Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells.

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT(2) receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT(2A) and 5-HT(2C) receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT(2A) receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT(2A) receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT(2) receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT(2A) and 5-HT(2C) receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT(2A) receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT(2A) receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells

Benchmarking of three-dimensional multicomponent lattice Boltzmann equation

We present a challenging validation of phase field multi-component lattice Boltzmann equation (MCLBE) simulation against the Re = 0 Stokes flow regime Taylor-Einstein theory of dilute suspension viscosity. By applying a number of recent advances in the understanding and the elimination of the interfacial micro-current artefact, extending to 3D a class of stability-enhancing multiple relaxation time collision models (which require no explicit collision matrix, note) and developing new interfacial interpolation schemes, we are able to obtain data which show that MCLBE may be applied in new flow regimes. Our data represent one of the most stringent tests yet attempted on LBE-one which received wisdom would preclude on grounds of overwhelming artefact flow

Further validation to support clinical translation of [18

BACKGROUND: This study aims to further evaluate the specificity and selectivity of [(18)F]FTC-146 and obtain additional data to support its clinical translation. METHODS: The binding of [(19)F]FTC-146 to vesicular acetylcholine transporter (VAChT) was evaluated using [(3)H]vesamicol and PC12(A123.7) cells in an in vitro binding assay. The uptake and kinetics of [(18)F]FTC-146 in S1R-knockout mice (S1R-KO) compared to wild-type (WT) littermates was assessed using dynamic positron emission tomography (PET) imaging. Ex vivo autoradiography and histology were conducted using a separate cohort of S1R-KO/WT mice, and radiation dosimetry was calculated from WT mouse data (extrapolated for human dosing). Toxicity studies in Sprague–Dawley rats were performed with a dose equivalent to 250× the anticipated clinical dose of [(19)F]FTC-146 mass. RESULTS AND DISCUSSION: VAChT binding assay results verified that [(19)F]FTC-146 displays negligible affinity for VAChT (K(i) = 450 ± 80 nM) compared to S1R. PET images demonstrated significantly higher tracer uptake in WT vs. S1R-KO brain (4.57 ± 1.07 vs. 1.34 ± 0.4 %ID/g at 20–25 min, n = 4, p < 0.05). In S1R-KO mice, it was shown that rapid brain uptake and clearance 10 min post-injection, which are consistent with previous S1R-blocking studies in mice. Three- to fourfold higher tracer uptake was observed in WT relative to S1R-KO mouse brains by ex vivo autoradiography. S1R staining coincided well with the autoradiographic data in all examined brain regions (r(2) = 0.85–0.95). Biodistribution results further demonstrated high [(18)F]FTC-146 accumulation in WT relative to KO mouse brain and provided quantitative information concerning tracer uptake in S1R-rich organs (e.g., heart, lung, pancreas) for WT mice vs. age-matched S1R-KO mice. The maximum allowed dose per scan in humans as extrapolated from mouse dosimetry was 33.19 mCi (1228.03 MBq). No significant toxicity was observed even at a 250X dose of the maximum carrier mass [(19)F]FTC-146 expected to be injected for human studies. CONCLUSIONS: Together, these data indicate that [(18)F]FTC-146 binds specifically to S1Rs and is a highly promising radiotracer ready for clinical translation to investigate S1R-related diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-015-0122-2) contains supplementary material, which is available to authorized users

Further validation to support clinical translation of [(18)F]FTC-146 for imaging sigma-1 receptors.

BackgroundThis study aims to further evaluate the specificity and selectivity of [(18)F]FTC-146 and obtain additional data to support its clinical translation.MethodsThe binding of [(19)F]FTC-146 to vesicular acetylcholine transporter (VAChT) was evaluated using [(3)H]vesamicol and PC12(A123.7) cells in an in vitro binding assay. The uptake and kinetics of [(18)F]FTC-146 in S1R-knockout mice (S1R-KO) compared to wild-type (WT) littermates was assessed using dynamic positron emission tomography (PET) imaging. Ex vivo autoradiography and histology were conducted using a separate cohort of S1R-KO/WT mice, and radiation dosimetry was calculated from WT mouse data (extrapolated for human dosing). Toxicity studies in Sprague-Dawley rats were performed with a dose equivalent to 250× the anticipated clinical dose of [(19)F]FTC-146 mass.Results and discussionVAChT binding assay results verified that [(19)F]FTC-146 displays negligible affinity for VAChT (K i = 450 ± 80 nM) compared to S1R. PET images demonstrated significantly higher tracer uptake in WT vs. S1R-KO brain (4.57 ± 1.07 vs. 1.34 ± 0.4 %ID/g at 20-25 min, n = 4, p &lt; 0.05). In S1R-KO mice, it was shown that rapid brain uptake and clearance 10 min post-injection, which are consistent with previous S1R-blocking studies in mice. Three- to fourfold higher tracer uptake was observed in WT relative to S1R-KO mouse brains by ex vivo autoradiography. S1R staining coincided well with the autoradiographic data in all examined brain regions (r (2) = 0.85-0.95). Biodistribution results further demonstrated high [(18)F]FTC-146 accumulation in WT relative to KO mouse brain and provided quantitative information concerning tracer uptake in S1R-rich organs (e.g., heart, lung, pancreas) for WT mice vs. age-matched S1R-KO mice. The maximum allowed dose per scan in humans as extrapolated from mouse dosimetry was 33.19 mCi (1228.03 MBq). No significant toxicity was observed even at a 250X dose of the maximum carrier mass [(19)F]FTC-146 expected to be injected for human studies.ConclusionsTogether, these data indicate that [(18)F]FTC-146 binds specifically to S1Rs and is a highly promising radiotracer ready for clinical translation to investigate S1R-related diseases

Additional file 6: Table S3. of Further validation to support clinical translation of [18F]FTC-146 for imaging sigma-1 receptors

Summary of [19F]FTC-146 toxicity study in rats. (DOC 34 kb

Additional file 2: Figure S2. of Further validation to support clinical translation of [18F]FTC-146 for imaging sigma-1 receptors

Representative MR images showing how regions of interest (ROIs) were drawn for 1 = cortex; 2 = caudate putamen; 3 = hippocampus, 4 = cerebellum during PET/MR image analysis. Whole brain ROIs were drawn using the skull from the CT image as a guide. (DOC 573 kb

Additional file 1: Figure S1. of Further validation to support clinical translation of [18F]FTC-146 for imaging sigma-1 receptors

FTC-146, SN-56 and Vesamicol binding affinity to VAChT. (DOC 171 kb