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CtBP2 interacts with TGIF to promote the progression of esophageal squamous cell cancer through the Wnt/β‑catenin pathway
Qianqian Ju
Maorong Jiang
Wenxin Huang
Qingbo Yang
Zhenghong Luo
Hui Shi
Affiliations: School of Basic Medical Sciences, Nanjing Medical University, Nanjing, Jiangsu 211166, P.R. China, Key Laboratory for Neuroregeneration, Medical College of Nantong University, Nantong, Jiangsu 226001, P.R. China, Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, P.R. China, Department of Thoracic Surgery, Shanghai Tenth People's Hospital, Shanghai 200072, P.R. China
Published online on: December 8, 2021 https://doi.org/10.3892/or.2021.8240
Copyright: © Ju et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
C‑terminal‑binding protein 2 (CtBP2), a transcriptional co‑repressor, plays a main role in tumorigenesis and in the development of multiple tumors. Transforming growth interacting factor (TGIF) is involved in a number of cellular signal transduction pathways and is related to tumor occurrence and development. In the present study, the proteins interacting with CtBP2 were identified and the mechanisms underlying the biological activity of CtBP2 in esophageal squamous cell carcinoma (ESCC) were investigated. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to search for known proteins interacting with CtBP2, and co‑immunoprecipitation (Co‑IP) assay was performed to validate the interactions. Reverse transcription‑quantitative PCR (RT‑qPCR), immunohistochemistry (IHC) and western blot analysis were performed to examine the expression levels of CtBP2 and TGIF in ESCC. The correlation between CtBP2 and TGIF was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) by Pearson's correlation analysis, and the co‑localization of CtBP2 with TGIF in the ECA109 cells was identified using immunofluorescence staining. XAV939 treatment, CCK‑8, 5‑ethynyl‑2'‑deoxyuridine (EdU) staining, wound healing and Transwell assays were performed to investigate the signaling pathways involved in the biological activity of CtBP2 in ECA109 cells. According to the results obtained from STRING and Co‑IP analysis, an interaction between CtBP2 and TGIF was indicated, and these proteins were co‑localized in the nucleus. CtBP2 and TGIF mRNA and protein expression levels were robustly and simultaneously increased in both ESCC tissues and cell lines. There was a direct correlation between CtBP2 and TGIF expression levels in ESCC tissues, and both were significantly associated with metastasis and survival. The TGIF and CtBP2 expression levels were significantly increased or decreased simultaneously, in ECA109 cells transfected with LV‑CtBP2 or sh‑CtBP2, and vice versa. According to the results of CCK‑8 assay, EdU staining and Transwell assay, CtBP2 promoted the proliferation, migration and invasion of ECA109 cells through the Wnt/β‑catenin pathway. On the whole, the present study demonstrates that CtBP2 interacts with TGIF and promotes the malignant progression of ESCC through the Wnt/β‑catenin pathway.
A high incidence of esophageal cancer (EC) has been reported in China (1,2). Recent surveys have demonstrated that there are ~450,000 new cases of EC each year worldwide, with more than half of these cases originating from China, ranking sixth among the list malignant tumors in China (3,4). EC is an invasive from of cancer, with the two main pathological types being esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA) (5). ESCC has been reported as the main histological type of EC in China, accounting for >95% of reported cases (6,7). The 5-year survival rate of patients with EC is only ~10%, and the recurrence and mortality rates remain high (8). Radiotherapy, chemotherapy and targeted therapy have been developed in recent years for the treatment of EC. However, the clinical efficacy and prognosis of patients have not been satisfactory (9,10). This is due to the lack of an in-depth understanding of the pathogenesis of EC (11). Therefore, a more detailed elucidation of the pathogenesis of EC at the molecular level may lead to the discovery of an ideal molecular target or other effective therapeutic drugs for EC.
