membrane samples prepared from luteinized rat ovaries. These alterations preceded detection of a significant fall in plasma progesterone concentration. The rise in superoxide radical was not accompanied by changes in activities of free radical scavenging enzymes. Within the first hour of prostaglandin treatment, there was also a significant increase in the activity of phospholipase A2 and ATP-dependent calcium uptake in the membrane samples. These experiments indicate that one of the initial sites affected by the luteolytic process appears to be the plasma membrane. The changes include a transient rise in production of superoxide radicals, which may cause membrane changes that are responsible for disrupting corpus luteum function in the rat.

Involvement of phospholipase A activity in the plasma membrane of the rat corpus luteum during luteolysis.

Plasma membrane samples prepared from corpora lutea (CL) of control and prostaglandin F2 alpha-treated rats were incubated with radiolabeled phosphatidylcholine for 90 min at 40 C with 1 mM CaCl2 to test for the presence of phospholipase A2 activity. At the end of the incubation period, labeled arachidonic acid cleaved from the 2 position of the phospholipid moiety had accumulated to 10.7 +/- 2.4% (mean +/- SE) of the initially added radioactivity in the prostaglandin F2 alpha-treated samples, with 3.7 +/- 1.2% appearing in the controls. Arachidonic acid production was inhibited by calcium chelation and by the phospholipase A2 inhibitor bromophenacyl bromide, indicating heightened activity of phospholipase A2 in CL plasma membranes undergoing regression. Unexpectedly, radiolabeled lysophosphatidylcholine was produced in regressed membrane samples at a similar rate, suggesting the induction of phospholipase A1 activity as well. To determine if the membrane rigidification that occurs with regressed membrane samples under the same incubation conditions is caused by the hydrolysis products of phospholipase activity, fluorescence polarization experiments with the probe trans-parinaric acid were conducted. Washing of incubated membrane samples with fatty acid-free BSA, which selectively removes free fatty acids and lysophosphatides from the bilayer, resulted in a restoration of the fluidity to levels recorded at the onset of the incubation. These results suggest that the previously described decreases in CL plasma membrane fluidity and hCG binding in vitro during luteolysis are caused by a synergistic effect of calcium ion and hydrolysis products of phospholipase A activity.

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.

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.

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.

The effects of prolactin and prostaglandin F2 alpha on plasma membrane changes during luteolysis in the rat.

The ability of PRL to modify prostaglandin F2 alpha (PGF2 alpha)-induced membrane changes during functional corpus luteum regression was examined in the pseudopregnant rat. Fluorescence polarization studies conducted 24 h after sc injection of PGF2 alpha revealed a marked and significant increase in the polarization parameter, which is suggestive of reduced plasma membrane fluidity. At the same time, there was a decrease in hCG binding and a significant increase in apparent phospholipase-A2 activity during incubation of dispersed rat luteal cells. Each of these changes was attenuated when the animals were pretreated with PRL 30 min before PGF2 alpha. The decrease in plasma progesterone caused by PGF2 alpha treatment was also inhibited by PRL. PGF2 alpha also stimulated a significant polarization increase in dispersed cells prepared from ovaries removed 1 h after injection of this luteolytic agent, although this effect could not be demonstrated in plasma membrane samples. These results indicate that PRL and PGF2 alpha affect the same membrane pathway in the rat corpus luteum and that this pathway appears to be closely coupled to luteal cell function.

Alterations in the cellular membranes of regressing rat corpora lutea.

Wide angle x-ray diffraction and fluorescence polarization were used to determine the structural properties of membranes from rat luteal cells. Examination of a plasma membrane fraction by x-ray diffraction revealed a significant increase in the gel phase melting temperature during luteal regression. The membrane fluidity of this fraction as well as that of a preparation of microsomes was also studied by fluorescence polarization. Using a fluorescent probe, membrane fluidity was observed to decrease during luteolysis. The temporal correlation between structural changes in the membrane and decreased progesterone secretion suggests that alterations in the physical properties of cellular membranes may be involved in the process of luteal cell regression.

A new perspective on the mechanism of corpus luteum regression.

Wide angle x-ray diffraction has been used to examine the phase behavior of microsomal membranes from regressing corpora lutea of prepubertal pseudopregnant rats. During periods of optimal progesterone secretion, all of the membrane lipid was in the liquid-crystalline phase at physiological temperature and, therefore, was fluid. However, mixtures of liquid-crystalline and gel phase lipid were observed under identical conditions in microsomal membrane preparations from animals undergoing spontaneous or prostaglandin F2 alpha-induced regression. This was accompanied by a parallel rise in the lipid phase transition temperature. In addition, the proportion of lipid in the gel phase increased with time after prostaglandin F2 alpha treatment. These results indicate that the mechanism of corpus luteum regression may involve phase changes in the phospholipid bilayer of cellular membranes. The resulting presence of gel phase lipid in the membrane matrices could contribute to the loss of tissue function.

Evidence of membrane changes during regression in the bovine corpus luteum.

Microsomal membranes prepared from bovine corpora lutea (CL) were examined by wide angle x-ray diffraction to determine if there were structural changes in the cellular membranes during regression. In samples prepared from CL removed at midcycle, all of the membrane lipid was in the liquid crystalline phase at body temperature. However, examination of microsomes prepared from regressing luteal tissue revealed a phase transition in which a portion of the lipid bilayer was in a gel phase at body temperature. Coincident with this physical change was a decline in CL function. These results indicate that there are structural changes that occur at the submicroscopic level in cellular membranes during luteal regression, which appear to be related to the loss in cellular function.

A study of the interaction between progesterone and membrane lipids.

The ability of progesterone to associate with phospholipid was examined in a model membrane system. Molecular interaction was assessed by measuring the enthalpy of the phase transition of dipalmitoylphosphatidylcholine liposomes by differential scanning calorimetry. The response was compared to cholesterol, a constituent of cellular membranes. Unlike cholesterol, progesterone caused minimal disruption of the phospholipid bilayer phase properties at concentrations ranging from 5-33 mol %. However, it interacted with the phospholipid to a greater degree when cholesterol was included in the liposomes. These results indicate that progesterone can intercalate into phospholipid bilayers containing cholesterol, and raise the prospect that there may be some diffusion of the hormone across the plasma membrane.

Compositional and physical properties of microsomal membrane lipids from regressing rat corpora lutea.

Wide angle x-ray diffraction has revealed that during corpus luteum regression there is a liquid-crystalline to gel phase transition in the phospholipid molecules of the cellular membranes. In the present study we have examined the lipid composition of these membranes and looked for evidence of membrane protein involvement in this change. Lipid analysis of smooth microsomal membranes prepared from rat corpora lutea revealed no significant change in the cholesterol to phospholipid ratio or in the ratio of unsaturated to saturated fatty acids with advancing luteolysis. In addition, there was no clear trend for these changes in the relative proportions of the major fatty acids. Liposomes were prepared from smooth microsomal fractions of regressing rat corpora lutea, and examination of these lipid vesicles by x-ray diffraction revealed that the temperature of the liquid-crystalline to gel phase transition was much lower (approximately 25-30 C) than that for the corresponding microsomes. These observations are consistent with the view that membrane proteins contribute to the ordering of lipid that results in a mixture of liquid-crystalline and gel phases in membranes from regressed corpora lutea.

Oxygen radicals and the control of ovarian corpus luteum function.

The superoxide radical (SOR) and other reactive oxygen species form in cells during the course of respiration as well as in response to various stimuli. Although well known for their damaging effects, these agents can also work beneficially to control cell function. The present review examines the evidence that oxygen radicals and H2O2 may regulate steroid hormone biosynthesis in the ovarian corpus luteum. Recent findings indicate that luteal cells can employ reactive oxygen species at specific sites in controlling the production of progesterone over the course of the reproductive cycle and in inhibiting its synthesis during regression at the end of the cycle. These studies indicate that oxygen radicals and related compounds may function as intracellular regulators of steroidogenesis in the corpus luteum.

Changes in superoxide radical and lipid peroxide formation in the brain, heart and liver during the lifetime of the rat.

The free radical theory of aging was examined by measuring the formation of superoxide radical (SOR) and the level of lipid peroxides in various tissues of the aging rat. A significant increase in SOR production was seen in mitochondria prepared from the brain and the heart as rats aged. An elevation in the level of lipid peroxidation was also found in whole tissue homogenates of the brain and the liver. Vitamin E concentrations in the blood rose rapidly in young rats and remained steady except for a non significant drop in old animals. These results suggest that age-related degeneration of various tissues in the rat may be due to a rise in free radical production in the mitochondria.

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.