The mechanisms and mediators of tooth eruption--models for developmental biologists

Tooth eruption is a localized process in the jaws which exhibits precise timing and bilateral symmetry. It involves resorption and formation of bone on opposite sides of the erupting tooth and these activities depend on the dental follicle, a thin connective tissue investment of the developing and erupting tooth. Biochemical studies have shown that during eruption cells, proteins and enzymes change in the dental follicle and several growth factors and proteins known to accelerate or retard eruption have been identified. This review discusses these aspects of tooth eruption and proposes testable hypotheses and strategies that can make studies of tooth eruption new experimental opportunities for developmental biologists

A Single Antibody based ELISA for the N-terminal sequence of BAG-75, a New Biomarker for Bone Formation [abstract]

Biomedical Tissue Engineering, Biomaterials, & Medical Devices Poster SessionBone acidic glycoprotein-75 (BAG-75) is a secreted product of osteoblastic cells localized predominantly to areas of new bone formation. We have identified the N-terminal sequence of BAG-75 as LPVARYQNTEEEE and shown that anti-peptide antibodies against residues #3-13 only recognize the 75 kDa precursor and apparent 50 kDa fragment in serum and in osteoblastic cultures. Formation of the 50 kDa fragment is blocked by AEBSF, a serine protease inhibitor which we also showed blocks mineralization in osteoblastic cultures. Measurement of BAG-75 and its fragment concentration in serum represents a new method to estimate the rate of new bone formation in vivo. Our purpose was to establish an anti-VARYQNTEEEE peptide antibody based ELISA test to measure cross-reactive proteins released from bone into blood. Western blotting was performed using young rat serum from different ages, rats subjected to ovariectomy (OVX) or sham surgery, and normal human serum. Immunoreactive 50 kDa fragment peaked at 18 days after birth which parallels bone formation. Ovariectomized rats displayed a peak of 50 kDa immunoreactivty at 21 days after surgery which corresponds to a spike in bone formation in this model (~2.5-fold above controls). Comparable assays for osteocalcin showed only a 39% increase. Also, human serum contains a 50 kDa protein which cross-reacts with anti-VARYQNTEEEE antibodies. We then established a competitive 96-well ELISA using anti-peptide antibody and new sera at 21 days from ovariectomized or sham rats, a model for stimulated bone formation. VARYQNTEEEE peptide conjugated to keyhole limpet hemocyanin (KLH) was used as the bound antigen. KLH-peptide amount, primary antibody concentration, secondary antibody concentration, and blocking agent were optimized in a series of experiments. Optimal conditions were determined to be 2 µg input KLH-peptide per well, 1/5,000 dilution of primary anti-VARYQNTEEEE antibody, 1/10,000 dilution of secondary antibody, and gelatin as a blocking agent. Sera from OVX rats and sham-operated controls were compared to the standard curve (r = 0.9923) created with free KLH-peptide as competitor to determine the equivalent amount of KLH-peptide present. OVX sera (n=3) contained an average 2.6 x 10-4 (+/- 1.4 x 10-4) µg peptide equivalent versus 1.05 x 10-4 (+/- 0.68 x 10-4) µg for sham sera (n=3). The difference was not significant (t-test, p=0.157), however, doubling the sample size is predicted to yield significance. Conclusions: A. Cross-reactive 75 kDa and 50 kDa proteins are present in human and rat serum and increase in concentration when bone formation is stimulated. B. A new, single antibody based ELISA assay was established to quantitate antigen released from bone into blood. C. In contrast to other commercial bone formation assays (collagen peptides and osteocalcin), the size of cross-reactive protein (>50 kDa) should preclude kidney filtration and facilitate measurement. D. This serum biomarker undergoes a 2-3 fold average increase within 3 weeks after simulation of bone. This test may be useful to monitor the early response to stimulatory therapy in osteoporosis patients or to repressive glucocorticoid therapy in sarcoidosis patients. Currently, a 1% change in bone mineral density requires 12-18 months to detect by x-ray methods

Activation of a Metabolic Gene Regulatory Network Downstream of mTOR Complex 1

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease

The ALCHEMI atlas: principal component analysis reveals starburst evolution in NGC 253