The transcriptional co-repressor C-terminal binding protein (CtBP) has been originally named thus, since it binds to the five amino acid domains (PLDLS) at the C-terminus of the adenovirus early region 1A (E1A) protein (12–14). The CtBP protein, including the CtBP1 and CtBP2 protein isoforms, is an evolutionarily conserved transcriptional repressor that has been reported to specifically bind to DNA or protein targets and function as a bridge molecule between DNA-binding proteins and transcriptional repressors, thereby inhibiting gene transcription (15). In addition, CtBP has been revealed to promote epithelial-mesenchymal transition (EMT) by inhibiting E-cadherin expression (14,16). Furthermore, it may function as a transcriptional co-repressor, negatively regulating certain tumor suppressors, thereby promoting the occurrence and development of tumors (17,18). Recent studies have revealed that the CtBP family plays a crucial role in the occurrence and development of breast, colon, ovarian and prostate cancer (19–21).
A previous study by the authors found that the expression of CtBP2 was significantly increased in ESCC tissues, and was positively associated with the tumor histological grade, and negatively associated with p16 tumor suppressor gene expression (22). In addition, CtBP2 has been demonstrated to promote ECA109 cell proliferation and migration, and reduce cell susceptibility to cisplatin (23). Moreover, cyclin H/cyclin-dependent kinase 7 (CCNH/CDK7) has been previously reported to competitively bind to homeodomain-interacting protein kinase 2 (HIPK2) and CtBP2, thereby inhibiting the phosphorylation and dimerization of CtBP2, ultimately regulating its stability in breast cancer cells (24). CtBP2 has been also revealed to promote the proliferation and migration, as well as inhibit the apoptosis of ESCC cells, through the regulation of its downstream target molecule, basic fibroblast growth factor (FGF2) (25). These results suggest that CtBP2 is involved in the occurrence and development of ESCC; however, the underlying mechanisms remain unknown. Therefore, it was hypothesized that CtBP2, as a transcriptional co-repressor, may also interact with other proteins and participate in the development of ESCC.
The transforming growth-interacting factor (TGIF) gene is located on chromosome 18p11 and encodes a nuclear protein composed of 272 amino acid residues with a molecular weight of ~30 kDa (26). It belongs to the family of three amino acid loop extensions (TALEs), which are expressed in various cells and tissues (27,28). TGIF has been reported to be involved in a number of cellular signal transduction pathways, particularly the TGF-β pathway (29). It has been demonstrated that TGIF can bind to Smad2 and Smad3, possibly changing chromatin structure from loose to dense through the recruitment of histone deacetylase (HDAC), thereby inhibiting the transcription of target genes mediated by TGF-β (29). TGIF can also inhibit the TGF-β signaling pathway through a Smad-independent mechanism, by recruiting HDAC (30). Previous research has confirmed that TGIF expression is increased in various tumors and may be related to the occurrence and development of tumors (26,31,32). In addition, it was revealed in a previously published study that high levels of TGIF were associated with high levels of Wnt signaling pathway components (Axin1, Axin2 and β-catenin) and a poor survival rate of patients with triple-negative breast cancer (33). Therefore, it was hypothesized that an in-depth study of the regulatory mechanisms between CtBP2 and TGIF may be of utmost significance, in order to clarify the roles of CtBP2 and TGIF in the occurrence and development of ESCC.
Patients and tissue samples
A total of 108 patients with ESCC were identified and enrolled from 2015 to 2019 at the Affiliated Hospital of Nantong University (Nantong, China). None of the patients had previously received radiotherapy, chemotherapy, or immunotherapy prior to surgery. All fresh tissues (ESCC tissues and matched adjacent tissues) were collected following surgical resection, were immediately washed with sterile phosphate-buffered saline (PBS) and immediately fixed in 4% paraformaldehyde (PFA) for 12 h before being embedded in paraffin or stored at −80°C. Patient written informed consent was obtained before the commencement of the study, according to the guidelines of the Ethics Committee of the Affiliated Hospital of Nantong University, and ethics approval was also provided from the respective ethics committee (2015 L132).
Cells and cell culture
The human ESCC cell lines, ECA109 (CC-Y1150), TE-1 (TCHu 89) and KYSE-150 (TCHu236), and the human normal esophageal epithelial cell line, HEEC (CL0420), were provided by the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and were cultured in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FCS (Shanghai Shuangru Biotechnology Co., Ltd.) and 1% penicillin/streptomycin antibiotic solution (Beijing Solarbio Science & Technology Co., Ltd.). All cell lines were cultured at 37°C with 5% CO2.
