14 research outputs found

    The Effects of Pharmacological Inhibition of Histone Deacetylase 3 (HDAC3) in Huntington’s Disease Mice

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    <div><p>An important epigenetic modification in Huntington’s disease (HD) research is histone acetylation, which is regulated by histone acetyltransferase and histone deacetylase (HDAC) enzymes. HDAC inhibitors have proven effective in HD model systems, and recent work is now focused on functional dissection of the individual HDAC enzymes in these effects. Histone deacetylase 3 (HDAC3), a member of the class I subfamily of HDACs, has previously been implicated in neuronal toxicity and huntingtin-induced cell death. Hence, we tested the effects of RGFP966 ((<i>E</i>)-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1<i>H</i>-pyrazol-4-yl)acrylamide), a benzamide-type HDAC inhibitor that selectively targets HDAC3, in the N171-82Q transgenic mouse model of HD. We found that RGFP966 at doses of 10 and 25 mg/kg improves motor deficits on rotarod and in open field exploration, accompanied by neuroprotective effects on striatal volume. In light of previous studies implicating HDAC3 in immune function, we measured gene expression changes for 84 immune-related genes elicited by RGFP966 using quantitative PCR arrays. RGFP966 treatment did not cause widespread changes in cytokine/chemokine gene expression patterns, but did significantly alter the striatal expression of macrophage migration inhibitory factor (<i>Mif)</i>, a hormone immune modulator associated with glial cell activation, in N171-82Q transgenic mice, but not WT mice. Accordingly, RGFP966-treated mice showed decreased glial fibrillary acidic protein (GFAP) immunoreactivity, a marker of astrocyte activation, in the striatum of N171-82Q transgenic mice compared to vehicle-treated mice. These findings suggest that the beneficial actions of HDAC3 inhibition could be related, in part, with lowered <i>Mif</i> levels and its associated downstream effects.</p></div

    Real-time qPCR results showing altered expression of <i>Mif</i> and <i>Il1b</i> in striatum and cortex of RGFP966 treated WT and N171-82Q mice.

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    <p>Groups of mice were treated with RGFP966 (25 mg/kg) for 12 weeks beginning at 8 weeks of age. Bar graphs shown the mean +/- S.E.M. expression value from n = 5–6 mice per group normalized to the expression of <i>Hprt</i>. Significant differences of p<0.05 were measured by a two-tailed, unpaired Student’s <i>t</i> test and are indicated by an asterisk (*).</p

    The effects of RGFP966 (25 mg/kg) on striatal volume in WT and N171-82Q transgenic mice.

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    <p>Representative photomicrographs of the striatum at ~0.62 Bregma under each condition are shown on the left. Scale bar = 40 μm. Bar graphs show quantitation of striatal volume. The total area of the striatum was assessed using sections 125 μm apart spanning the striatum from ~bregma, 1.18 to 0.38 mm. One-way ANOVA (Dunnett’s post-test) revealed significant differences between vehicle-treated wild type and N171-82Q transgenic mice and also a significant difference between vehicle-treated and RGFP966-treated N171-82Q mice (*, P<0.05). Bars represent mean score ± SEM (n = 6 to 7 per group).</p

    Summary of cytokines array gene expression changes in striatum due to RGFP966 treatment.

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    <p>A. Heatmap of expression values for 84 cytokine/chemokine genes showing two-way clustering of expression levels and treatment groups. Red denotes increased relative gene expression levels for the indicated groups, with green denoting decreased expression levels. B. Volcano plots of expression changes due to RGFP966 treatment showing three different comparisons, as indicated. Dotted line on y-axis denotes the significance cut-off of p-value<0.05, using one-way ANOVA. Dotted lines on x-axis denote a fold-change cut-off of > +/- 2.</p

    Stimulated Emission Depletion Microscopy Resolves Individual Nitrogen Vacancy Centers in Diamond Nanocrystals

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    Nitrogen-vacancy (NV) color centers in nanodiamonds are highly promising for bioimaging and sensing. However, resolving individual NV centers within nanodiamond particles and the controlled addressing and readout of their spin state has remained a major challenge. Spatially stochastic super-resolution techniques cannot provide this capability in principle, whereas coordinate-controlled super-resolution imaging methods, like stimulated emission depletion (STED) microscopy, have been predicted to fail in nanodiamonds. Here we show that, contrary to these predictions, STED can resolve single NV centers in 40–250 nm sized nanodiamonds with a resolution of ≈10 nm. Even multiple adjacent NVs located in single nanodiamonds can be imaged individually down to relative distances of ≈15 nm. Far-field optical super-resolution of NVs inside nanodiamonds is highly relevant for bioimaging applications of these fluorescent nanolabels. The targeted addressing and readout of individual NV<sup>–</sup> spins inside nanodiamonds by STED should also be of high significance for quantum sensing and information applications

    Onset of Multiferroicity in Prototypical Single-Spin Cycloid BiFeO<sub>3</sub> Thin Films

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    In the room-temperature magnetoelectric multiferroic BiFeO3, the noncollinear antiferromagnetic state is coupled to the ferroelectric order, opening applications for low-power electric-field-controlled magnetic devices. While several strategies have been explored to simplify the ferroelectric landscape, here we directly stabilize a single-domain ferroelectric and spin cycloid state in epitaxial BiFeO3 (111) thin films grown on orthorhombic DyScO3 (011). Comparing them with films grown on SrTiO3 (111), we identify anisotropic in-plane strain as a powerful handle for tailoring the single antiferromagnetic state. In this single-domain multiferroic state, we establish the thickness limit of the coexisting electric and magnetic orders and directly visualize the suppression of the spin cycloid induced by the magnetoelectric interaction below the ultrathin limit of 1.4 nm. This as-grown single-domain multiferroic configuration in BiFeO3 thin films opens an avenue both for fundamental investigations and for electrically controlled noncollinear antiferromagnetic spintronics

    An HDAC3 selective inhibitor triggers apoptosis associated with increased DNA damage and cell cycle defects.

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    <p>(A) Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and apoptosis assessed by Annexin V staining and flow cytometry. Cells were also labeled with propidium iodide to assess DNA content. Untreated (UT) and DMSO treated cells were used as controls. Shown is a representative graph from an experiment performed in duplicate that is consistent with other biological replicates. (B) Western blot analysis of γH2aX levels in Hut78 cells treated with DMSO, 10 nM Depsi, 10 µM 233, or 10 µM 966 for 8 hrs. Untreated and DMSO treated cells were used as controls. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side by side comparison of DMSO and Depsipeptide. (C) Cell cycle status was analyzed using BrdU incorporation and propidium iodide to assess DNA content by flow cytometry. Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and pulsed for an hour and a half with BrdU prior to cell harvest and analysis. Shown are representative flow cytometry plots from an experiment performed in duplicate that is consistent with other biological replicates. (D) Graphical representation of BrdU incorporation from the experiment described in (C). (E) Graphical representation of the percent of S phase cells that did not incorporate BrdU (shown by box in panel (C)). Statistical analysis for both the Annexin V and BrdU experiments was performed using a two-tail T-test and comparing the HDI treated cells to the DMSO treated cells resulting in the following p-values: (A) Depsi: p = 0.0002, 233: p = 0.003, and 966: p = 0.0003. (D) Depsi: p = 0.003, 233: p = 0.01, and 966: p = 0.08. (E) Depsi: p = 0.003, 233: p = 0.003, and 966: p = 0.004.</p

    Dual treatment with RGFP966 and CTCL drugs has an additive effect on cell growth.

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    <p>Growth curves of dual treatment on HH cells or Hut78 cells. Cells were treated once at hour 0 with DMSO, 10 nM Depsipeptide (Depsi), 2 µM 966, or a combination of 2 µM 966 and either Bexarotene, Methotrexate, or ATRA. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. (A) HH cells (left) or Hut78 cells (right) were treated with 20 µM or 75 µM Bexarotene alone or in combination with 966. (B) Cells were treated with 0.1 µM Methotrexate alone or in combination with 966. DMSO and 1 M Na<sub>2</sub>CO<sub>3</sub> served as vehicle controls. (C) Cells were treated with 2 µM ATRA alone or in combination with 966. ATRA was administered at hour 0 and re-dosed at 48 hours after treatment. For (A–C), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI, CTCL drug, or dual treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, Bexarotene: p = 0.003, and 966 plus Bexarotene: p = 0.002. For the Hut78 cells (right), Depsi: p = 0.001, 966: p = 0.08, Bexarotene: p = 0.01, and 966 plus Bexarotene: p = 0.009. (B) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, Methotrexate: p = 0.003, and 966 plus Methotrexate: p = 0.003. For the Hut78 cells (right) Depsi: p = 0.001, 966: p = 0.01, Methotrexate: p = 0.01, and 966 plus Methotrexate: p = 0.004. (C) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, ATRA: p = 0.002, and 966 plus ATRA: p = 0.0007. For the Hut78 cells (right) Depsi: p = 0.001, 966: p = 0.01, ATRA: p = 0.02, and 966 plus ATRA: p = 0.004.</p

    iPOND analysis reveals HDAC3 association with replication forks in Hut78 CTCL cells.

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    <p>Hut78 cells were pulsed for 15 minutes with EdU followed by either no thymidine chase or a 60 minute thymidine chase. The protein-DNA complexes were then cross-linked, nascent DNA was conjugated to biotin using click chemistry, and then protein-DNA complexes were purified using Streptavidin beads. The eluted proteins were then analyzed using western blot analysis. A no click reaction sample (No Clk) that did not include biotin azide was used as a negative control. 0.1% input samples were included for positive controls of each protein analyzed. PCNA served as a positive control for a replication fork bound protein and H2B served as a loading control and positive control for a chromatin bound protein.</p
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