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Angiogenesis, Metastasis, and the Cellular Microenvironment
Progesterone and 1,25-Dihydroxyvitamin D3 Inhibit Endometrial Cancer Cell Growth by Upregulating Semaphorin 3B and Semaphorin 3F
Huyen Nguyen, Vessela S. Ivanova, Leyla Kavandi, Gustavo C. Rodriguez, George L. Maxwell and Viqar Syed
Huyen Nguyen
Vessela S. Ivanova
Leyla Kavandi
Gustavo C. Rodriguez
George L. Maxwell
Viqar Syed
DOI: 10.1158/1541-7786.MCR-11-0213 Published November 2011
Class 3 semaphorins (SEMA), SEMA3B and SEMA3F, are secreted proteins that regulate angiogenesis, tumor growth, and metastasis by binding to their transmembrane receptor complex consisting of plexins and neuropilins (NP). Expression of SEMAs and their receptors was assessed in tissue microarrays by immunohistochemistry. SEMA3B, SEMA3F, and plexin A3 were expressed strongly in normal endometrial tissues, whereas grade-dependent decreases were found in endometrial carcinomas. No change was observed in the expression of plexin A1, NP1, and NP2 in normal versus endometrial cancer tissues. Endometrial cancer cells showed decreased expression of SEMA3B, SEMA3F, and plexin A3 compared with their normal counterparts. Treatment of cancer cells with progesterone (P4) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for a period of 72 hours induced a significant upregulation of SEMA3B and SEMA3F as well as inhibited growth of cancer cells by increasing caspase-3 activity. Cotreatment of cell lines with P4 or 1,25(OH)2D3 and their respective antagonists confirmed the specificity of their actions. Transfection of siRNA-targeting SEMA3B and SEMA3F in endometrial cancer cells attenuated P4 or 1,25(OH)2D3-induced growth inhibition. Restoration of SEMA3B or SEMA3F expression in cancer cells caused growth inhibition, reduced soft agar colony formation, and cell invasiveness by inhibiting expression of matrix metalloproteinase-2 (MMP-2), MMP-9, integrin αvβ3, and proangiogenic genes and by upregulating antiangiogenic genes. Thus, we have identified two new P4 and 1,25(OH)2D3-regulated antitumor genes for endometrial cancer. These results suggest that the loss of SEMAs contribute to the malignant phenotype of endometrial cancer cells and that reexpression of SEMAs by ectopic expression or with anticancer agents P4 or 1,25(OH)2D3 can be a promising therapeutic treatment against endometrial cancer. Mol Cancer Res; 9(11); 1479–92. ©2011 AACR.
Endometrial cancer is the fourth most common gynecologic cancer among women in the Western world (1, 2). Although advances have been made in the field, the American Cancer Society (2) estimated that 43,406 new cases of endometrial cancer will be diagnosed in 2010, and approximately 7,950 women will die from this disease.
The pathogenesis of endometrial cancer involves a multistep process of genetic and molecular changes (3, 4). Studies have shown that a genomic aberration in human chromosome 3p is the most frequent and earliest genetic event in endometrial tumorigenesis (5–8). Genetic changes result in the clonal expansion of cells with selective growth advantages, including the activation of proto-oncogenes and the inactivation of tumor suppressor genes (9).
SEMAs are glycoproteins that were originally described as mediators of neuronal guidance (10, 11). Class 3 SEMAs (SEMA3) serve as key regulators of cellular processes, such as cell survival, proliferation, apoptosis, and migration (11, 12). Both SEMA3F and SEMA3B are mapped to chromosome 3p21.3, a region of frequent loss in lung, breast, and endometrial cancers (13–15). Most SEMAs directly bind to plexins which elicit intracellular signals (16). However, secreted SEMA3s are recruited to plexins by neuropilin (NP) receptors (17, 18). NP1 preferentially binds to SEMA3B, whereas NP2 has higher affinity for SEMA3F (19). We (20), and others (21–23) have shown that SEMA3B and SEMA3F are either absent or are expressed at very low levels in ovarian, lung, and breast cancer cells. When expression is restored, they function as potent inhibitors of tumor growth and angiogenesis. There is no published information on the status and regulation of SEMA3B and SEMA3F in endometrial cancer.
Clinical, epidemiologic, and basic science evidence suggest that progestins and vitamin D may be highly effective endometrial cancer preventive agents. Routine use of progestin lowers endometrial cancer risk, and the protective effect increases with increasing progestin potency. In premenopausal women, use of estrogen–progestin combination oral contraceptives (OC) for a period of at least 12 months confers a 30% to 50% reduction in the risk of endometrial cancer, a protective effect which lasts for 10 to 20 years after discontinuation of use (24, 25). In addition, progestin-potent OCs have enhanced endometrial cancer protective effects compared with OCs containing weak progestins (26, 27). Finally, high-dose progestin therapy has been shown to reverse preexisting PTEN-inactivated endometrial latent precursors, as well as endometrial hyperplasia and even low-grade endometrial cancer in some women (28, 29). With regard to vitamin D, several lines of evidence suggest a chemopreventive role in the endometrium: (i) there is a significant inverse correlation between UVB exposure and endometrial cancer risk (30); (ii) the endometrium has been shown to express the 1-alpha hydroxylase enzyme (CYP27B1) and is thereby capable of synthesizing 1,25 (OH)2 D3 from circulating 25 (OH)2 D3; (iii) vitamin D has been shown to modulate the PTEN pathway and inhibit carcinogenesis in PTEN-related neoplasms (31); (iv) it has been shown that high dietary intake of vitamin D is associated with a decrease in endometrial cancer risk (OR = 0.43; 95% CI: 0.23–0.80; P = 0.01; ref. 32), and (v) vitamin D has been shown to inhibit carcinogenesis in a mouse model of endometrial cancer (33).
Although the biological mechanism(s) that underlies the protective effect of progestins and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has not been well characterized, a growing body of evidence has implicated apoptotic signaling events as important mediators of the chemopreventive effects in the endometrium (29, 34).
The purpose of the study was to characterize the status of tumor suppressors SEMA3B and SEMA3F in endometrial cancer and to determine whether the chemopreventive effects of progesterone and 1,25(OH)2D3 were mediated by modulation of SEMA3B and SEMA3F levels in endometrial cancer cells. Understanding how SEMAs regulate cell invasion and metastasis could lead to the development of new anticancer therapies for endometrial cancer patients.
Our results showed a decrease in the expression of SEMA3B and SEMA3F in endometrial tumors and in cancer cell lines as compared with the normal endometrium. Furthermore, restoration of SEMA3 expression by ectopic expression or by P4 or 1,25(OH)2D3 treatment resulted in the reduction of cell proliferation, colony formation, and invasion that was associated with decreased expression of matrix metalloproteinases (MMP), integrins, αvβ3, and proangiogenic genes and increased expression of antiantiangiogenic genes, implicating a role of SEMAs in the inhibition of tumorigenesis.
Endometrial tissue arrays, EMC962, consisting of normal (12 cases per 24 cores) and cancerous (36 cases per 72 cores) tissues of the endometrium in duplicates were obtained from US Biomax. Immunohistochemistry was done on formalin-fixed, paraffin-embedded endometrial cancer tissue arrays. The array slides were deparaffinized and antigen retrieval was carried out by using a Retriever 2100 (Electron Microscopy Sciences) in 10 mmol/L of sodium citrate buffer (pH 6). The staining kits used were Vectastain Elite ABC kit and DAB (Vector Laboratories Inc.). The primary antibodies were SEMA3B (1:100; Abcam Inc.), SEMA3F (1:50; Millipore), plexin-A1 (1:50; Cell Signaling Technology), plexin-A3 (1:50; Santa Cruz), NP1 (1:100; Abcam Inc.), and NP2 (1:50; Abcam Inc.). Images were captured using a Nikon Eclipse E-800 microscope. Expression levels within tumor tissue was quantified across cores as a product of the staining intensity (0 = negative, 1 = weak, 2 = moderate, and 3 = strong) × percentage cells stained. Staining intensity was quantified under high magnification (×20). Expression patterns were correlated with a range of clinical parameters such as tumor classifications (tumor stage and grade).
Cell lines and culture conditions
Normal epithelial endometrial (EM-E6/E7/TERT) cells established and characterized by Kyo and colleagues (35) and endometrial cancer cell lines (Ishikawa, HEC-1B, and RL-95) from American Type Culture Collection were maintained and propagated in Dulbecco's modified Eagle's medium (DMEM; EM-E6/E7/TERT), DMEM:F12, (Ishikawa), Eagle MEM (HEC-1B), and DMEM:F12 (RL-95) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin (Life Technologies), and 10 μg/mL insulin (Sigma, Chemical Co.) at 37°C in a humidified atmosphere of 5% CO2, 95% air.
Treatment of endometrial cell lines
The antiproliferative effects of progesterone (P4, 99.9% pure; Sigma) and 1,25(OH)2D3 (Sigma) on HEC-1B, Ishikawa, and RL-95 cells were determined using CellTiter96 AQueous One Solution cell viability assay. Cells (2 × 103 per well) were plated into 96-well plates and incubated overnight to allow cell adherence. The media were removed, and progesterone (12.5–200 μmol/L) or 1,25(OH)2D3 (50–400 nmol/L) was added in a total volume of 200 μL. Cells were treated for 72 hours. To avoid the toxicity associated with higher doses of progesterone in most experiments, we used the lowest dose of progesterone that has a potent effect on the growth of endometrial cancer cells. Because high doses of 1,25(OH)2D3 are associated with hypercalcemia in vivo, we therefore used a concentration of 1,25(OH)2D3 that would not be thought to induce hypercalcemia in vivo (36).
For a set of experiments, HEC-1B, Ishikawa, and RL-95 cells were harvested when 80% confluent and washed with PBS. Then 2 × 105 cells were seeded per T-25 flask and allowed to attach for 24 hours. The cells were exposed to either progesterone receptor antagonist mifepristone at 100 nmol/L or the vitamin D receptor (VDR) antagonist telmisartan (Sigma) at 10 μmol/L 1 hour prior to treatment with P4 (25 μmol/L) or 1,25(OH)2D3 (100 nmol/L), respectively, for 72 hours. Cells were collected and protein was extracted.
Transfection of SEMA3B and SEMA3F in endometrial cancer cells
The vectors containing SEMA3B and SEMA3F were kindly provided by Dr. John D. Minna (Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX) and Dr. Gera Neufeld (Cancer Research and Vascular Biology Center, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel). Endometrial cancer (HEC-1B and Ishikawa) cells (8 × 104) were seeded in 6-well plates and transfected the following day with the pcDNA3 vector alone or with pcDNA3 vector containing SEMA3F or SEMA3B (0.25–1.0 μg) using the Lipofectamine 2000 reagent (Invitrogen). To study whether SEMA3B and SEMA3F have an additive effect on proliferation, cells were cotransfected with SEMA3B (0.5 μg/mL) and SEMA3F (0.5 μg/mL). The effect of SEMA overexpression was assessed on cell proliferation, soft agar colony formation, and invasion. To establish that SEMAs are mediators of P4 and 1,25(OH)2D3-induced growth inhibition, cells were transfected with siRNAs targeting SEMA3B, SEMA3F (5 nmol/L; Santa Cruz Biotechnology, Inc.) or nontargeting siRNA and treated 24 hours later with P4 or 1,25(OH)2D3 for 72 hours. To better understand the role of SEMA3B and SEMA3F in the inhibition of proliferation, cells were transfected with siRNAs directed against NP1 and NP2 (5 nmol/L; Santa Cruz Biotechnology, Inc.) or scrambled siRNA and treated 24 hours later with SEMA3F-CM or SEMA3B-CM for 72 hours. The overall transfection efficiency for endometrial cells assessed by X-Gal staining assay against pSV-β-galactosidase vector–transfected cells was 70% to 74%. Cellular extracts were prepared to analyze the expression of SEMAs, and some cells were used for cell viability assay.
Cell viability assay
Viability of P4 or 1,25(OH)2D3-treated or SEMA-transfected cells was evaluated using the CellTiter96 AQueous One Solution cell viability assay (Promega) according to the instructions of the manufacturer. On the fourth day, 20 μL of CellTiter 96 Aqueous One solution reagent were added into each well of the 96-well assay plate containing the cells in 100 μL of culture medium. Absorbance was measured at 490 nm using a microtiter plate reader. Relative cell viability was expressed as percent change of treated/transfected cells over control/empty vector–transfected cells.
Invasion assay
The Biocoat Matrigel Invasion Chambers (BD Biosciences) were used to assess the effects of the ectopic expression of SEMA3B or SEMA3F on endometrial cancer cell invasiveness. Matrigel chambers were rehydrated at 37°C for 2 hours. Endometrial cancer cells (2.5 × 104 cells), transfected with an empty vector or a vector carrying an expression plasmid of SEMA3B or SEMA3F, were seeded in the inserts of the Matrigel Invasion Chambers. Serum added to the bottom chamber was used as the chemoattractant. At 24 hours after plating, noninvading cells were removed, and invading cells were counted in 5 fields per slide as previously described (37). The assay was run in triplicate.
Soft agar assay
The effects of ectopic expression of SEMA3B and SEMA3F on anchorage-independent growth were measured by the ability of transfected cells to form colonies on soft agar. HEC-1B and Ishikawa cells were transfected with SEMA3B or SEMA3F vectors (1 μg/mL), or the empty vectors as described above. After 18 hours, transfected cells were cultured at 5,000 cells per 100-mm plate (4 plates per sample) in 0.3% agar above an underlayer of 0.6% Noble agar, both containing complete medium (38). Number of colonies was counted after 4 weeks of culture.
Cellular extracts were prepared as previously described (37). Briefly, cell lysates were prepared in radioimmunoprecipitation assay buffer, and proteins extracted (20 μg) from each cell culture were separated by electrophoresis on 7.5% or 10% SDS-polyacrylamide gels. The blots were incubated overnight at 4°C in blocking solution with either SEMA3B (1:5,000; Abcam Inc.), SEMA3F (1:5,000; Millipore), NP1 (1:1,000; Abcam Inc.), NP2 (1:1000; Abcam Inc.), plexin-A1 (1:1,000; Cell Signaling Technology), plexin-A3 (1:250; Novus Biologicals), MMP-2 (1:200; Abcam Inc.), MMP-9 (1:1,000; Cell Signaling), αvβ3 (1:1,000; Cell Signaling), or β-actin antibody (1:25,000; Sigma-Aldrich). After washing with PBS Tween-20 (PBST), the membranes were then incubated with the respective secondary antibodies at a 1:3,000 dilution in 5% nonfat dry milk in PBS Tween-20 for 2 hours at room temperature. After washing, bound antibodies were detected by using an enhanced chemiluminescence detection system (Pierce).
Gelatin zymography
Subconfluent monolayers of endometrial cancer cells, transfected with SEMA3B, SEMA3F, or empty vector were grown in T-25 tissue culture flasks and serum starved in serum-free media overnight. Cells were cultured for an additional 24 hours. Cell culture conditioned media (CM) were collected, cleared by centrifugation, and concentrated 5-fold using Centricon centrifugal filters (Millipore). CM, equivalent to 200 μg of endometrial cancer cells, were analyzed with gelatin zymography. CM were electrophoresed on a 10% SDS–polyacrylamide gel containing 0.1% to 0.2% gelatin. Gels were washed twice with washing buffer (50 mmol/L Tris–HCl, pH 7.5, 100 mmol/L NaCl, and 2.5% Triton X-100). Gels were then treated with incubation buffer (50 mmol/L Tris–HCl, pH 7.5, 150 mmol/L NaCl, 10 mmol/L CaCl2, 0.02% NaN3, and 1 μmol/L ZnCl2) at 37°C for 18 to 36 hours, stained (0.05% Coomassie blue, 10% isopropanol, 10% acetic acid) and destained (10% isopropanol, 10% acetic acid). MMPs were detected as transparent bands on the blue background of Coomassie blue–stained slab gels.
Caspase-3 activity assay
Endometrial cells were treated with P4 or 1,25(OH)2D3 or transfected with siRNA of SEMAs. After 72 hours of incubation, the detached cells and the adherent cells were collected from each culture and suspended in ice-cold lysis buffer provided with the Caspases Assay kit (MLB International). Following sonification, the cell lysates were centrifuged for 20 minutes at 14,000 × g at 4°C. The resulting supernatants were analyzed for protein concentrations using a protein determination kit (Pierce) and stored at −20°C until use. Colorimetric enzymatic activity assays for caspase-3 were done according to the manufacturer's instructions.
Angiogenesis RT2 profiler PCR array
Total RNA was isolated from control, SEMA3B, or SEMA3F-overexpressing cells using TRI-reagent (Sigma). After DNase treatment, RNA was further cleaned using the Qiagen RNeasy Mini kit (Qiagen). cDNA was synthesized by RT2 First Strand kit (SABiosciences) per the company's instructions. Gene expression profiling using the Angiogenesis RT2 Profiler PCR Array (SA Biosciences) was conducted. This platform is designed to profile the expression of 84 key genes in angiogenesis (for a comprehensive list of genes included in this array see http://www.sabiosciences.com). Quantitative reverse transcriptase PCR (RT-PCR) was conducted using the 7500 RT-PCR System (AB Applied Biosciences) following the array manufacturer's instructions. Relative gene expression was determined using the ΔΔCT method. Data was further analyzed with the PCR Array Data Analysis Web Portal (http://www.SABiosciences.com/pcrarraydataanalysis.php).
Data normalization and analysis
Five endogenous control genes: beta-2-microglobulin (B2M), hypoxanthine phosphoribosyltransferase (HPRT1), ribosomal protein L13a (RPL13A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin (ACTB) present on the PCR array were used for normalization. Each replicate cycle threshold (CT) was normalized to the average CT of 5 endogenous controls on a per plate basis. The comparative CT method was used to calculate the relative quantification of gene expression. The following formula was used to calculate the relative amount of the transcripts in the treated samples to the control group, both of which were normalized to the endogenous controls: ΔΔCT = ΔCT (treated) − ΔCT (control) for RNA samples. ΔCT is the log2 difference in CT between the target gene and the endogenous controls by subtracting the average CT of controls from each replicate. The fold-change for each treated sample relative to the control sample = 2 − ΔΔCT. Sensitivity detection and identification of expressed genes PCR array quantification was based on the CT number. CT was defined as 35 for the ΔCT calculation when the signal was below detectable limits. A list of differentially expressed genes was identified using a 2-tailed student t test. Changes in gene expression between SEMA-transfected cells and vector controls were illustrated as a fold increase/decrease. The criteria were a P value < 0.05 and a mean difference equal to or greater than a 2-fold change in expression levels after treatment. The statistical calculation was based on the web-based program for Profiler TM PCR Array Data Analysis. Alterations in mRNA levels that fitted the criteria above were considered to be up- or downregulated. The experiments were repeated 3 times.
Statistical analysis was carried out using ANOVA, followed by Tukey's post hoc test. Values are presented as the mean ± SD and are considered significant at a P value of less than 0.05.
Attenuation of SEMA3B, SEMA3F, and plexin A3 expression in stage III endometrial cancer tissues
In vivo expression of SEMA3B, SEMA3F, plexin A1, plexin A3, NP1, and NP2 was analyzed by immunohistochemistry in TMA (US Biomax Inc.). The TMA consisted of 24 normal and 72 malignant tissues. There were 22 tissues from grade I, 26 from grade II, and 16 from grade III cancer. The staining of TMAs was correlated with clinicopathologic parameters of patients (Table 1). Stained TMAs were examined by 3 individuals in a blinded manner. The results indicated that in normal endometrial tissues, SEMA3F and SEMA3B were detected in the epithelial cells in the glands as well as in the stromal cells (Fig. 1, panels 2 and 7). Expression of SEMA3F and SEMA3B was markedly decreased (P < 0.05, Table 1) with progression of tumors to advanced grades (Fig. 1, panels 5 and 10). SEMA receptors, plexin A1 and plexin A3, were expressed in the gland and stromal cells of normal endometrial tissues (Fig. 1, panels 12 and 17). Gradual decrease in plexin A3 expression was seen in cancer specimens (Fig. 1, panels 18–20). Complete loss of plexin A3 was evident in grade III endometrial cancer (Fig. 1, panel 20). No changes in expression of NP1 and NP2 were noticed between normal and different grades of endometrial cancer tissues. Taken together, these data suggested that loss of SEMA3B, SEMA3F, and plexin A3 expression is associated with endometrial carcinogenesis.
Expression of SEMA3B, SEMA3F, plexin A1, plexin A3, NP1, and NP2 in human endometrial tumors. Immunohistochemical analysis of endometrial tumors from TMA using SEMA3B, SEMA3F, plexin A1, plexin A3, NP1, and NP2 antibodies was done. Expression of SEMA3B (2) and SEMA3F (7) was significantly high in normal endometrial tissues. Gradual decrease of SEMA3B (3–4) and SEMA3F (8–9) expression was seen in grades I–II. Grade III (5 and 10) showed no to weak expression of SEMA3B and SEMA3F. Negative controls for SEMA3B (1) and SEMA3F (6) are shown. Expression of plexin A1 (12) and plexin A3 (17) was significantly high in normal endometrial tissues. No change in plexin A1 expression was noticed in different grades of endometrial cancer. Gradual decrease of plexin A3 expression was seen in grades I to III (18–20). Negative controls for plexin A1 (11) and plexin A3 (16) are shown. No alterations of NP1 (22) and NP2 (27) in normal and different grades of endometrial cancer tissues were seen. Original magnification, 400×.
Correlation between clinicopathologic features of patients and staining intensity of SEMA3B, SEMA3F, plexins, and NPs
Loss of SEMA3B, SEMA3F, and their receptor plexin A3 expression in endometrial cancer cells
To confirm that SEMA3B, SEMA3F, and their receptors are expressed in cultured endometrial carcinoma cells, expression levels of these proteins were determined by immunoblotting. Because SEMAs are secretory proteins, the expression of SEMA3B and SEMA3F was examined in CM and cellular extracts of a panel of normal and malignant endometrial cell lines. Analysis of endogenous SEMA3B and SEMA3F protein expression in 1 normal endometrial (EM-E6/E7/TERT) and 3 endometrial cancer cell lines (HEC-1B, Ishikawa, and RL-95) showed decreased SEMA3B (active form 83-kDa and 50-kDa cleaved form) and SEMA3F (90 kDa) expression in CM and extract of cancer cells compared with the normal cell line (Fig. 2A). SEMA3 family members act through a membrane-associated receptor complex composed of plexin A1 or plexin A3, in addition to NP1 and NP2 receptors. Therefore, we also analyzed the expression of these receptors in normal and malignant endometrial cells. Normal endometrial epithelial cells (EM-E6/E7/TERT) showed higher expression of plexin A3 (220 kDa) compared with Ishikawa, HEC-1B, and RL-95 cells, whereas the expression of plexin A1 (211 kDa) was not altered (Fig. 2A). No marked changes were observed in the expression of NP1 (130 kDa) and NP2 (116 kDa) in normal and malignant cells (Fig. 2A). Mouse E16 cerebellum and rat brain lysates were used as positive controls for SEMA3B and SEMA3F, respectively (Fig. 2B).
Upregulation of SEMA3B and SEMA3F in endometrial cancer cells by P4 and 1,25(OH)2D3. A, normal immortalized EM-E6/E7/TERT and endometrial cancer cells (Ishikawa, HEC-1B, and RL-95) were evaluated by Western blot for basal expression of SEMA3B, SEMA3F, plexin A1, plexin A3, NP1, and NP2. Cells and CM were harvested, and 20 μg of protein from the whole-cell extract or CM were loaded in each lane. The blot was probed with the indicated antibody. β-Actin was used as a loading control. B, mouse E16 cerebellum and rat brain lysates were used as positive controls for SEMA3B and SEMA3F, respectively. Lysates probed with only secondary antibody (rabbit IgG) were used as negative controls. C, effect of P4 and 1,25(OH)2D3 on endometrial cancer cell (Ishikawa, HEC-1B, and RL-95) growth and apoptosis. The cells were treated with P4 (12.5–200 μmol/L) or 1,25(OH)2D3 (50–400 nmol/L) for 72 hours, and the viability of cells was determined by MTT assay. Data shown are mean ± SD of 3 separate experiments in which each treatment was repeated in 8 wells. The cells were treated with vehicle only or specified concentrations of P4 and 1,25(OH)2D3 for 72 hours, harvested, and cell lysates were prepared for caspase-3 enzyme activity. The data are representative of 3 independent experiments with similar results. *, statistically significant changes in cell viability/apoptosis, compared with those seen in control cells (P < 0.05). D, the effect of P4 (25 μmol/L) and 1,25(OH)2D3 (100 nmol/L) on the expression of SEMA3B and SEMA3F was studied in endometrial cancer cell lines. Cells were cultured with P4 or 1,25(OH)2D3 in the presence or absence of antagonists (mifepristone for P4 and telmisartan for 1,25(OH)2D3) for 72 hours. Protein was extracted from cell cultures and loaded in each lane and blots were probed with SEMA3B and SEMA3F. β-Actin was used as a loading control.
P4 and 1,25(OH)2D3-induced growth inhibition of endometrial cancer cells is associated with enhanced expression of SEMA3B and SEMA3F
The effects of P4 and 1,25(OH)2D3 on cell proliferation in 2 endometrial cancer lines (HEC-1B and Ishikawa) were investigated over a wide concentration range. Cells were treated with either P4 (12.5–200 μmol/L) or 1,25(OH)2D3 (50–400 nmol/L) for 72 hours and cell proliferation was assessed by MTS assay. Although both P4 and 1,25(OH)2D3 showed dose-dependent decreases in cell viability, P4 illustrated a more pronounced effect on cell growth compared with 1,25(OH)2D3 (Fig. 2C). To assess whether P4 or 1,25(OH)2D3–induced growth inhibition is mediated through induction of apoptosis, we evaluated the activity of caspase-3 in cellular extracts. A concentration-dependent increase in caspase-3 activity was found in cells following P4 or 1,25(OH)2D3 treatments (Fig. 2C).
As all 3 endometrial cancer cell lines showed low expression of SEMA3B and SEMA3F compared with the normal cell line, we next sought to determine whether exposure of cells to endometrial cancer preventive agents P4 or 1,25(OH)2D3 could restore the expression of SEMA3B and SEMA3F in cells. Treatment with P4 (25 μmol/L) or 1,25(OH)2D3 (100 nmol/L) for 72 hours caused a marked increase in SEMA3B (active form 83 kDa and 50 kDa cleaved form) and SEMA3F expression in both normal and malignant cell lines (Fig. 2D). To ascertain whether the observed P4 or 1,25(OH)2D3-stimulated SEMA3B or SEMA3F expression was mediated through a receptor-mediated pathway, endometrial cancer cell lines were pretreated with P4 antagonist mifepristone (100 nmol/L) or the VDR antagonist telmisartan (10 μmol/L) 1 hour prior to addition of P4 or 1,25(OH)2D3. Addition of mifepristone or telmisartan blocked P4- and 1,25(OH)2D3-stimulated expression of SEMA3B and SEMA3F down to levels comparable to that in the controls in all the cell lines. However, in HEC-1B SEMA3F expression levels did not go down to control levels in the presence of antagonists (Fig. 2D). Mifepristone and telmisartan alone had no effect on SEMA3B and SEMA3F expression (not shown).
P4- and 1,25(OH)2D3-induced cell growth inhibition of endometrial cells is attenuated by knockdown of SEMA3B and SEMA3F
To show that P4 and 1,25(OH)2D3 inhibit endometrial cancer cell growth by upregulating SEMA3B and SEMA3F, we knocked down the expression of these genes separately or conjointly using their respective siRNAs in 2 endometrial cancer cell lines, then treated them with P4 and 1,25(OH)2D3 and analyzed the effect on cell growth. A significant loss of SEMA3B and SEMA3F protein expression was observed in cell cultures transfected with respective siRNAs (Fig. 3A). No change in SEMA3B and SEMA3F expression was observed in control cultures compared with scrambled siRNA controls (Fig. 3A). Mock-transfected cells treated with P4 and 1,25(OH)2D3 showed a marked increase in SEMA3B and SEMA3F expression (Fig. 3A). Treatment of cells with P4 and 1,25(OH)2D3 for 72 hours showed a significant decrease in cell viability in control cells compared with cells in which SEMA3B and SEMA3F were separately or conjointly silenced by siRNA (Fig. 3B). After 72 hours of 1,25(OH)2D3 exposure, 55% to 58% of cells were viable in the control group. However, the number of viable cells increased significantly when SEMA3B (72%–80%), SEMA3F (70%–75%), or both SEMAs (85%–88%) were silenced (Fig. 3B), indicating that SEMAs mediate the growth inhibitory effects of 1,25(OH)2D3. Progesterone treatment of cells resulted in 35% to 40% viable cells in the control group. Knockdown of SEMA3B (55%–65%), SEMA3F (55%–60%), or both SEMAs (83%–87%) resulted in an increased number of viable cells and attenuation of P4 and1,25(OH)2D3-induced growth (Fig. 3B). SEMA3B and SEMA3F siRNAs have no effect on control cells (Fig. 3B). To investigate whether P4 and 1,25(OH)2D3 activate apoptosis, we measured the activation of caspase-3 in cells by a colorimetric substrate assay. Treatment of Ishikawa and HEC-1B cell cultures with 1,25(OH)2D3 and P4 induced significant decreases in cell growth and increases in caspase-3 activity (Fig. 3B), when compared with vehicle-treated cultures. The proapoptotic effects of 1,25(OH)2D3 and P4 were attenuated in cells transfected with siRNAs directed against SEMA3B and SEMA3F (Fig. 3B), suggesting that 1,25(OH)2D3 and P4 inhibit growth of endometrial cancer cells by upregulating SEMA3B and SEMA3F and inducing apoptosis.
Silencing of SEMA3B and SEMA3F attenuate progesterone and 1,25(OH)2D3-induced cell growth inhibition. The cell lines (HEC-1B and Ishikawa) were transfected with vector containing SEMA3B or SEMA3F alone, in combination or with the respective empty vectors. Transfected and mock-transfected cells were treated with P4 (25 mmol/L) or 1,25(OH)2D3 (100 nmol/L) for 72 hours. A, protein was extracted from cell cultures for analyses of SEMA3B and SEMA3F expression by Western blotting. B, cell viability was assessed by MTS assay and caspase-3 activity was determined by enzyme activity assay. Experiment was repeated 3 times. *, Statistically significant changes in cell viability/apoposis, compared with those seen in control cells (P < 0.05).
Ectopic expression of SEMA3B and SEMA3F inhibit in vitro tumorigenicity
Consistent with the data from the human endometrial cancer specimens, SEMA3B and SEMA3F expression was lost/reduced in all endometrial cancer cell lines tested, although these proteins were abundantly expressed in normal endometrial cells. To gain insight into the functional role of SEMA3B and SEMA3F downregulation in endometrial cancer cells, SEMA3B and SEMA3F expression was restored in the cancer cell lines (HEC-1B and Ishikawa) by transfection with the vectors containing SEMA3B and SEMA3F. An empty vector without the SEMA3 gene insert was used as a control (mock). Expression of SEMA3B and SEMA3F in the transfected cells was confirmed by Western blotting (Fig 4A and B). Because the acquisition of cell migration and colony-forming ability positively correlates with tumorigenicity, we sought to determine whether SEMA3B and SEMA3F restoration might alter these characteristics of endometrial cancer cells. We investigated the effects of SEMA3B and SEMA3F reexpression on cell proliferation (Fig. 4C) and migration (Fig. 4D) using cell viability and cell invasion assays. Seventy-two hours after transfection, cell proliferation and migration was assessed. As shown in Fig. 4C, a marked concentration-dependent decrease in cell proliferation was observed in SEMA3B- and SEMA3F-transfected cells compared with mock transfected cells. Of note, coexpression of SEMA3B and SEMA3F inhibited cell proliferation in a syngersitic manner (Fig. 4C). The number of cells migrating to the other side of the Matrigel was significantly reduced when SEMA3B and SEMA3F expression was restored in cancer cells (Fig. 4D). The effect of SEMA3B and SEMA3F restoration on the colony formation capacity of cancer cells was also analyzed (Fig. 4D). Upregulation of SEMA3B and SEMA3F in endometrial cancer cells showed a 30% to 40% and 60% to 70% respective decrease in colony-forming ability on culture plates as compared with mock-transfected cells. Taken together, these results show that SEMA3B and SEMA3F restoration inhibit cell proliferation, migration, and colony-forming ability, indicating a reduction of in vitro tumorigenicity. Similar results were obtained when SEMA3B and SEMA3F in the cells were upregulated by vitamin D or progesterone treatment (results not shown).
The effect of SEMA3B and SEMA3F overexpression on endometrial cancer cell malignant phenotypes. The effect of SEMA3B and SEMA3F overexpression on the viability, colony formation, and migration was investigated in 2 endometrial cancer cell lines. The cell lines (HEC-1B and Ishikawa) were transfected with a vector containing SEMA3B or SEMA3F. Control cells were transfected with the respective empty vectors. The second control was cells without transfection. Protein from control and transfected HEC-1B (A) and Ishikawa (B) was extracted and analyzed for the expression of SEMA3B and SEMA3F by Western blot. A significant difference between the protein levels in transfected cell lines and those observed in control cell cultures is shown in the figure. C, after 3 days of transfection, cell viability was measured by MTS assay, and the viability of transfected cells was expressed as percentage growth compared with that of control cells (100%). Columns, mean of 3 independent experiments, bars, ± SD. Statistically significant decreases in cell growth compared with those seen in control cells are indicated by *, P < 0.05. D, SEMA3B and SEMA3F-transfected and control cells were cultured on soft agar, and 4 weeks later, colonies were counted. The value shown is the percentage compared with that of empty vector–transfected control cells (100%). Columns, mean of 3 independent experiments; bars, ± SD. *, Statistically significant changes in colony formation, compared with those seen in control cells (P < 0.05). Endometrial cancer cells transfected with SEMA3B and SEMA3F were plated on Matrigel to assess cell invasion. After 22 hours, cells that migrated through the Matrigel were counted. Columns, mean of 3 independent experiments; bars, ± SD. *, Statistically significant changes in cell invasion, compared with those seen in control cells (P < 0.05).
Silencing of neuropilin-2 in endometrial cells results in loss of responsiveness to SEMA3F but not to SEMA3B
SEMA3s act in a paracrine fashion by binding to either to NP1 or NP2, or both. NPs complex with the plexins to mediate downstream signaling. To better understand the requirement of NP1 and NP2 to mediate the effects of SEMA3B and SEMA3F, respectively, Ishikawa and HEC-1B cells were transfected with either NP1- or NP2–specific siRNA or scrambled siRNA as a control. As shown in Fig. 5A, NP1 and NP2 proteins were markedly reduced in both endometrial cancer cell lines with respective siRNA. Downregulation of NP2 significantly reduced the ability of cells to respond to SEMA3F-CM and attenuated inhibition of tumor cell proliferation, whereas cells transfected with NP1 or control siRNA (Fig. 5B) retained the responsiveness to SEMA3F and inhibited cell proliferation (Fig. 5B). Interestingly, silencing NP1 or NP2 retained the responsiveness of cells to SEMA3B-CM, and a marked decrease in cell proliferation was seen compared with cells exposed to control-CM. However, when both NP1 and NP2 were silenced, cells were not responsive to either SEMA3F-CM or to SEMA3B-CM (Fig. 5B). Together, these studies indicate that treatment with soluble SEMA3F inhibits endometrial cell growth in a NP2–dependent manner, whereas soluble SEMA3B mediates it effect by employing both NP1 and NP2.
Expression of NP2 is required for SEMA3F and SEMA3B inhibition of MMPs. Endometrial cancer cells (HEC-1B and Ishikawa) were transfected with a scrambled siRNA, siRNA for NP1, siRNA for NP2 or both. Transfected cells were exposed to SEMA3F-CM, or SEMA3B-CM or control-CM for 72 hours. A, cells extracts were analyzed by Western blotting for the expression of MMP-9 and MMP-2. β-Actin was used as a loading control. B, cell viability was assessed by MTS assay. Columns, mean; bars, SD. *, P < 0.05, significant comparisons between the different treatments shown. C, the gelatinolytic activity of MMP-2 and MMP-9 in CM of endometrial cancer cells. Endometrial cancer cells overexpressing SEMA3B or SEMA3F or mock empty vector–transfected cells were serum starved for 12 hours and then incubated in the serum-free culture medium for 24 hours. CM were collected and then subjected to gelatin zymography. Representative picture from 3 independent experiments is shown. D, Western blot for MMP-9 and MMP-2 in HEC-1B and Ishikawa cells after NP1 siRNA, NP2 siRNA, both NP1 and NP2 siRNAs, or control siRNA knockdown followed by treatment with SEMA3F-CM or SEMA3B-CM for 72 hours. E, total cellular protein extracts were analyzed for αVβ3 integrin using specific antibodies. β-Actin was used as a loading control.
Upregulation of SEMA3B and SEMA3F inhibit matrix metalloproteinases, integrins, and alter pro- and antiangiogenic factors in endometrial cancer cells
Increased expression of MMPs is associated with cancer cell invasiveness and metastatic potential (39, 40). To explore the mechanism by which SEMA3 inhibits tumorigenesis, we evaluated the expression levels of MMPs in cells overexpressing SEMA3B and SEMA3F. Zymography done with CM from endometrial cancer HEC-1B and Ishikawa cells overexpressing SEMA3B or SEMA3F indicated reduced MMPs activity in endometrial cancer cell lines (Fig. 5C). Because both SEMA3B and SEMA3F inhibited MMPs, we explored the relationship between SEMAs and the specific receptors that transduce their signals. Expression of MMP-9 was significantly lower in NP1–downregulated endometrial cancer cells exposed to SEMA3F-CM or SEMA3B-CM compared with cells exposed to control-CM. No significant changes in the expression of MMP-9 and MMP-2 were detected in NP2–silenced HEC-1B and Ishikawa cells treated with SEMA3F-CM and SEMA3B-CM compared with control cells (Fig. 5D).
We then determined whether the expression of integrin αvβ3, which has been shown as a key mediator of cell–matrix attachment and motility (12–14) was regulated in the cancer cells by SEMA3 alteration. We found that SEMA3B and SEMA3F restoration by ectopic expression resulted in the reduction of integrin αvβ3 (Fig. 5E). Next, we sought to evaluate the effect of SEMA3B and SEMA3F upregulation on the expression of angiogenic factors in vitro. Tumor-associated angiogenesis is a complex process, involving many proangiogenic and antiangiogenic factors. RNAs extracted from control, SEMA3B- and SEMA3F-overexpressing cells were subjected to an angiogenesis RT2 Profiler RT-PCR array. The changes in mRNA levels of angiogenesis-related genes in SEMA3B- and SEMA3F-upregulated endometrial cancer cells are shown in Table 2. A 2-fold change in mRNA expression was considered as significant when compared with the mock-transfected control. It is evident from the results that SEMA3 overexpression led to decreased expression of proangiogenic factors (e.g., IL-8, VEGF, FGFR, ECGF, and MMP-9; Table 2), and increased expression of antiangiogenic factors (e.g., TIMP-3 and TGF-β, CXCL10; Table 2).
Fold change in angiogenesis-related genes in endometrial cancer cells overexpressing SEMA3B and SEMA3F
Endometrial cancer is a common and invasive gynecologic cancer in women, and the incidence and mortality is moderately increasing in the United States, whereas rapidly increasing in Asia. It would therefore be beneficial to develop molecular biomarkers, which could be used to detect endometrial cancer, predict disease progression, monitor therapeutic response, and, perhaps, be used as therapeutic targets.
Recently, the role of SEMA3s in the pathogenesis of multiple types of cancer has been investigated in preclinical studies, and SEMA3B and SEMA3F have been implicated in the inhibition of tumor cell growth, chemotaxis, and angiogenesis in breast, ovarian, and lung cancer cells (20–22, 41). In this study, we investigated the status, functional role, and regulation of SEMA3B and SEMA3F in human endometrial carcinogenesis either by ectopic expression or following exposure of cells to P4 and 1,25(OH)2D3.
Our study showed a significant reduction in the expression of both SEMAs and their receptor plexin A3 in grade III endometrial cancer compared with grade I or normal endometrial tissues. To our knowledge, this is the first article revealing the inverse correlation of SEMAs and plexin A3 levels with tumor aggressiveness. Our results are consistent with the pattern previously shown of loss of SEMA3B and SEMA3F expression in ovarian carcinomas compared with benign and borderline tumors (20, 42). We also investigated the expression of both SEMAs and their receptors in human endometrial cancer cell lines and found higher levels of SEMA3B and SEMA3F in the cell lysates and CM of normal cells than in cancer cells, indicating a potential role for class 3 SEMAs in endometrial cancer. Coexpression of SEMAs and their receptors by tumor cells suggests that SEMAs may act through an autocrine or paracrine mechanism in these cells. SEMAs secreted by endometrial tumor cells may, therefore, have a direct effect on tumor growth, as seen in breast cancer cells (43).
We are the first to report that SEMA3B and SEMA3F are strongly induced by P4 and 1,25(OH)2D3 in endometrial cancer cells. Although the precise regulatory mechanisms remain unclear, upregulation of SEMAs was blocked by the antagonists mifepristone or telmisartan, providing evidence for the specificity of P4 and 1,25(OH)2D3 action in cancer cells. Telmisartan was shown to strongly antagonize VDR (44). Our findings are consistent with the previous study revealing strong induction of SEMA3B mRNA by 1,25(OH)2D3 in multiple osteoblastic cell lines (MG-63, ST-2, MC3T3) and primary osteoblastic cells, suggesting a potential role for SEMA3B in modulating osteoclastogenesis and bone resorption (45).
Our results show that siRNA-mediated downregulation of SEMA3B and SEMA3F expression attenuated progesterone and 1,25(OH)2D3-induced cell growth, strongly suggesting that SEMA3B and SEMA3F expression is required to mediate growth inhibitory function induced by progesterone and 1,25(OH)2D3 in endometrial cancer cells. Interestingly, the combined suppression of SEMA3B and SEMA3F expression in cancer cells resulted in a greater attenuation of progesterone and 1,25(OH)2D3-induced cell growth inhibition compared with SEMA3B and SEMA3F alone, implicating that combined progesterone and 1,25(OH)2D3 treatment of cells may have a cumulative growth inhibitory effect on cancer.
Studies reported herein highlight the roles of SEMA3B and SEMA3F as negative regulators of tumor growth in endometrial cancer. SEMA3F binds to NP2 and SEMA3B binds to both NP1 and NP2, leading to growth suppression of tumor cells in vitro. We found that SEMA3B and SEMA3F expression was low in endometrial cancer cell lines. Constitutive activation of SEMA3B and SEMA3F expression in endometrial cancer cell lines, HEC-1B and Ishikawa, changes their in vitro attributes of malignancy. Ectopic expression of SEMA3B and SEMA3F reduced cell proliferation in a synergistic manner and attenuated invasion and anchorage-independent growth. These observations strongly show the negative influences of SEMA3B and SEMA3F on endometrial tumor progression. Our results are in agreement with earlier studies proposing that SEMA3B and SEMA3F act as tumor suppressors (20–22). Downregulation of SEMA expression in human tumors is attributed to LOH and promoter hypermethylation (46). Consistently, SEMA3B and SEMA3F overexpression in tumor cell lines induced apoptosis, inhibited cell proliferation, and colony formation in soft agar (15, 20, 22). In general, the tumor suppressing role of SEMA3B and SEMA3F is supported by in vitro and in vivo data; however, Rolny and colleagues (47) showed that SEMA3B-induced IL-8 secretion from tumor cells promotes cancer progression and metastasis.
To search for the underlying mechanisms by which SEMAs upregulation enact their antitumor effects, we used Human Angiogenesis RT2 Profiler PCR Array and found a significant downregulation of many pivotal angiogenic genes in SEMA3B and SEMA3F-overexpressing Ishikawa and HEC-1B cells compared with their receptive control cells. It is of interest to note that the 2 different cell cultures responded differently in terms of the regulation of angiogenesis-related molecules, probably because of the differences in their genetic profiles. In many types of cancers, several genes such as LAMA-5, ID, NOTCH4, and MMP-9 have been observed to be highly expressed and are shown to play a key role in promoting angiogenesis (48, 49). Our results showed downregulation of several proangiogenic genes (FGF2, FGFR3, MDK, MMP-2, and MMP-9) and upregulation of antiangiogenesis genes (TIMP1, TIMP2, and TIMP3) in cancer cells ectopically expressing SEMA3B and SEMA3F. These results suggest that the reexpression of SEMA3B and SEMA3F affect angiogenesis. In our previous study (20), we have shown that the reexpression of SEMA3F, but not SEMA3B, in ovarian cancer cells inhibited tube formation of endothelial cells, implicating that SEMA3F plays a role in tumor angiogenesis.
The mechanism of tumor suppression by SEMAs is not fully understood. The data presented here shows that restoration of SEMA3B and SEMA3F in endometrial cancer cells reduces their invasive properties by inhibiting MMP-2, MMP-9, αvβ3, and proangiogenic genes and by upregulating antiangiogenic genes. The results of the studies reported here showed that exogenously added SEMA3F and SEMA3B inhibit MMPs by acting through NP2 receptors in cancer cells, leading to decreased cell survival. MMPs are key players in the degradation of ECM and basement membranes and are thus important in tumor invasion. Gelatinases, in particular, are prognostic factors in many solid tumors (50). Previous reports showed that MMP-2 and MMP-9 were expressed in a high percentage of primary endometrial carcinomas and that their expression was associated closely with tumor aggressiveness (50, 51). In our study, the downregulation of MMP-2 and MMP-9 may contribute to reduced invasiveness in SEMA3-restored cells. Interestingly, inhibitor of MMP, that is, TIMP3, which is also a key player in tumor cell invasion, angiogenesis, and cell growth processes (51), has been found upregulated upon SEMA restoration. This data supports our observation that SEMA can regulate both active gelatinases and their inhibitor in the cellular invasion pathway.
In conclusion, this is the first article to show that SEMA3B and SEMA3F expression is downregulated in endometrial cancer tissues and that SEMA upregulation inhibited cell growth and invasion in endometrial cancer cells through downregulation of MMP-9, MMP-2, αvβ3, and proangiogenic genes and by upregulation of antiangiogenic genes. These findings also suggest that the differential expression levels of SEMAs in various stages of endometrial cancer may serve as endometrial tumor markers and that the restoration of SEMAs may be beneficial as a therapeutic tool for endometrial cancer.
This study was supported, in part, by NIH grant CA123558 (to V. Syed) and startup funds from the Uniformed Services University of the Health Science, Bethesda, Maryland.
The authors thank Dr. Satoru Kyo, Kanazawa University, School of Medicine, Kanazawa, Japan, for providing EM-E6/E7/TERT cell line and also Dr. John D. Minna, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, and Dr. Gera Neufeld, Cancer Research and Vascular Biology Center, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel, for providing SEMA3B and SEMA3F expression plasmids, respectively.
Note: The opinions and assertions expressed herein are those of the authors and should not be construed as reflecting those of the Uniformed Services University of the Health Sciences or the U.S. Department of Defense.
Received May 9, 2011.
Revision received July 15, 2011.
Accepted September 13, 2011.
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You are going to email the following Progesterone and 1,25-Dihydroxyvitamin D3 Inhibit Endometrial Cancer Cell Growth by Upregulating Semaphorin 3B and Semaphorin 3F
Mol Cancer Res November 1 2011 (9) (11) 1479-1492; DOI: 10.1158/1541-7786.MCR-11-0213
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text:
Cops Investigating Break-In Attacked by Fleas
SOUTH BEND, Ind. – Four officers investigating a burglary were attacked, not by a fleeing burglar, but a swarm of fleas in a filth-ridden vacant house.
The tiny, biting attackers were so overwhelming that the South Bend patrolmen had to be decontaminated and ended up being sent home early from their shifts.
"They were all over the place—in our socks and even in our shorts. It was disgusting," said Cpl. Ken Stuart.
To avoid infesting their squad cars, the police station or relatives, Stuart, Cpl. Chris Slager and Patrolman Paul Strabavy endured a lengthy flea decontamination process.
A van took them back to the station, where the men showered with flea/lice shampoo and soap. A wife of one of the officers brought them spare clothes.
As many as seven officers helped with the decontamination on Sunday.
"The guys were very angry. The last thing they wanted to deal with was fleas," said Sgt. Chuck Stokes. "That killed the whole shift."
Stokes said the house's tenants had recently been evicted, but returned periodically to feed a dog tied up in the backyard and allowed it to run around inside the garbage-filled house.

redpajama_set_name: RedPajamaCommonCrawl


The Miami Heat brought Miami Vice colors to their jerseys and inspired us to do the same for our lacrosse shorts but with lightning bolts. These shorts are super vibrant and will stand out pretty much anywhere.
Sweat wicking, stay dry and cool while playing or relaxing. Dry off naturally within 5 minutes.
Knee length lacrosse shorts. Look sweet on and off the field. Lacrosse doesn't end on the field, take your style with you everywhere.
Miami Vice colors totally increase shot accuracy by at least 100%, the hundreds of lightning bolts combine their shot power to aid you.


Ascenda BioSciences is committed to providing quality laboratory services. We take our quality very seriously because we know that the information we provide affects patient treatment and overall outcomes!
When she came to NC for Christmas vacation I tested her with a GI plus kit that showed Giardia. She was treated with Flaggy for two weeks and all her symptoms subsided.
We get fast results with actionable plans for our patients. ….and not to mention the clinical support and explanation of tests is phenomenal.
The Ascenda BioSciences test gives the best of both worlds. It offers an expanded stool panel and is also covered by most insurance companies. I am thankful to have an affordable option to offer my complex patients that provides actionable results.


The best way to confirm an infestation is to find a live louse. Use a magnifying glass and a fine-toothed comb to search. Nits more than a quarter-inch from the scalp are almost always dead, and usually the infestation no longer needs treatment. If you're unsure, ask a doctor or health department official.
Head lice have claws specially designed to cling to human hair. To survive, they huddle close to your scalp, eating blood several times a day. A single bug, called a louse, can live up to 30 days on your head but dies within a day or two if it falls off.
Head lice infest younger children in child care or school first because they play closely together. An estimated 6 million to 12 million cases occur each year among kids ages 3 to 11. But adults can get head lice, too. If one child in your house is infested, it's best to check everyone.
Nits are head lice eggs, and they're small, oval-shaped, and tough to spot. People who detect them often think they're dandruff, scabs, or droplets of hairspray. If they're not treated, they'll hatch into nymphs, or young lice, within eight to nine days. Within about another seven days, they'll become adult lice.
There's no proof mayonnaise, olive oil, margarine, or butter kills lice. However, nondrug methods can supplement pesticide treatment. Wash clothing, linens, combs, and brushes with hot water. Vacuum floors and furniture. But don't spend too much time or money; lice can't live long if they're not on a human host.
Some medicated shampoos and creams are available without a prescription. Called pediculicides, many contain the ingredients pyrethrin (such as the brand Rid) or permethrin (Nix). Read the instructions carefully. Ask your doctor before using them if you're pregnant or if you or your child has allergies or asthma.
Prescription-strength creams, lotions, and shampoos kill lice when milder products can't. Some may be safer for pregnant or breast-feeding women. These treatments may contain the ingredients ivermectin (Sklice) or benzyl alcohol (Ulesfia). Some may require the use of a fine-toothed nit comb after each application to remove remaining nits.
Head lice spread through direct head-to-head contact. For kids, this often occurs at school, sports activities, camp, or slumber parties. Less commonly, lice travel on hats, scarves, coats, brushes, or combs. The cleanliness of your hair, home, or school has nothing to do with the risk of getting them.
Most elementary and grade schools have rules regarding head lice infestations. For instance, some don't allow infected children at school until they're free of nits. To prevent the spread of lice, tell children to avoid contact with the hair of infected friends or classmates.


Dingsplatz! What's that? It's something new from Zentangle headquarters. Maria created a video about the technique and shared it with Certified Zentangle Teachers first (one of the perks!). If you've heard about Dingbatz, it's got certain similarities but in a circular fashion. If you want more info, find a local CZT or wait until the video is eventually released to everyone.
My Dingsplatz was done on one of my handcolored tiles. I used a blue Sakura Micron Pigma pen. The shading was done with colored pencils and highlights were added with a white chalk pencil.
Dingsplatz are fun! I can see doing lots more in my future!


We are extremely pleased and proud!
8 years ago we started with the development of 1 PRIMARY WOUND DRESSING® and just now, the Federal Office of Public Health has decided to create a new category on the "Mittel- und Gegenständeliste" (MiGeL), thereby enabling full reimbursement of 1 PRIMARY WOUND DRESSING® by the compulsory basic insurance.
As of January 1, 2017, reimbursement for 1 PRIMARY WOUND DRESSING® is granted (MiGeL number 34.70.01.00.1) with a reimbursement amount of CHF 25.00 for 10ml.
The path to full reimbursement was a long, stony way. Not least due to the support from many wound experts the authorities have been convinced that 1 PRIMARY WOUND DRESSING® is a wound care product, which enables a clinically effective, useful and cost effective treatment of acute and chronic wounds. We are very proud to have been able to demonstrate that even in today's environment, which is dominated by cost savings and increasing regulatory requirements, it is possible to find a path to market and full reimbursement for an innovative product if a great team is engaged with a lot of passion supported by doctors and wound experts.


Tucked away on a hillside in Patrick County, VA sits a little cabin with a beautiful view of the mountains in the far off distance. Inside, it's chilly. Outside, it's freezing. But inside, something magical was happening… little Ethan was digging into his first birthday cake. It's been a year since I first met Ethan as a precious newborn and have loved watching him grow this past year. Every three months, he seemed to change so much! And now, here I am shooting his 1 year session. Since Ethan is a winter baby, we thought that the "Winter ONEderland" theme would be perfect! His mom and I bounced ideas back and forth and decided that "snow" would be too cute…so this is what happened!
« Anything less than the best is a felony….


text:
Q: SparkThriftServer crashed after 11 days Using Spark 2.3.0 (running on ec2 in standalone mode) with Hadoop 2.7.6 and aws sdk 1.7.4, sparksql queries parquet files living on S3. Queries have been running fine over the last week but all of a sudden STS crashed while small query was running, same small query on same data that has been running  before.
Does anyone know the cause of this error?
18/10/11 04:37:04 ERROR Utils: uncaught error in thread spark-listener-group-eventLog, stopping SparkContext
java.lang.OutOfMemoryError
            at java.io.ByteArrayOutputStream.hugeCapacity(ByteArrayOutputStream.java:123)
            at java.io.ByteArrayOutputStream.grow(ByteArrayOutputStream.java:117)
            at java.io.ByteArrayOutputStream.ensureCapacity(ByteArrayOutputStream.java:93)
            at java.io.ByteArrayOutputStream.write(ByteArrayOutputStream.java:153)
            at org.apache.hadoop.fs.s3a.S3AFastOutputStream.write(S3AFastOutputStream.java:194)
            at org.apache.hadoop.fs.FSDataOutputStream$PositionCache.write(FSDataOutputStream.java:58)
            at java.io.DataOutputStream.write(DataOutputStream.java:107)
            at java.io.BufferedOutputStream.write(BufferedOutputStream.java:122)
            at sun.nio.cs.StreamEncoder.writeBytes(StreamEncoder.java:221)
            at sun.nio.cs.StreamEncoder.implWrite(StreamEncoder.java:282)
            at sun.nio.cs.StreamEncoder.write(StreamEncoder.java:125)
            at java.io.OutputStreamWriter.write(OutputStreamWriter.java:207)
            at java.io.BufferedWriter.flushBuffer(BufferedWriter.java:129)
            at java.io.BufferedWriter.write(BufferedWriter.java:230)
            at java.io.PrintWriter.write(PrintWriter.java:456)
            at java.io.PrintWriter.write(PrintWriter.java:473)
            at java.io.PrintWriter.print(PrintWriter.java:603)
            at java.io.PrintWriter.println(PrintWriter.java:739)
            at org.apache.spark.scheduler.EventLoggingListener$$anonfun$logEvent$1.apply(EventLoggingListener.scala:143)
            at org.apache.spark.scheduler.EventLoggingListener$$anonfun$logEvent$1.apply(EventLoggingListener.scala:143)
            at scala.Option.foreach(Option.scala:257)
            at org.apache.spark.scheduler.EventLoggingListener.logEvent(EventLoggingListener.scala:143)
            at org.apache.spark.scheduler.EventLoggingListener.onTaskEnd(EventLoggingListener.scala:164)
            at org.apache.spark.scheduler.SparkListenerBus$class.doPostEvent(SparkListenerBus.scala:45)
            at org.apache.spark.scheduler.AsyncEventQueue.doPostEvent(AsyncEventQueue.scala:37)
            at org.apache.spark.scheduler.AsyncEventQueue.doPostEvent(AsyncEventQueue.scala:37)
            at org.apache.spark.util.ListenerBus$class.postToAll(ListenerBus.scala:82)
            at org.apache.spark.scheduler.AsyncEventQueue.org$apache$spark$scheduler$AsyncEventQueue$$super$postToAll(AsyncEventQueue.scala:89)
            at org.apache.spark.scheduler.AsyncEventQueue$$anonfun$org$apache$spark$scheduler$AsyncEventQueue$$dispatch$1.apply(AsyncEventQueue.scala:89)
            at scala.util.DynamicVariable.withValue(DynamicVariable.scala:58)
            at org.apache.spark.scheduler.AsyncEventQueue.org$apache$spark$scheduler$AsyncEventQueue$$dispatch(AsyncEventQueue.scala:83)
            at org.apache.spark.scheduler.AsyncEventQueue$$anon$1$$anonfun$run$1.apply$mcV$sp(AsyncEventQueue.scala:79)
            at org.apache.spark.util.Utils$.tryOrStopSparkContext(Utils.scala:1265)
            at org.apache.spark.scheduler.AsyncEventQueue$$anon$1.run(AsyncEventQueue.scala:78)
18/10/11 04:37:04 INFO TaskSetManager: Finished task 28.0 in stage 13058.0 (TID 553499) in 252 ms on 10.13.5.60 (executor 0) (16/63)
18/10/11 04:37:04 INFO TaskSetManager: Finished task 23.0 in stage 13058.0 (TID 553494) in 253 ms on 10.13.5.60 (executor 0) (17/63)
18/10/11 04:37:04 ERROR Utils: throw uncaught fatal error in thread spark-listener-group-eventLog
java.lang.OutOfMemoryError
18/10/11 04:37:04 INFO HiveServer2: Shutting down HiveServer2
18/10/11 04:37:04 INFO ThriftCLIService: Thrift server has stopped
18/10/11 04:37:04 INFO AbstractService: Service:ThriftBinaryCLIService is stopped.
18/10/11 04:37:04 INFO AbstractService: Service:OperationManager is stopped.
18/10/11 04:37:04 INFO AbstractService: Service:SessionManager is stopped.

redpajama_set_name: RedPajamaStackExchange


St Matthew's is a church for the community.
It does not have access to funds from benefactors or other church bodies whether nationally or at local or diocesan level.
With the exception of any grants we may be able to obtain, we rely on the generosity of individuals to fund everything we do, so we need your help to achieve this vision.
You can make a donation – either on a regular basis, or as a one-off gift. Even a modest monthly sum over several years makes quite a difference. A tax payer can effectively add 25% to their gift through Gift Aid. (Higher rate taxpayers themselves receive a further tax rebate!).
You can leave a legacy to St. Matthew's in your will. Deciding to leave a legacy doesn't require the drafting of a new will if an individual already has a will – it can be amended with a simple codicil. Our Treasurer can send you a brochure giving further information.


text:
Amy Lashley 'Travels Of A Homebody' Wanamaker
May 2, 2011 LonesomeHighway.com
These songs are delivered with a quiet confidence and highlight Amy Lashley's observations and crystalline voice. Recorded with a simple quartet of players led by multi-instrumentalist Thomm Jutz and produced by singer/songwriter Otis Gibbs they give these songs all the space they need to tell their tales with a back-porch intimacy. As she says herself in Homebody Blues "all I want is simplicity". Her songs are observations of life and of events, as with Emmett Till which tells of the death, by lynching, of a 14 year old African American boy at the hands of white mob in 1955. An event that still resonates and also inspired a song on Emmylou Harris' new album. Amy Lashley apparently doesn't like to play live so we have this insight into her music instead and it serves her well. The songs are well written and tell their stories well from the aforementioned historical songs to the story of being from the wrong side of the street in Wrong Side Of Gallatin. There are elements of country blues and string bands in evidence here but it sounds very much from the here and now even of the constituent parts are timeless. Livin' On Beans On Cornbread has a kind of jauntiness and acceptance that makes it almost joyful. The album closes with Older Brother that again has the bedrock of Pat McInerney's unobtrusive but essential drumming underpinning it. All this makes this the album one that deserves repeated playing as you get to know its intimacy and heart. It brings you inside the hearth and heart of a homebody. Which in this case is a pretty good place to be.
Tags Amy Lashley, Travels Of A Homebody
← The O's 'Between The Two' IdolStacy Grubb 'Hurricane' Self-Release →


I slept through my alarm. A lot.
Lately, people keep talking about illegal deforestation of the Amazon. Sadly, the first thing I think of is whether or not that'll effect my Prime membership.
Yesterday was a day of accomplishing a whole lotta not much. I made big giant brush strokes with not a lot of paint on the brush, so to speak.
The Blacksmith and I worked on plans for the Forge, your one stop shop for all things nerd-gym related. Wanna actually be a warrior like Conan? Want to be a ranger like Legolas or Aragorn? Want to be a ninja assassin? We can help.
That's why I need a costume. I'm also working on a name for my character. I'm leaning toward "Longstride". That sounds kinda bitchin', right? What do you think? Ideas?
I also primed 8 more canvases. Granted they're smaller and will more than likely we attached together to make bigger paintings, but still. More paintings are in the works.
I also worked on getting my press up and running. It really does seem like every step I take forward, I find that I'm taking two steps back. In reality, I am just discovering the million little things that I've taken for granted that are needed to print. I am used to working in a functioning shop. A shop which mine is not. Yet. Baby steps.
And then there was work. More leading horses to water. And not a lot of drinking.
« A Vaguely Remembered Conversation.
Name suggestions – OddBeard (too piratey?), FellBow the Highstrung, Ol' Bentfinger, Ranger Danger, Lamestride the Elf Taster.


computer desk drawer computer desk with 2 drawers glass top white frame computer desk with lockable drawer. . . . . . . . . . . . . . .
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fallout 4 looks mirror for viewers the appearance of the human hunting dogs may feel shockingly 4 fallout 4 mirror reflection. . . . . . . . . . . . . . .


Do you often feel overwhelmed with sudden surge of emotions when in public or in other people�s company, which you cannot explain or reason out? Do you get deeply moved and influenced while seeing something horrible in real life or even in television? Do you always know what the people really mean? If the answers to these questions is a �Yes�, then you are someone who is called an �Empath�. An Empath is a psychic who almost feels and suffers other peoples� pains or emotions to an extent that he/she might even suffer from a stranger�s ailments without knowing the cause and reason for it. In the present world of human indifference and insensitivity, a natural Empath might feel like out of place. Being an Empath can be very exhaustive as we keep on absorbing and feeling others� emotions and energies, especially when we find our surroundings full of negativities.
But for a professional psychic, being an Empath is of great significance, as this innate psychic tool helps him to connect and relate to people, their genuine problems and pains. This helps the psychic to find out the accurate spiritual solution by doing the perfect psychic reading for the client or the help-seeker. Empathy is a virtue which is associated with humanity and higher subconscious of man. An Empath possesses the developed version of this principle which can be used for spreading compassion, love and understanding through psychic communication. It is also accompanied with the psychic power of healing. It is thus a blessing in disguise which if overlooked can bring disharmony within us, because there is always a spiritual reason behind possessing this gift. Also an Empath can be identified with his love, knack and natural inclination towards any form of art, like writing, painting, music etc. Having the power of emotional expression, an Empath has the power to move a mass with motivational speaking and preaching.
This is indeed the toughest of psychic potential to come to terms with. But still there are people who are in tune with this psychic ability through regular meditations and practicing inner calm and harmony. Learn to feel one with the universal emotions and that way you can also detach from them without much influence. Try to realize and analyze those emotions and feelings which are not being caused by you and learn to know their sources, accept and acknowledge them and then release the emotions.
Remember, if you are an Empath, do not feel burdened with the thought, rather feel empowered as you have been chosen to create positive universal vibrations by projecting and absorbing emotions that can bring people and our world together.
If you want to share your comment on this article, please visit Psychic forum.
Can I Be A Professional Psychic?


Our respite care services help caregivers who look after the elderly and people with disabilities or special needs.
Respite care helps ward off the burnout that can leave care providers stressed out, depressed, and exhausted. It does not necessarily mean resenting your care duties or wanting someone else to do your job. This simply gives you relief from the pressures and demands of caregiving when you are tending to a loved one who requires regular monitoring and frequent assistance. More often, a respite break is organized to offer the carer a holiday from their care-related duties. It comes in handy when friends or the patient's family members are the regular caregivers, allowing them to look after their health and well-being.
For more information about respite care or anything related to Home Care Services in Maryland and Virginia, please call us at 888-958-3524. Feel free to send us a message if you have concerns, queries, or suggestions.


Undoubtedly, Acropolis is the most impressive historical site of ancient Greece, and the main attraction of Acropolis is Parthenon. Though ancient Greece was but a conglomerate of independent state-cities, Parthenon was then the most important temple, both from architectural and religious points of view.
Parthenon was built by architects Kallikrates and Iktinos in 447-432 B.C., in the reign of Pericles, which was the greatest ruler (strategist) of Athens. Of course, the main temple of Athens was dedicated to god patroness of the city Athene-Pallades. Near Parthenon there was erected a huge sculpture of the goddess reaching the height of 7 meters, which was then called Athene Promachos (Athene the Warrior).
Parthenon commands Athens. The temple is surrounded by columns. According to rules of ancient Greek architecture, the long side of colonnade must have twice as many columns as the short side plus 1, that is 8 and 17.
The colonnade is crowned with the famous frieze of Parthenon. There were relief profiles of 192 warriors fallen in the Battle of Marathon with Persians. On the main eastern pediment there was depicted «Nativity of Athens». And on the western pediment there was depicted «Contest of Athens and Poseidon on the power over Attica». Inside the temple, where light reached only through door openings, there was set the sculpture of Athene Parthenos made by Phidias. Its decoration required more than one tonne of gold.
In 662 A.D., after decay of ancient civilization and strengthening of Christianity, Parthenon was turned into the Church of the Most Holy Mother of God, and the sculpture of Athene was taken to Constantinople where it was destroyed during the fire. When Turkey conquered Athens in 1460, Parthenon was converted into a mosque. During the war with Venice there was a powder store in the temple and it was hit by one of the shots. As a result of powerful explosion, all the central part of Parthenon was demolished. And after that the temple wasn't restored.
However, Parthenon and the entire Acropolis most suffered from English philarchaists. With the help of lord Elgin (English ambassador in Turkey) most part of the temple's sculptures was taken to the British Museum. Some sculptures were also taken to the Louvre and Copenhagen. Now the authorities of Greece demand their returning.


I'll never grow tired of watching sweet interactions between cats and dogs. You never know how things are going to end when you put a big dog and a kitten in the same room together, but it's clear that the pit bull in this video has nothing but love for her feline friend. I have a feeling that these two pets are going to share a beautiful friendship together.
Watch the video and share your reaction with us in the comments section below!
Looks Like A Normal Layer Cake, But Minutes Later? I'm SPEECHLESS!!


text:
Graham, Bruce | Article about Graham, Bruce by The Free Dictionary
https://encyclopedia2.thefreedictionary.com/Graham%2c+Bruce
Graham, Bruce
Designer of Chicago's John Hancock Center and Sears Tower (now Willis Tower), he led the Chicago office of Skidmore, Owings & Merrill from the 1960s through the 1980s with a staff that included Gordon Bunshaft, Walter Netsch, and structural engineer Faziur Khan. Graham is responsible for projects in London, Barcelona, Egypt, Korea, Guatemala, Houston, Los Angeles, Boston, Atlanta, Kansas City, Milwaukee, Wichita, Nashville, Tulsa, Madison, and many other locales. Their revolutionary design, engineered by Khan, placed the structural supporting elements on the outside, greatly reducing construction costs and providing vast open interiors. His 1957 Inland Steel building, Chicago, IL featured the use of structural columns on the building's perimeter and a separate adjoining tower for elevators and other services to open up the interiors. With William Hartmann, another partner at SOM, Graham developed the Chicago 21 Plan of 1973 for the Chicago Central Area Committee, which called for alterations and additions to Chicago's lakefront as the city entered the 21st century, including rebuilding the dilapidated Navy Pier into a tourist destination and the creation of the museum campus.
Graham, Bruce
Michigan Algorithm Decoder
He won the Premier League, two FA Cups, the League Cup and the Uefa Cup Winners' Cup at Highbury, under George Graham, Bruce Rioch and Arsene Wenger.
Ian Wright to hold Dubai Q&A
It is difficult to imagine a more stark contrast," said Dixon, who grew up as a Manchester City fan, but who found glory in his 14 years at Arsenal under George Graham, Bruce Rioch and then Wenger.
DIXON FEELING TIME IS RIGHT FOR GUNNERS
Body art figured in issues 6 and 8, with photographs, stills, and occasional typewritten descriptions of performances by Vito Acconci, Dan Graham, Bruce Nauman, Oppenheim, and the Viennese Actionists.
Continental Schrift: the story of Interfunktionen
Other guys like George Graham, Bruce Rioch and Mick McCarthy have done well at the club and I want to keep that going.
I JUST CAN'T BEAR TO SAY GOODBYE TO MY PLAYERS; Jimmy to give stars the slip
installations by Glen Seator, Paul McCarthy, Chris Burden, Diana Thater, Dan Graham, Bruce Nauman, and Jason Rhoades.
"1997 Biennial Exhibition:' Whitney Museum of American art
That rules out George Graham, Bruce Rioch and Kenny Dalglish and leaves just one viable choice - Bruce.
Bruce's my Maine man; YOUR SHOUT
Roy Hodgson, George Graham, Bruce Rioch and Ossie Ardiles are just a few of the names being bandied about as the replacement for the unfortunate Todd.
FOOTBALL: Crisis County reeling as fans lose patience; DERBY 1 IPSWICH 3: A TOWN CRIER FOR AILING RAMS
Graffman, Gary
graft copolymer
Grafting Machine
Graftio, Genrikh
Graftio, Genrikh Osipovich
Grafton Lakes State Park
Grafton Notch State Park
Grafton, Augustus Henry Fitzroy, 3d duke of
Grafton, Richard
graftonite
Graham Cave State Park
Graham Island
Graham Land
Graham of Claverhouse
Graham Sutherland
Graham, Bill
Graham, Ernest R.
Graham, George
Graham, James
Graham, John Jr.
Graham, Jorie
Graham, Katharine
Graham, Katharine Meyer
Graham, Otto
Graham, Otto Everett, Jr.
Graham, Patricia Albjerg
Graham, Philip Leslie
Graham, Shirley
Graham, Susan
Graham, Sylvester
Graham-Cole test
Grahame
Grahamella
grahamite
Graham's Island State Park
Graham's law of diffusion
Graham's pendulum
Graiae
Graham Sylvester
Graham System
Graham Taylor
Graham Taylor Photography Services
Graham Vivian Sutherland
Graham Waldon Management Consultants
Graham William Franklin
Graham's Dyke
Graham's law
Graham, Ben
Graham, Benjamin
Graham, Billy
Graham, Evarts Ambrose
Graham, Martha
Graham, Thomas
Graham-Harvey Measure 1
Graham-Kapowsin High School