Lentiviral transduction
To knockdown or overexpress CtBP2 and TGIF, recombinant lentiviral vectors (sh-CtBP2, sh-TGIF, LV-CtBP2 and LV-TGIF, respectively) were constructed (Vigen Biotechnology) (23). The coding sequence of CtBP2 or TGIF was cloned as an overexpression vector, into a GV492 vector (Vigen Biotechnology). The shRNA of CtBP2 or TGIF, whose target sequence was 5′-GCGCCTTGGTCAGTAATAG-3′ or 5′-AGCTTCTAGTGGATGTTGC-3′, was cloned into a GV248 vector, respectively. The lentiviral particles were obtained from Vigen Biotechnology Co. Ltd. Briefly, 293T packaging cells (The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences) were co-transfected with shutter plasmids and packaging vectors using polyethyleneimine (PEI; Shanghai life ilab Biotechnology; http://life-ilab.com/) and incubated in 5% CO2, 37°C incubator for overnight. The following day, the 293T cells were supplemented with fresh medium. The supernatant was collected at 48 and 72 h and post-transfection filtered through 0.45-µm filter, and then concentrated with lentivirus concentration solution. Briefly, the supernatant from 293T cells co-transfected with pMD2G, psPAX2 and shutter plasmids was collected and centrifuged at 2,000 × g for 10 min and then filtered through 0.45-µm filters to remove cells and debris. In total, four volumes of clarified supernatant were mixed with one volume of concentration reagent. The mixture was incubated at 4°C overnight followed by centrifugation at 1,500 × g for 45 min at 4°C. Following centrifugation, the off-white pellet was re-suspended by PBS. The ECA109 cells were then transfected with recombinant lentiviral vectors and negative control viruses at a multiplicity of infection (MOI) of 5. The following formula was used to calculate to volume of virus to be added: Virus volume = MOI × cell number/virus titer. In addition, 1 µg/ml polybrene was added, to improve the transduction efficiency. The mixture was centrifuged at 800 × g for 50 min at 32°C. Following centrifugation, the cells were seeded into 6-well culture dish and incubated at 37°C overnight. Transfected ECA109 cells were selected using 2.5 µg/ml puromycin for 1 week. The efficiency of lentivirus-mediated knockdown or overexpression of CtBP2 or TGIF was verified using reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis.
Total RNA extraction and reverse transcription were performed as previously described (23). Total RNA from cells or tumor tissues was extracted using TRIzol® LS reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and was quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized by reverse transcription using the PrimeScript RT reagent kit (Takara Bio, Inc.). qPCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio Inc.) on a LightCycler 96 system (Roche Diagnostics). Primers were synthesized by Sangon Biotech Co., Ltd. The thermocycling conditions were as follows: Firstly 95°C for 10 min, followed by 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec, for 40 cycles. Data were analyzed using the 2−ΔΔCq method with GAPDH as the internal reference control (34). All results are expressed as the mean ± standard deviation of three independent experiments. The following primer sequences were used: GAPDH sense, 5′-GACCTGACCTGCCGTCTA-3′ and antisense, 5′-AGGAGTGGGTGTCGCTGT-3′; CtBP2 sense, 5′-CTGAGTTCCTGGCCTTTCTG-3′ and antisense, 5′-GACTTGATATCCGCGTCCTC-3′; TGIF sense, 5′-GGATGAGGACAGCATGGACA-3′ and antisense, 5′-AGGCATTGTAACGGTGCTCA-3′.
Protein extraction and western blot analysis
Cells or tissues were lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology), as previously described (23). The protein concentration of each sample was determined by BCA assay (Thermo Scientific, Inc.) according to the manufacturer's instructions. Proteins were separated via 10% SDS-PAGE (50 µg protein/lane), and then blotted onto PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked with Tris-buffered saline and 0.1% Tween-20 (TBST) supplemented with 5% non-fat milk for 2 h at room temperature, and the PVDF membranes were incubated with primary antibodies including, rabbit anti-TGIF (ab52955; 1:1,000; Abcam), mouse anti-CtBP2 (sc-17759; 1:200; Santa Cruz Biotechnology, Inc.) and rabbit anti-β-catenin (ab68183; 1:1,000; Abcam) overnight at 4°C. Rabbit anti-β-actin (ab8227; 1:1,000; Abcam) was used as an internal control. The membranes were then incubated with HRP-conjugated goat anti-rabbit IgG (A8919; 1:1,000) or rabbit anti-mouse IgG (A9044; 1:1000) (Sigma-Aldrich; Merck KGaA) secondary antibodies at room temperature for 2 h. Finally, western blot images were visualized by incubation with enhanced chemiluminescence detection reagent (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, 34577) (Thermo Fisher Scientific, Inc.) at room temperature for 5 min, and the bands were quantified using ImageJ (v1.48) software (National Institutes of Health).
Immunohistochemical staining was performed as previously described (22). Briefly, 4% PFA-fixed ESCC tissue or normal tissue sections were deparaffinized in xylene, rehydrated in a graded alcohol series, and finally washed three times with 0.1 M PBS. The slides were submerged in Tris-EDTA buffer (100°C, 20 min) for antigen retrieval and cooled naturally at room temperature. The IHC kit was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (cat. no. SP-9000). The sections were blocked with 10% goat serum (containing 0.1% triton-100, reagent 2) for 30 min at 37°C following treatment with reagent 1 (endogenous peroxidase blockers) for 5 min, followed by incubation with CtBP2 (sc-17759; 1:200; Santa Cruz Biotechnology, Inc.) or TGIF (ab52955; 1:1,000; Abcam) antibody overnight at 4°C. After washing three times with 0.1 M PBS, the sections were incubated with reagent 3 [HRP-conjugated goat anti-rabbit IgG (A8919; 1:1,000) or HRP-conjugated rabbit anti-mouse IgG (A9044; 1:1000, Sigma-Aldrich; Merck KGaA) secondary antibodies] for 1 h at 37°C, and subsequently incubated with reagent 4 at 37°C for 1 h. Subsequently, hematoxylin (Beyotime Institute of Biotechnology) was used for re-staining at room temperature for 2 h, and finally the sections were dehydrated in graded alcohol until they were transparent in xylene. The scoring criteria (semi-quantitative method) were comprehensive and determined by the staining intensity and proportion of positively stained cells. The staining intensity score was as follows: 0 points for no staining, 1 point for weak staining (light yellow), 2 points for moderate staining (yellowish-brown), and 3 points for strong staining (brown). The score for the proportion of positive cells was as follows: 0 for ≤5%, 1 for 5–25%, 2 for 25–50%, 3 for 50–75%, 4 for >75%. A final score was obtained by multiplying the two scores, 0–4 indicated negative, 4–8 was weakly positive, and >8 was strongly positive. CtBP2 and TGIF expression in ESCC tissues and normal tissues of patients were detected according to the aforementioned method and score. Low expression was observed in the negative and weak positive groups, whereas high expression was observed in the strongly positive group. After staining, five fields were randomly selected in each section (magnification, ×40) under a microscope (Axio Imager 2; Carl Zeiss AG). The digitized images of immunohistochemistry were quantitatively analyzed using Image-Pro Plus 6.0 software (IPP 6.0; Media Cybernetics, Inc.).
H&E staining
H&E staining was used to distinguish tissue morphology as per the manufacturer's instructions (Beyotime Institute of Biotechnology). ESCC tissue or normal tissue sections (4% PFA-fixed) were deparaffinized in xylene, then rehydrated in a graded alcohol series. Nuclear staining was performed with hematoxylin at room temperature for 10 min following by flushing with running water to yield the color blue. The sections were then differentiated with 1% hydrochloric acid ethanol for 3 sec and then washing with running water was continued. The cytoplasm was stained with eosin at room temperature for 30 sec, followed by 95% ethanol twice for 5 min, 100% ethanol twice for 5 min, 100% ethanol + xylene for 5 min (1:1), xylene for 5 min twice, and finally sealed with neutral gum. H&E staining was observed under a microscope (Axio Imager 2; Carl Zeiss AG).
Staining reagents included CtBP2 (sc-17759; 1:200; Santa Cruz Biotechnology, Inc.) and TGIF (ab52955; 1:1,000; Abcam) primary antibodies, anti-mouse (SAB3701092) or anti-rabbit (F4890) secondary antibodies (MilliporeSigma), and Hoechst 33342 (Beyotime Institute of Biotechnology). The cells were digested with trypsin and pipetted vigorously to make a single-cell suspension. The cells were placed on glass coverslips (24-well plates), incubated at 37°C and fixed with 4% PFA for 20 min at 15–25°C. The glass coverslips were then washed three times with PBS for 10 min each and blocked with 10% bovine serum albumin (BSA) for 2 h at 15–25°C. Subsequently, all cells were incubated with primary antibody (1:200) overnight at 4°C. The following day, the cells were incubated with secondary antibody (1:2,000) for 2 h at 15–25°C. Finally, the cells were stained with Hoechst 33258 (Beyotime Institute of Biotechnology) at room temperature for 10 min, fixed with anti-fade solution and imaged under a fluorescence microscope (Zeiss AG).
Co-immunoprecipitation (Co-IP) assay
The ECA109 cells were transfected with lentiviruses expressing CtBP2 or TGIF. At 1 week post-transfection, the cells were subjected to co-immunoprecipitation assay using a commercial kit (Prod#26149; Thermo Fisher Scientific, Inc.). Briefly, firstly, for antibody immobilization, ultrapure water, 20X Coupling Buffer and 10 µg affinity-purified antibody were added directly to the agarose (50% protein A/G agarose with ratio of 100 µl for a 1 ml sample) in the spin column. Secondly, for the lysis of the cell cultures, the culture medium was carefully removed from the cells. The cells were then washed once with 1X modified Dulbecco's PBS. This was followed by the addition of 400 µl per well ice-cold IP lysis/wash buffer (2X 50 ml, 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol; pH 7.4) to the cells cultured in a 6-well plate. The cells were then incubated on ice for 5 min with periodic mixing. The lysate was then transferred to a microcentrifuge tube and centrifuged at ~13,000 × g at 4°C for 10 min to pellet the cell debris. The agarose and cell proteins were then mixed and appropriate experimental controls were prepared, followed by rocking overnight at 4°C. The column was then centrifuged at 1,000 × g at 4°C for 1 min. The protocol uses the IP Lysis/Wash Buffer (1M NaCl) for coupling and washing the immune complex. The spin column was then placed in a new collection tube. This was followed by the addition of 50 µl elution buffer and centrifugation. The tube was centrifuged at 1,000 × g at 4°C for 1 min and the flow-through was collected and analyze for protein. The resulting immuno-complex was analyzed using western blot analysis, with an anti-TGIF (ab52955; 1:1,000; Abcam) or anti-CtBP2 (sc-17759; 1:200; Santa Cruz Biotechnology, Inc.) antibody. To eliminate heavy chain signals, a light chain-specific secondary antibody (cat. no. 58802; Cell Signaling Technology, Inc.) was used.
CCK-8 assay
Cell viability was measured using CCK-8 assay (Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions in three independent experiments. The transfected ECA109 cells at a density of 5×103 cells/well were seeded in 96-well plates with 100 µl 10% FBS. After culturing the cells for 24, 48 and 72 h with (10 and 100 nM, and 1, 10 and 100 µM) XAV939 (Wnt signaling pathway inhibitor; Merck KGaA) at 37°C, 10 µl CCK-8 reagent were added to each well and incubated at 37°C for a further 4 h. Cell viability was then measured based on the absorbance at 450 nm wavelength (OD450) using a microplate reader (BioTek Instruments, Inc.).
5-Ethynyl-2′-deoxyuridine (EdU) staining
Cell proliferation of the transfected ECA109 cells upon XAV939 treatment was investigated using EdU staining assay. Briefly, 1×105 transfected ECA109 cells were resuspended in 200 µl DMEM, and then seeded into 24-well plates. Following incubation for 48 h with 10 µM XAV939, the cells were stained with EdU (Beyotime Institute of Biotechnology) at room temperature for 30 min against exposure to light. The cells were re-stained with Hoechst 33342 for 10 min in the dark, followed by washing with PBS. The number of EdU-positive cells was photographed, and five randomly selected fields were counted, using a fluorescence microscope (Carl Zeiss AG) (magnification, ×200). The experiments were performed in triplicate.
Wound healing assay
The migration of the transfected ECA109 cells upon XAV939 treatment was investigated using a wound healing assay, as previously described (35). To ensure the consistency of each initial scratch, a scratch chamber was used to carry out the experiment. Briefly, 5×104 transfected ECA109 cells were seeded into the scratch chamber (70 µl cell suspension for each side of the scratch chamber), and then cultured overnight to adhere, followed by the removal of the scratch chamber. The cells were then washed twice with PBS, in order to remove any non-adherent cells. Before scratching, the cells were cultured in high-glucose DMEM containing 10% FBS and supplemented with 1% penicillin/streptomycin mixture (36). To ensure the consistency of each initial scratch, ibidi Culture-Inserts (ibidi GmbH) were used to carry out the experiment. Briefly, 5×104 transfected ECA109 cells were seeded into the ibidi Culture-Inserts (70 µl cell suspension for each side of the scratch chamber), and then cultured overnight to adhere, followed by the removal of the ibidi Culture-Inserts. Following the removal of the ibidi Culture-Inserts, the scratch wound was created. After scratching was complete, the cells were cultured in high-glucose DMEM containing 2% FBS and supplemented with 1% penicillin/streptomycin mixture. The migration (wound closure) of the indicated cells was monitored and photographed randomly at 0, 24 and 48 h. The wound healing rate (%) = (x h scratch area - 0 h scratch area)/0 h scratch area ×100. Independent experiments were performed at least three times.
Transwell assay
The cell migratory and invasive abilities were examined using a Transwell assay, as previously described (35). Briefly, for the migration assay, 1×105 transfected ECA109 cells were resuspended in 200 µl DMEM, and then seeded into the upper chamber of the Transwell (8 µm pore size; Corning, Inc.) in 24-well plates. For the invasion assay, the upper chambers were coated with Matrigel matrix (50 µl, BD Biosciences) before seeding the cells, and 500 µl of 10% FBS were added to the lower chamber. Following incubation at 37°C for 48 h with 10 µM XAV939, the non-migrating cells were removed using a cotton swab. However, the migrated or invaded cells on the underside were fixed with 4% PFA and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) at room temperature for ~40 min. The number of migrating and invading cells was photographed, and cells in five randomly selected fields were counted using a phase contrast microscope (Leica DM IL LED) (magnification, ×200). All experiments were performed in triplicate.
Bioinformatics analysis
The interactions between CtBP2 and predictive proteins were analyzed using the STRING database (https://string-db.org). Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn) was used to analyze the correlation between the expression of two interesting genes in a given tissue (37).
Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, Inc.) or SPSS 23.0 (IBM Corp.). χ2 tests were performed to assess the clinical association between CtBP2 and TGIF expression and other tumor characteristics. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. A Cox proportional hazards regression model was established to assess the factors independently associated with patient survival. Differences between two groups were compared using an unpaired or paired Student's t-test. To compare more than two groups, one-way ANOVA with post hoc Holm-Sidak correction for multiple comparisons was performed. All experimental data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference and all tests performed were two-sided.
Interaction between CtBP2 and TGIF
The STRING database was used for the detection of known protein-protein interactions and predictive protein-protein interactions. The CtBP2 protein network is illustrated in Fig. 1A. As a predictive result, an interaction was detected between CtBP2 and TGIF.
Interaction between CtBP2 and TGIF. (A) Visualization of CtBP2 protein network based on STRING database analysis. (B) Western blot analysis of immuno-complexes. The cells were lysed, and immunoprecipitation was performed with anti-TGIF or anti-CtBP2 antibody. The immuno-complexes were then subjected to western blot analysis. IgG was used as the negative control. IP, immunoprecipitation; CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor; STRING, Search Tool for the Retrieval of Interacting Genes.
Co-IP was performed to validate the interaction between CtBP2 and TGIF. Following ECA109 cell transfection with the with the CtBP2 or TGIF lentiviral transduction vector, the cells were subjected to Co-IP assay with anti-CtBP2 or anti-TGIF antibody. Immuno-complexes of CtBP2 and TGIF were observed using western blot analysis (Fig. 1B).
Expression levels of CtBP2 and TGIF in ESCC tissues and cells
RT-qPCR and western blot analysis were performed to examine the expression levels of CtBP2 and TGIF in ESCC tissues and cells. The mRNA expression levels of CtBP2 and TGIF were robustly increased simultaneously in the tumor tissues of representative patients with ESCC (Fig. 2A). Western blot analysis and IHC were performed to further confirm the CtBP2 and TGIF expression levels in ESCC tissues. Compared with the adjacent normal tissues, the expression levels of CtBP2 and TGIF were significantly upregulated simultaneously in ESCC tissues (Fig. 2B-E). RT-qPCR and western blot analysis were performed to evaluate the mRNA and protein expression levels of CtBP2 and TGIF in the ESCC cell lines. In comparison with the human normal esophageal epithelial cell line, HEEC, the mRNA and protein expression levels of both CtBP2 and TGIF were increased simultaneously in the ESCC cell lines. In ECA109 cells, the levels of CtBP2 and TGIF were the highest (Fig. 2F-H). These results revealed that CtBP2 and TGIF expression levels were significantly simultaneously increased in ESCC tissues and cells.
Expression levels of CtBP2 and TGIF in tissues or cells of ESCC. (A) mRNA expression levels of CtBP2 and TGIF in ESCC tissues were examined by RT-qPCR. (B) Expression levels of CtBP2 and TGIF in ESCC tissues were tested by western blot analysis. N, normal tissues; T, tumor tissues. (C) Statistical analysis of western blot analysis results. (D) Expression levels of CtBP2 and TGIF in ESCC tissues were tested by IHC. PBS was used as the negative control. Scale bar=200 µm. (E) Statistical analysis of IHC results. (F) Expression levels of CtBP2 and TGIF in the human normal esophageal epithelial cell line, HEEC, and the human ESCC cell lines, ECA109, TE-1 and KYSE-150, were examined using western blot analysis. (G) mRNA expression levels of CtBP2 and TGIF in HEEC and ECA109, TE-1 and KYSE-150 cells were examined using RT-qPCR. (H) Statistical analysis of the western blots in panel F. Data are presented as the mean ± SD, and the Student's t-test and one-way ANOVA were performed. *P<0.05, **P<0.01 and ***P<0.001. CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor; ESCC, esophageal squamous cell carcinoma; IHC, immunohistochemistry; RT-qPCR, reverse transcription quantitative -PCR.
Association between CtBP2 and TGIF expression and clinicopathologic characteristics of patients with ESCC
The correlation between CtBP2 and TGIF expression levels in ESCC based on GEPIA by Pearson's correlation analysis is depicted in Fig. 3A. There was a direct correlation between the CtBP2 and TGIF expression levels in ESCC tissues, with a correlation coefficient of R=0.45 (P<0.05).
Correlation between CtBP2 and TGIF. (A) Scatter plot of the correlation between CtBP2 and TGIF expression levels in ESCC tissues based on GEPIA. There was a direct correlation between CtBP2 and TGIF expression in ESCC tissues with a correlation coefficient of R=0.45 (P<0.05). (B) Co-localization of CtBP2 and TGIF expression in ECA109 cells was examined by using immunofluorescence. Scale bar, 50 µm. CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor; ESCC, esophageal squamous cell carcinoma; GEPIA, Gene Expression Profiling Interactive Analysis.
Subsequently, since there was a correlation between CtBP2 and TGIF expression levels in ESCC, their co-localization in the ESCC cell line, ECA109, was examined. For this purpose, immunofluorescence staining was performed to evaluate CtBP2 and TGIF expression co-localization in ECA109 cells. The results demonstrated that CtBP2 and TGIF expression was co-localized in the nucleus of ECA109 cells (Fig. 3B).
