Interfrontal Bone Among Inbred Strains of Mice and QTL Mapping

The interfrontal bone (IF) is a minor skeletal trait residing between the frontal bones. IF is considered a quasi-continuous trait. Genetic and environmental factors appear to play roles in its development. The mechanism(s) underlying IF bone development are poorly understood. We sought to survey inbred strains of mice for the prevalence of IF and to perform QTL mapping studies. Archived mouse skulls from a mouse phenome project (MPP) were available for this study. 27 inbred strains were investigated with 6–20 mice examined for each strain. Skulls were viewed dorsally and the IF measured using a zoom stereomicroscope equipped with a calibrated reticle. A two generation cross between C3H/HeJ and C57BL/6J mice was performed to generate a panel of 468 F2 mice. F2 mice were phenotyped for presence or absence of IF bone and among mice with the IF bone maximum widths and lengths were measured. F2 mice were genotyped for 573 SNP markers informative between the two strains and subjected to linkage map construction and interval QTL mapping. Results: Strain dependent differences in the prevalence of IF bones were observed. Overall, 77.8% or 21/27, of the inbred strains examined had IF bones. Six strains (C3H/HeJ, MOLF/EiJ, NZW/LacJ, SPRET/EiJ, SWR/J, and WSB/EiJ) lack IF bones. Among the strains with IF bones, the prevalence ranged from 100% for C57BL/6J, C57/LJ, CBA/J, and NZB/B1NJ and down to 5% for strains such as CAST/Ei. QTL mapping for IF bone length and widths identifies for each trait one strong QTL detected on chromosome 14 along with several other significant QTLs on chromosomes 3, 4, 7, and 11. Strain dependent differences in IF will facilitate investigation of genetic factors contributing to IF development. IF bone formation may be a model to understand intrasutural bone formation

Ex vivo evaluation of new 2D and 3D dental radiographic technology for detecting caries

Proximal dental caries remains a prevalent disease with only modest detection rates by current diagnostic systems. Many new systems are available without controlled validation of diagnostic efficacy. The objective of this study was to evaluate the diagnostic efficacy of three potentially promising new imaging systems

Fine mapping of dental fluorosis quantitative trait loci in mice

Genetic factors underlie dental fluorosis (DF) susceptibility/resistance. The A/J (DF susceptible) and 129P3/J (DF resistant) strains have been previously used to detect quantitative trait loci (QTL) associated with DF on chromosomes (Chr) 2 and 11. In the present study increased marker density genotyping followed by interval mapping was performed to narrow the QTL intervals and improve the LOD scores. Narrower intervals on Chr 2 where LOD ≥ 6.0 (57–84 cM or ~51 Mb), LOD ≥ 7.0 (62–79 cM or ~32 Mb), and LOD ≥ 8.0 (65–74 cM or ~17 Mb); and on Chr 11 where LOD ≥ 6.0 the interval was 18–51 cM (~53 Mb), LOD ≥ 7.0 (28–48 cM or ~34 Mb), and LOD ≥ 8.0 (31–45 cM or~22 Mb) were obtained. Haplotype analysis between A/J and 129P3/J further reduced QTL intervals. Accn1 was selected as a candidate gene based upon its location near the peak LOD score on Chr 11 and distant homology with the C. elegans fluoride resistance gene flr1. DF severity between Accn1−/− and wildtype mice was not different. The loss of ACCN1 function does not modify DF severity in mice. Narrowing the DF QTL intervals will facilitate additional candidate gene selections and interrogation

The AP-2 adaptor β2 appendage scaffolds alternate cargo endocytosis

The independently folded appendages of the large α and β2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The β2 subunit appendage contains a common binding site for β-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing β2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 α subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the β2 chain with the closely related endogenous β1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both β1 and β2 subunit transcripts recapitulates the strong α subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive β2-yellow fluorescent protein (YFP) expressed in the β1 + β2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the β appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a β2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a β-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with β2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and β-arrestin binding to a site upon the β2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and β-arrestin depend on a privileged β2 appendage site for proper cargo recruitment to clathrin bud sites

Genetic background influences fluoride's effects on osteoclastogenesis

Excessive fluoride (F) can lead to abnormal bone biology. Numerous studies have focused on the anabolic action of F yet little is known regarding any action on osteoclastogenesis. Little is known regarding the influence of an individual’s genetic background on the responses of bone cells to F. Four-week old C57BL/6J (B6) and C3H/HeJ (C3H) female mice were treated with NaF in the drinking water (0ppm, 50ppm and 100ppm F ion) for 3 weeks. Bone marrow cells were harvested for osteoclastogenesis and hematopoietic colony-forming cell assays. Sera were analyzed for biochemical and bone markers. Femurs, tibiae and lumbar vertebrae were subjected to microCT analysis. Tibiae and femurs were subjected to histology and biomechanical testing, respectively. The results demonstrated new actions of F on osteoclastogenesis and hematopoietic cell differentiation. Strain specific responses were observed. The anabolic action of F was favored in B6 mice exhibiting dose dependent increases in serum ALP activity (p < 0.001); in proximal tibia trabecular and vertebral BMD (tibia at 50&100ppm, p = 0.001; vertebrae at 50&100ppm, p = 0.023&0.019, respectively); and decrease in intact PTH and sRANKL (p = 0.045 and p < 0.001, respectively). F treatment in B6 mice also resulted in increased numbers of CFU-GEMM colonies (p = 0.025). Strain specific accumulations in bone [F] were observed. For C3H mice, dose dependent increases were observed in osteoclast potential (p < 0.001), in situ trabecular osteoclast number (p = 0.007), hematopoietic colony forming units (CFU-GEMM: p < 0.001, CFU-GM: p = 0.006, CFU-M: p < 0.001), and serum markers for osteoclastogenesis (intact PTH: p = 0.004, RANKL: p = 0.022, TRAP5b: p < 0.001). A concordant decrease in serum OPG (p = 0.005) was also observed. Fluoride treatment had no significant effects on bone morphology, BMD and serum PYD crosslinks in C3H suggesting a lack of significant bone resorption. Mechanical properties were also unaltered in C3H. In conclusion, short term F treatment at physiological levels has strain specific effects in mice. The expected anabolic effects were observed in B6 and novel actions hallmarked by enhanced osteoclastogenesis shifts in hematopoietic cell differentiation in the C3H strain

Detection of Dental Fluorosis-Associated Quantitative Trait Loci on Mouse Chromosomes 2 and 11

Systemic exposure to greater than optimal fluoride (F) can lead to dental fluorosis (DF). Parental A/J (DF-susceptible) and 129P3/J (DF-resistant) inbred mice were used for histological studies and to generate F2 progeny. Mice were treated with 0 or 50 ppm F in their drinking water for 60 days. A clinical criterion (modified Thylstrup and Fejerskov categorical scale) was used to assess the severity of DF for each individual F2 animal. Parental strains were subjected to histological examination of maturing enamel. F treatment resulted in accumulation of amelogenins in the maturing enamel of A/J mice. Quantitative trait loci (QTL) detection was performed using phenotypic extreme F2 animals genotyped for 354 single nucleotide polymorphism-based markers distributed throughout the mouse genome followed by χ2 analysis. Significant evidence of association was observed on chromosomes 2 and 11 for a series of consecutive markers (p < 0.0001). Further analyses were performed to examine whether the phenotypic effects were found in both male and female F2 mice or whether there was evidence for gender-specific effects. Analyses performed using the markers on chromosomes 2 and 11 which were significant in the mixed-gender mice were also significant when analyses were limited to only the male or female mice. The QTL detected on chromosomes 2 and 11 which influence the variation in response to fluorosis have their effect in mice of both genders. Finally, the QTL in both chromosomes appear to have an additive effect

Putative Stem Cells in Human Dental Pulp with Irreversible Pulpitis: An Exploratory Study

Although human dental pulp stem cells isolated from healthy teeth have been extensively characterized, it is unknown whether stem cells also exist in clinically compromised teeth with irreversible pulpitis. Here we explored whether cells retrieved from clinically compromised dental pulp have stem cell-like properties

Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats

Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F−) exposure generates reactive oxygen species (ROS) that can cause ER-stress. We therefore screened oxidative stress arrays to identify genes regulated by F− exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F− treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F− (~1.5 & 2.0 fold for the 50 or 100 ppm F− treatment groups respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F− treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E enriched diets within a given F− treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F− and activation of this gene may adversely affect ameloblast function

Fluoride Modulates Parathyroid Hormone Secretion in vivo and in vitro

The study objective was to investigate fluoride’s effects on iPTH secretion. Thryo-parathyroid complexes (TPCs) from C3H (n=18) and B6 (n=18) mice were cultured in Ca2+ optimized medium. TPCs were treated with 0, 250 or 500µM NaF for 24hrs and secreted iPTH assayed by ELISA. C3H (n=78) and B6 (n=78) mice were gavaged once with distilled or with fluoride (0.001mg [F−]/g body weight) water. At serial time points (0.5–96hrs) serum iPTH, fluoride, total calcium, phosphorus and magnesium levels were determined. Expression of genes involved in mineral regulation via bone-parathyroid-kidney (BPK) axis such as: Pth, Casr, Vdr, Pthlh, Fgf23, αKlotho, Fgf1rc, Tnfs11, Pth1r, Slc34a1, Slc9a3r1, Clcn5 and Pdzk1 were determined in TPCs, humerii and kidneys at 24hrs. An in vitro decrease in iPTH was seen in C3H and B6 TPC at 500µM (p<0.001). In vivo levels of serum fluoride peaked at 0.5hr in both C3H (p=0.002) and B6 (p=0.01). In C3H, iPTH decreased at 24hrs (p<0.0001) returning to baseline at 48hrs. In B6, iPTH increased at 12hrs (p<0.001) returning to baseline at 24hrs. Serum total calcium, phosphorus and magnesium did not change significantly. Pth, Casr, αKlotho, Fgf1rc, Vdr and Pthlh were significantly up-regulated in C3H TPC as compared to B6. Conclusions, fluoride’s effects on TPC in vitro were equivalent between the two mouse strains. However, fluoride demonstrated an early strain dependent effect on iPTH secretion in vivo. Both strains demonstrated a differences in the expression of genes involved in BPK axis suggesting a possible role in physiologic handling of fluoride

Exoplanet Diversity in the Era of Space-based Direct Imaging Missions

This whitepaper discusses the diversity of exoplanets that could be detected by future observations, so that comparative exoplanetology can be performed in the upcoming era of large space-based flagship missions. The primary focus will be on characterizing Earth-like worlds around Sun-like stars. However, we will also be able to characterize companion planets in the system simultaneously. This will not only provide a contextual picture with regards to our Solar system, but also presents a unique opportunity to observe size dependent planetary atmospheres at different orbital distances. We propose a preliminary scheme based on chemical behavior of gases and condensates in a planet's atmosphere that classifies them with respect to planetary radius and incident stellar flux.Comment: A white paper submitted to the National Academy of Sciences Exoplanet Science Strateg