If you daily drive your '79-'04 Mustang, chances are you've grown accustomed to the clunks and bumps coming from the rear over anything but smooth asphalt. The original design of the Fox chassis rear suspension is an engineering and financial compromise of grand proportions. You can almost imagine the Ford engineers arguing for a parallel-link four-bar rear to the bean counters, who in turn probably just kept shooting them down by spouting budget numbers and deadlines. Nevertheless, the non-parallel four-link suspension found its way under the new Fox chassis and we had to live with its short comings for 25 years—with only minor improvements/Band Aids—until the '05 Mustang debuted with its all-new parallel three-link design and fixed Panhard rod.
The design of the Fox four-link utilizes four control arms, two lower arms parallel to each other that locate the axle in the wheel housing and two upper control arms mounted at an angle to the lower arm that locate the axle laterally. These upper arms are the main issue with the Fox rear suspension, as they were designed to do two jobs: locate the axle AND prevent the axle from moving side to side. The control arms themselves are made from stamped/formed steel in a U-shaped configuration with standard rubber bushings at each end. The shape/design of the stock control arms, along with the softness of the OE rubber bushings, allows for a lot of play in the rear suspension as the bushings, as well as the control arms themselves, flex under load. Adding years of service and often well north of 100,000 miles means worn bushings and even fatigue cracking of the control arms is common.
While there are upgraded suspension offerings on the market that really improve the Mustang's four-link setup by enhancing stability, traction, and handling, many require chassis modifications, welding, and so on, not to mention their relatively steep price points.
If you're looking for a budget solution to help your Mustang's rear suspension improve on the OE design without needing anything more than basic hand tools to install, then you should consider upgrading to aftermarket tubular control arms with urethane bushings. The aftermarket arms offer increased strength and rigidity through their solid tube design that resists flexing. The urethane bushings included with these aftermarket arms also reduce flex via their higher durometer materials.
There are literally a dozen or more manufacturers of tubular control arms for the '79-'04 Mustang with as many options, including billet aluminum, powder-coated steel, fixed end bushings, adjustable spherical rod-ends, and much more. While all have their benefits and detractions, for the majority of our readers looking for a nice basic replacement set of control arms, the standard powdercoated control arm set from Mustangs Unlimited certainly fits the bill.
Our Mustang coupe has seen a lot of miles and hard driving, so it had the typical Mustang rear axle clunk over speed bumps, railroad crossings, and the like. A quick call to Mustangs Unlimited for their '79-'98 control arm kit (PN 215563R, $139.95) means that we'll have improved traction, better handling, and best of all no more clunk!
13. To finish the installation, simply return the coil springs to their home between the chassis and lower control arms, then raise the axle housing to relocate and secure the bottom mounting points of the shock absorbers. Although not required for urethane bushings, it's good practice to do final tightening of the suspension mounting bolts with the full weight of the vehicle on the suspension. Reinstall the antisway bar using the stock hardware. Lastly, to return the parking brake cable brackets to their rightful position, you'll have to bend the locating tabs as shown so they'll sit flush on the new lower control arms using the antisway bar attaching bolt. Alternatively, you can drill and tap a hole for the bracket, though many people simply leave them off.
Check out this custom 1980 Ford Fairmont Futura restomod, with a 5.0 liter high output Ford Mustang engine, from Muscle Mustangs & Fast Fords Magazine.


text:
Oriental Bank, otherwise known as Oriental Bank Plc. or Oriental Bank Cambodia, is a commercial bank in Cambodia. The bank was established in 2021 by founder, managing director and CEO — Datuk Phan Ying Tong and other partners. Oriental Bank received their approval in principal from the National Bank of Cambodia on the ninth of December 2020  The banking operation license was received from the National Bank of Cambodia in January of 2022, and were officially presented to them on the 28th of February 2022, by the Governor of the National Bank of Cambodia, H.E Dr. Chea Chanto.

See also
List of banks in Cambodia

References

External links

Banks of Cambodia
Banks established in 2021
Cambodian companies established in 2021

redpajama_set_name: RedPajamaWikipedia


text:
Q: Dynamic Menu Contribution on View ToolBar thru plugin.xml We are trying to fill the Toolbar of the View using MenuService as documented in this tutorial
http://appddeevvmeanderings.blogspot.in/2007/10/eclipse-defining-your-own-toolbar-menu.html
In the above tutorial, he creates a Toolbar in a View and reads the Commands from the plugin.xml
toolbar = new ToolBarManager(SWT.FLAT);
       getMenuService().populateContributionManager(toolbar, "toolbar:yourapp.toolbarX");
       toolbar.createControl(p);

In our code instead of creating a ToolBarManager we take the ToolBarManager of the View.
toolbar = getViewSite().getActionBars().getToolBarManager();
           getMenuService().populateContributionManager((ToolBarManager)toolbar, "toolbar:YourID?after=additions");
//         ((ToolBarManager)toolbar).createControl(parent);
           toolbar.update(true);
           getViewSite().getActionBars().updateActionBars();

This doesn't work. Any help would be highly appreciated. I would want to add this to the Toolbar of the View and not to my own custom ToolBar. It seems to be working in Eclipse 3.8 and not in Eclipse 4x onwards.


Thus far, one has established a basic model of society with which to identify failures in guaranteeing and maximising satisfaction and stability of society. Summarily, society, comprised of population, labour, capital and agreement, and its economy, the relations between them, pursues civilization as a means of guaranteed and optimal satisfaction, productivity and profit of the citizenry, thereby ensuring sustainability, referred to as civilization. Failures in achieving civilization vary but are considered fundamentally the fault of human faculty and its inabilities. Supplementary development of human faculty lacks adequate improvement to ensure civilization, and so one considers Transhumanism and the augmentation of human faculty to advance further. In this respect, one combines the organic with the synthetic beyond mere mixed labour and, in doing so, changes organic labour to synthetic, as previously stated. As such, citizens become synthetic labourers and, providing adequate increase to their economic utility, retain their solvency, thus, hypothetically, allowing for civilization.
Before applying Transhumanism as a solution to societal problems, one must detail the principles required in establishing it. To do so, one considers the central ideal of this philosophy and extends this to encompass the entirety of an economy, drawing principles from the philosophy's imposition onto a civil contract. At its most fundamental, Transhumanism aims to augment human faculties beyond what is humanly possible. Improving physical, mental and autonomous faculties in such a manner is nontrivial; physical and mental faculties are perhaps the easiest faculties one can imagine augmenting, with autonomy augmentations being more obscure, pertaining to such concepts as emotional maturity, self-awareness, sentience and self-actualization. Current technology lacks in improving these faculties to beyond human capability, however, the progression of research can be accelerated, particularly if given the additional motivation of social reform and civilization.
capital flow into research and development of augmentations. This cycle of improved efficacy drives increases to productivity and profit, which is then partly invested back into developing further augmentations; this succession of continuous improvements that is intended to continually increase productivity and wealth across society.
This is of course both a simplification and idealization, however to remain generalized, one does not attempt further specification. Nonetheless, even a speculative theory still requires care, given the nature of research. It may not always be possible to gauge the feasibility of an augmentation and its productivity, which can lead to overlong or expensive research and development, and potentially underperforming augmentations. Additionally, disruption to capital flow through lack of affordability, overpricing or other uncivil phenomena also poses risks to such a system. Ultimately, such a theory of value is only a suggestion, and it will be incumbent on a society's economy, leadership and social contract to provide terms that optimize this theory for maximal civilization. Any such arrangement attempting this kind of economy is thus referred to as the innovation theory of labour.
Furthermore, considerations must be made for agreement to augmentation, and its effect on population. It is assumed that agreement generally follows solvency, making augmentation agreeable; however human nature is compounded by identity, which, at time of writing, relies heavily on the human body. Augmentation, especially physical, is consequently a source of contention; citizens currently live their lives within the human body, founding their identity on it, with other citizens associating the body's appearance with that individual. There is no outright adjustment to policy that reconciles Transhumanism with this quality of humanity; the most agreeable solution to this are augmentations that improve on human nature entirely, and do not detract from one's experience, even in how those augmentations appear. Although generations that mature with technology are usually more accustomed to such phenomena, Transhumanist policy must initially accommodate this quirk of autonomy; such a civil contract must meet citizen demand for augmentations that are at least equivalent to a human body and guarantee solvency for citizens that decline augmentation. Otherwise, one risks disagreement, and subsequent uncivilization, threatening implementation of Transhumanist policy.
avoid unnecessary disagreement. Providing a majority of the citizenry opts for augmentation, society should see a substantial improvement to its economy, allowing for civilization. However, the terms of this civil contract will depend on the specifics of the society attempting it, as will how the citizenry and leadership reconcile these terms with regards to maintaining these fundamental principles.