Molecular lines are powerful diagnostics of the physical and chemical properties of the interstellar medium (ISM). These ISM properties, which affect future star formation, are expected to differ in starburst galaxies from those of more quiescent galaxies. We investigate the ISM properties in the central molecular zone of the nearby starburst galaxy NGC 253 using the ultra-wide millimeter spectral scan survey from the ALMA Large Program ALCHEMI. We present an atlas of velocity-integrated images at a 1".6 resolution of 148 unblended transitions from 44 species, including the first extragalactic detection of HCNH$^+$ and the first interferometric images of C$_3$H$^+$, NO, HCS$^+$. We conduct a principal component analysis (PCA) on these images to extract correlated chemical species and to identify key groups of diagnostic transitions. To the best of our knowledge, our dataset is currently the largest astronomical set of molecular lines to which PCA has been applied. The PCA can categorize transitions coming from different physical components in NGC 253 such as i) young starburst tracers characterized by high-excitation transitions of HC$_3$N and complex organic molecules (COMs) versus tracers of on-going star formation (radio recombination lines) and high-excitation transitions of CCH and CN tracing PDRs, ii) tracers of cloud-collision-induced shocks (low-excitation transitions of CH$_3$OH, HNCO, HOCO$^+$, and OCS) versus shocks from star-formation-induced outflows (high-excitation transitions of SiO), as well as iii) outflows showing emission from HOC$^+$, CCH, H$_3$O$^+$, CO isotopologues, HCN, HCO$^+$, CS, and CN. Our findings show these intensities vary with galactic dynamics, star formation activities, and stellar feedback.Comment: 65 pages, 39 figures. Accepted for publication in ApJ

Bivariate genome-wide association meta-analysis of pediatric musculoskeletal traits reveals pleiotropic effects at the SREBF1/TOM1L2 locus

Bone mineral density is known to be a heritable, polygenic trait whereas genetic variants contributing to lean mass variation remain largely unknown. We estimated the shared SNP heritability and performed a bivariate GWAS meta-analysis of total-body lean mass (TB-LM) and total-body less head bone mineral density (TBLH-BMD) regions in 10,414 children. The estimated SNP heritability is 43% for TBLH-BMD, and 39% for TB-LM, with a shared genetic component of 43%. We identify variants with pleiotropic effects in eight loci, including seven established bone mineral density loci: _WNT4, GALNT3, MEPE, CPED1/WNT16, TNFSF11, RIN3, and PPP6R3/LRP5_. Variants in the _TOM1L2/SREBF1_ locus exert opposing effects TB-LM and TBLH-BMD, and have a stronger association with the former trait. We show that _SREBF1_ is expressed in murine and human osteoblasts, as well as in human muscle tissue. This is the first bivariate GWAS meta-analysis to demonstrate genetic factors with pleiotropic effects on bone mineral density and lean mass

Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner

Bivariate genome-wide association meta-analysis of pediatric musculoskeletal traits reveals pleiotropic effects at the SREBF1/TOM1L2 locus

Bone mineral density is known to be a heritable, polygenic trait whereas genetic variants contributing to lean mass variation remain largely unknown. We estimated the shared SNP heritability and performed a bivariate GWAS meta-analysis of total-body lean mass (TB-LM) and total-body less head bone mineral density (TBLH-BMD) regions in 10,414 children. The estimated SNP heritability is 43% for TBLH-BMD, and 39% for TB-LM, with a shared genetic component of 43%. We identify variants with pleiotropic effects in eight loci, including seven established bone mineral density loci: _WNT4, GALNT3, MEPE, CPED1/WNT16, TNFSF11, RIN3, and PPP6R3/LRP5_. Variants in the _TOM1L2/SREBF1_ locus exert opposing effects TB-LM and TBLH-BMD, and have a stronger association with the former trait. We show that _SREBF1_ is expressed in murine and human osteoblasts, as well as in human muscle tissue. This is the first bivariate GWAS meta-analysis to demonstrate genetic factors with pleiotropic effects on bone mineral density and lean mass

MMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells

Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Abstract: Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria