Modeling Random Effects Using Global–Local Shrinkage Priors in Small Area Estimation

<p>Small area estimation is becoming increasingly popular for survey statisticians. One very important program is Small Area Income and Poverty Estimation undertaken by the United States Bureau of the Census, which aims at providing estimates related to income and poverty based on American Community Survey data at the state level and even at lower levels of geography. This article introduces global–local (GL) shrinkage priors for random effects in small area estimation to capture wide area level variation when the number of small areas is very large. These priors employ two levels of parameters, global and local parameters, to express variances of area-specific random effects so that both small and large random effects can be captured properly. We show via simulations and data analysis that use of the GL priors can improve estimation results in most cases. Supplementary materials for this article, including a standardized description of the materials available for reproducing the work, are available as an online supplement.</p

Increased Cytoplasmic Localization of p27^kip1 and Its Modulation of RhoA Activity during Progression of Chronic Myeloid Leukemia

<div><p>The role of p27^kip1 in Chronic Myeloid Leukemia (CML) has been well studied in relation to its function as a cell cycle inhibitor. However, its cytoplasmic function especially in CML remains to be seen. We studied the localization of p27^kip1 and its function during the progression of CML from chronic to blast phase. Our investigations revealed an increased localization of p27^kip1 in the cytoplasm of CD34⁺ cells in the blast phase compared to chronic phase. Cytoplasmic p27^kip1 was found to modulate RhoA activity in CD34⁺ stem and progenitor cells. Further, RhoA activity was shown to be dependent on cytoplasmic p27^kip1 which in turn was dependent on p210^Bcr-Abl kinase activity. Interestingly, RhoA activity was observed to affect cell survival in the presence of imatinib through the SAPK/JNK pathway. Accordingly, inhibition of SAPK/JNK pathway using SP600125 increased apoptosis of K562 cells in presence of imatinib. Our results, for the first time, thus reveal a crucial link between cytoplasmic p27^kip1, RhoA activity and SAPK/JNK signalling. To this effect we observed a correlation between increased cytoplasmic p27^kip1, increased RhoA protein levels, decreased RhoA-GTP levels and increased SAPK/JNK phosphorylation in blast phase CD34⁺ cells compared to chronic phase CD34⁺ cells.</p> </div

RhoA activity affects cell survival.

<p>(A) K562 cells were transfected with MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with 1µM imatinib for the indicated time points in hours. Cell lysates were assessed by western blot for the presence of pro-caspase3 and cleaved active caspase3. Beta actin was used as a loading control. Corresponding densitometric analysis indicates the mean±s.e.m of three independent experiments, (p<0.02). (B) K562 cells were transfected with MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with/without 1µM imatinib. Cell survival was assessed by Annexin V staining. The plot shows the percent Annexin V positive staining population in three independent experiments (*p<0.005, **p<0.008) (C) K562 cells were transfected with GFP tagged MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with/without 1µM imatinib for 24 hr. Cells were stained with anti cytochrome c antibody (red) and observed under the confocal microscope. The panel shows cytochrome c staining (red), GFP (green), differential interference contrast (DIC) image and a merged image of GFP and DIC. Release of cytochrome c from mitochondria gives a diffused cytochrome c staining as against the punctate staining pattern observed in cells with intact mitochondrial membrane permeability.</p

p210^Bcr-Abl activity affects RhoA GTP levels.

<p>(A) Western blot showing the RhoA-GTP and total RhoA levels in K562 cells treated with/without imatinib. Corresponding densitometric analysis was performed after normalisation against beta actin (n=3, *p<0.004). (B) Western blot showing the RhoA-GTP and total RhoA levels in CML cells treated with/without imatinib. Corresponding densitometric analysis was performed after normalisation against beta actin (n=3, *p<0.0014). (C) CD34⁺ stem and progenitor cells isolated from chronic phase CML bone marrow was nucleofected with the various constructs of p27^kip1. The levels of RhoA-GTP and total RhoA were assessed in the resulting cells by western blotting. A representative blot of three independent experiments is shown (*p<0.09). (D) The distribution of RhoA between the membrane and cytosolic fraction under conditions of nucleotide exchange in K562 cells as observed by western blotting. (E) Ba/F3 cells were electroporated with p210 ^Bcr-AblWT and p210 ^Bcr-AblT315I and then treated with/ without imatinib. The western blot shows the expression of p210^Bcr-Abl and Bcr proteins, the distribution of RhoA between the membrane and cytosolic fraction and corresponding levels of p27^kip1 and phosphorylated p27^kip1 S10 in these cells. Corresponding densitometric analysis shows the mean±s.e.m of three independent experiments (*p<0.09). (All the other plots have a p<0.002).</p

Inhibition of p210^Bcr-Abl leads to increased JNK activity.

<p>(A) K562 cells were treated with imatinib with 1µM for 24 hr and the levels of phosphorylated p38 MAPK, p38MAPK, phosphorylated SAPK/JNK, SAPK/JNK phosphorylated p42/44 MAPK, p42/44 MAPK was assessed. Beta actin was used as a loading control. (B) K562 cells were transfected with MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with/without 1µM imatinib for 24 hr. The levels of phosphorylated p38 MAPK, p38MAPK and phosphorylated SAPK/JNK, SAPK/JNK was assessed by western blotting. (C) K562 cells were treated with imatinib, C3 exozyme or both and the activity of SAPK/JNK pathway was assessed by looking into the phosphorylation of c-jun. (D) K562 cells were treated with imatinib, SP600125 or both and the level of cytochrome c released from the mitochondria was analysed in the mitochondria free cytosolic fraction. Tim23 was used to estimate mitochondrial contamination in the mitochondria free cytosolic preparations while beta actin was used as a loading control. (E) K562 cells were treated with the indicated concentrations of imatinib and SP600125 either singly or in combination for 24hrs. The percentage metabolic activity was assessed by MTT assay. Data shows the mean percent metabolic activity for individual treatments in three separate experiments. (F) K562 cells were treated with 1µM imatinib, 20µM SP600125 or both and apoptosis was measured by staining the cells with Annexin V and PI. Graph shows the change in Annexin V stained and Annexin V + PI stained population compared to untreated control cells (n=3, *p<0.037, **p<0.002). (G) Protein expression of phosphorylated SAPK/JNK, SAPK/JNK, phosphorylated c-jun and c-jun in CD34⁺ cells of chronic and blast phase CML. Beta actin was used as a loading control.</p

Cytoplasmic p27^kip1 is dependent on p210^Bcr-Abl.

<p>(A) K562 cells treated with the indicated doses (in µM) of imatinib for 24hrs were subjected to sub-cellular fractionation and the nuclear and cytoplasmic fractions were used to detect the levels of p27^kip1. Lamin and GAPDH were used as loading controls for nuclear and cytoplasmic fractions respectively. Densitometric analysis indicates the mean ± s.e.m of three independent experiments with p<0.007 (B) K562 cells were treated with imatinib with the indicated dose (in µM) for 24 hr and the levels of phosphorylated p27 ^kip1S10 and p27^kip1 was assessed by western blotting. Beta actin was used as a loading control. (C) Primary chronic phase CML cells were treated with/without imatinib and then subjected to nuclear and cytoplasmic fractionation. The fractions were used to detect the levels of p27^kip1. Histone 2B and GAPDH were used as loading controls for nuclear and cytoplasmic fractions respectively. The panel shows a densitometric analysis of three independent experiments with mean±s.e.m (p<0.01). (D) mRNA expression of p27^kip1 in chronic and blast phase CD34⁺ stem and progenitor cells of CML was assessed by qRT-PCR. The expression was normalised against HPRT1. Horizontal bar indicates the mean of expression for the individual phase. (E) Protein expression of p27 ^kip1S10 and p27^kip1 in chronic and blast phase CD34⁺ cells. Beta actin was used as a loading control. Densitometric analysis of the blots shows the mean ± s.e.m of 4 individual experiments (p<0.02). (F) Primary chronic and blast phase CML cells were subjected to nuclear and cytoplasmic fractionation. The fractions were used to detect the levels of p27^kip1. Histone 2B and GAPDH were used as loading controls for nuclear and cytoplasmic fractions respectively. The panel shows a representative blot of two independent experiments. Densitometric analysis of two independent experiments has been shown with mean±s.e.m . Lower panel shows the fold change in expression of p27^kip1 in the cytoplasm of Blast phase CML cells compared to Chronic phase CML cells.</p

The Discovery of <i>N</i>‑(1-Methyl-5-(trifluoromethyl)‑1<i>H</i>‑pyrazol-3-yl)-5-((6- ((methylamino)methyl)pyrimidin-4-yl)oxy)‑1<i>H</i>‑indole-1-carboxamide (Acrizanib), a VEGFR‑2 Inhibitor Specifically Designed for Topical Ocular Delivery, as a Therapy for Neovascular Age-Related Macular Degeneration

A noninvasive topical ocular therapy for the treatment of neovascular or “wet” age-related macular degeneration would provide a patient administered alternative to the current standard of care, which requires physician administered intravitreal injections. This manuscript describes a novel strategy for the use of in vivo models of choroidal neovascularization (CNV) as the primary means of developing SAR related to efficacy from topical administration. Ultimately, this effort led to the discovery of acrizanib (LHA510), a small-molecule VEGFR-2 inhibitor with potency and efficacy in rodent CNV models, limited systemic exposure after topical ocular administration, multiple formulation options, and an acceptable rabbit ocular PK profile