Response of thin-film SQUIDs to applied fields and vortex fields: Linear SQUIDs

In this paper we analyze the properties of a dc SQUID when the London penetration depth \lambda is larger than the superconducting film thickness d. We present equations that govern the static behavior for arbitrary values of \Lambda = \lambda^2/d relative to the linear dimensions of the SQUID. The SQUID's critical current I_c depends upon the effective flux \Phi, the magnetic flux through a contour surrounding the central hole plus a term proportional to the line integral of the current density around this contour. While it is well known that the SQUID inductance depends upon \Lambda, we show here that the focusing of magnetic flux from applied fields and vortex-generated fields into the central hole of the SQUID also depends upon \Lambda. We apply this formalism to the simplest case of a linear SQUID of width 2w, consisting of a coplanar pair of long superconducting strips of separation 2a, connected by two small Josephson junctions to a superconducting current-input lead at one end and by a superconducting lead at the other end. The central region of this SQUID shares many properties with a superconducting coplanar stripline. We calculate magnetic-field and current-density profiles, the inductance (including both geometric and kinetic inductances), magnetic moments, and the effective area as a function of \Lambda/w and a/w.Comment: 18 pages, 20 figures, revised for Phys. Rev. B, the main revisions being to denote the effective flux by \Phi rather than

Chemical Inhibition of Histone Deacetylases 1 and 2 Induces Fetal Hemoglobin through Activation of GATA2.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene

Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor models

ACY-241 is a novel, orally available and selective histone deacetylase (HDAC) 6 inhibitor in Phase 1b clinical development in multiple myeloma (NCT 02400242). Like the structurally related drug ACY-1215 (ricolinostat), ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor drug candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. We now show that combination treatment of xenograft models with paclitaxel and either ricolinostat or ACY-241 significantly suppresses solid tumor growth. In cell lines from multiple solid tumor lineages, combination treatment with ACY-241 and paclitaxel enhanced inhibition of proliferation and increased cell death relative to either single agent alone. Combination treatment with ACY-241 and paclitaxel also resulted in more frequent occurrence of mitotic cells with abnormal multipolar spindles and aberrant mitoses, consistent with the observed increase of aneuploid cells. At the molecular level, multipolar mitotic spindle formation was observed to be NuMA-dependent and γ-tubulin independent, suggesting that treatment-induced multipolar spindle formation does not depend on centrosomal amplification. The significantly enhanced efficacy of ACY-241 plus paclitaxel observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor versus pan-HDAC inhibitors, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors

Treatment with a selective histone deacetylase 6 inhibitor decreases lupus nephritis in NZB/W mice

To date, there are 18 histone deacetylase (HDAC) enzymes, divided into four classes, which alter protein function by removing acetyl groups from lysine residues. Prior studies report that non-selective HDAC inhibitors decrease disease in lupus mouse models. Concern for adverse side effects of non-selective HDAC inhibition supports investigation of selective-HDAC inhibition. We hypothesized that a selective HDAC-6 inhibitor (HDAC6i) will alleviate disease in a mouse model of lupus by increasing acetylation of alphatubulin. Intraperitoneal injections of the selective HDAC6i ACY-1083 (0.3 mg/kg, 1 mg/kg, or 3 mg/kg), vehicle control, or dexamethasone were administered to 21-week-old, female NZB/W mice, 5 days a week, for 13 weeks. Disease progression was evaluated by proteinuria, serum levels of anti-dsDNA antibody, cytokines and immunoglobulins, and post mortem evaluation of nephritis and T cell populations in the spleen. HDAC6i treatment decreased proteinuria, glomerular histopathology, IgG, and C3 scores when compared to vehicle-treated mice. Within glomeruli of HDAC6i-treated mice, there was increased acetylation of alpha-tubulin and decreased NF-κB. Additionally, HDAC6i decreased serum IL-12/IL-23 and Th17 cells in the spleen. Taken together, these results suggest HDAC6 inhibition may decrease lupus nephritis in NZB/W mice via mechanisms involving acetylation of alphatubulin and decreased NF-κB in glomeruli as well as inhibition of Th17 cells

Effect of HDAC1/2 inhibition on gene expression in erythroid progenitors.

<p>(A) Erythroid maturation stage of cells used in gene expression profiling experiments. BM cells were cultured in CS1 expansion media and then shifted to CS1 differentiation media for 5 days with vehicle or 1 μM ACY-957. At the end of the culture period, erythroid maturation stage was determined by flow cytometry and total RNA was isolated for analysis using Affymetrix GeneChips. Experiments were performed using cells from three independent donors. (B) Differentially expressed genes resulting from ACY-957 treatment using a filter of absolute fold change greater than 1.5 and a P-value less than 0.025. (C) Gene set enrichment analysis demonstrates that genes up-regulated by HDAC2 knockdown (‘Up in <i>HDAC2</i> KD’ gene set) are significantly overrepresented at the top of a ranked list of gene expression changes resulting from ACY-957 treatment. Significant enrichment is illustrated by the positive running enrichment score (ES) marked by the green line, normalized enrichment score (NES) = 2.5, and false discovery rate (FDR) P-value < 0.001. (D) Enrichment scores of the ‘Up in <i>HDAC1</i> KD’, ‘Up in <i>HDAC2</i> KD’, and ‘Down in <i>HDAC2</i> KD’ gene sets relative to all gene sets (2777 total) in the Molecular Signatures Database collection of Chemical and Genetic Perturbations. (E) GeneChip derived gene expression ratios of candidate <i>HBG</i> modulators following ACY-957 treatment, <i>HDAC1</i> knockdown, or <i>HDAC2</i> knockdown. Ratios expressed as treatment versus control. NS, not significant. (F) Gene expression of candidate <i>HBG</i> modulators by QPCR. BM cells were cultured in CS1 expansion media, and then shifted to CS1 differentiation media for 4 days in presence of vehicle or 1 μM ACY-957. Gene expression is shown relative to <i>ACTB</i> and normalized to day 0 (mean ± SD, n = 3 cell culture replicates). P-values were calculated for day 4 using a two-tailed <i>t</i> test. *P<0.05 and ***P<0.0005 compared to vehicle treatment. Data is representative of experiments using cells from two independent donors. (G) Induction of <i>GATA2</i> mRNA by ACY-957 in cells from the four SCD donors described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g003" target="_blank">Fig 3A</a>. P-values were calculated using a two-tailed, paired <i>t</i> test. *P<0.05 compared to vehicle treatment.</p

HDAC1/2 inhibition by ACY-957 leads to elevated levels of histone acetylation and GATA2 binding at <i>GATA2</i> regulatory regions.

<p>BM cells were cultured in CS1 expansion media and then shifted to CS1 differentiation media with vehicle or 1 μM ACY-957 until reaching a differentiation stage similar to cells used in the GeneChip experiments of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g005" target="_blank">Fig 5</a>. (A) Erythroid differentiation stage of cells used for ChIP in Fig 8C, 8E and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g009" target="_blank">Fig 9A</a>. (B) Erythroid differentiation stage of cells used for ChIP in Fig 8D and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g009" target="_blank">Fig 9B</a>. (C) HDAC1 and HDAC2 ChIP-Seq profiles at the <i>GATA2</i> locus in erythroid progenitors. Chromatin was immunoprecipitated and sequenced using antibodies against HDAC1 or HDAC2 (black histogram tracks). Sequencing read count (y-axis) is plotted as a function of genomic region bin (x-axis). Publically available ENCODE Consortium ChIP-Seq data for HDAC1, HDAC2, and GATA2 in K562 cells is shown (gray histogram tracks) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.ref049" target="_blank">49</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.ref050" target="_blank">50</a>]. The previously described <i>GATA2</i> enhancer regions (red text) map to GATA2 binding peaks in K562 cells. In both K562 and primary erythroid progenitors, HDAC1 and HDAC2 occupy a region bounded by the +9.5 kb and -3.9 kb enhancer regions (red dashed lines). (D) Histone acetylation at <i>GATA2</i> regulatory regions in vehicle and ACY-957 treated erythroid progenitors. Chromatin from each treatment was immunoprecipitated using anti-H3K9ac, anti-H2BK5ac, or anti-H3K27ac antibodies. A region near the INO80 gene (Ctrl), identified as a region of saturated acetylation across a wide variety of ENCODE cell lines, was used as a control (mean ± SD, n = 2 QPCR replicates for each of n = 3 IP replicates per antibody). P-values were calculated using a two-tailed <i>t</i> test. **P<0.005 and ***P<0.0005 for ACY-957 compared to vehicle control. (E) GATA2 binding at <i>GATA2</i> regulatory regions following ACY-957 treatment. IP with anti-GATA2 antibody and sequencing as described in ‘C’. ACY-957 treatment resulted in increased GATA2 occupancy (black histogram tracks) at the <i>GATA2</i> regulatory regions, indicated by solid red bars (wide view) or red arrows (magnified view). ACY-957-responsive regions localize to GATA2 binding peaks in K562 cells (gray histogram track).</p

Effect of HDAC inhibition on erythroid differentiation.

<p>BM cells were cultured in CS1 expansion media, and then placed in CS1 differentiation media for 8 days with vehicle or 1 μM ACY-957. Erythroid maturation stage was determined by flow cytometry using the cell surface markers TFRC and GYPA. Representative of experiments performed with cells from four independent donors.</p