A total of 108 patients with ESCC was enrolled, and the association between CtBP2 and TGIF expression and the patient clinicopathological characteristics was analyzed (Table I). The CtBP2 and TGIF expression levels were significantly associated with metastasis and survival (P<0.05).
Table I.
Association between CtBP2 and TGIF expression and the clinicopathological characteristics of patients with ESCC.
CtBP2
Age (years) 0.912 0.742
<60 10 24 14 20
≥60 21 53 28 46
Sex 0.154 0.55
Male 21 62 31 52
Female 10 15 11 14
Clinical stage 0.147 0.175
I 3 6 6 3
II 24 47 27 44
III 4 24 9 19
Histological differentiation 0.053 0.379
Well 5 15 6 14
Good 12 45 21 36
Poor 14 17 15 16
Tumor diameter (cm) 0.078 0.097
<3 12 17 15 14
≥3 19 60 27 52
T classification 0.695 0.312
T1 3 11 6 8
T2 11 22 16 17
N classification 0.108 0.375
N0 20 47 27 40
N2 0 11 2 9
N3 0 1 0 1
Metastasis 0.015a 0.014a
No 17 23 22 18
Yes 14 54 20 48
Depth 0.493 0.345
t0 1 1 0 2
Outcome 0.003a 0.001a
Mortality 18 21 28 11
Survival 13 56 14 55
{ label (or @symbol) needed for fn[@id='tfn1-or-47-02-08240'] } Statistical analyses were performed using the χ2 test.
a P<0.05 was considered to indicate a statistically significant difference. ESCC, esophageal squamous cell carcinoma; CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor.
Prognostic value of CtBP2 and TGIF expression
Kaplan-Meier analysis was performed to examine the association between CtBP2 or TGIF expression and the survival of patients with ESCC. A total of 108 patients with follow-up data were evaluated and IHC staining followed by Kaplan-Meier survival analysis was performed. The high vs. low expression of CtBP2 (Fig. 4A) or TGIF (Fig. 4B) differed significantly in the survival curves. Patients with a low expression of CtBP2 or TGIF had a longer cumulative survival. Univariate analysis, performed using the Cox proportional hazards regression model, revealed that CtBP2 (P=0.005), TGIF (P=0.001), clinical stage (P=0.001), tumor diameter (P=0.005), T classification (P=0.026) and N classification (P=0.001) were independent prognostic indicators of overall survival (Table II).
Survival curves of CtBP2 (A) or TGIF (B) expression in patients with ESCC. A total of 108 patients with follow-up data were evaluated and immunohistochemical staining followed by Kaplan-Meier survival analysis was performed. Patients with a low expression of CtBP2 or TGIF had a longer cumulative survival. CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor; ESCC, esophageal squamous cell carcinoma.
Table II.
Univariate analyses of various prognostic parameters in patients with ESCC using Cox regression analysis.
Univariate analysis
Hazard ratio
95% confidence interval
Age 1.473 0.867–2.503 0.152
Sex 0.763 0.43–1.354 0.355
Clinical stage 2.088 1.349–3.232 0.001a
Histological differentiation 0.959 0.683–1.346 0.807
Tumor diameter 2.381 1.299–4.364 0.005a
T classification 1.497 1.049–2.137 0.026a
N classification 1.684 1.240–2.287 0.001a
Metastasis 1.240 0.747–2.058 0.405
Depth 1.239 0.952–1.613 0.112
CtBP2 2.361 1.288–4.329 0.005a
TGIF 4.194 2.317–7.590 0.001a
{ label (or @symbol) needed for fn[@id='tfn3-or-47-02-08240'] } Statistical analyses were performed using the log-rank test.
Signaling pathway involved in the of biological activity of CtBP2 in ECA109 cells
To explore the biological activity of CtBP2 and TGIF in ECA109 cells, recombinant lentiviral vectors overexpressing CtBP2 and TGIF (LV-CtBP2 and LV-TGIF) or carrying shRNAs to knockdown (sh-CtBP2 and sh-TGIF) CtBP2 and TGIF expression were constructed. Since the recombinant lentiviral vectors contained the gene encoding the green fluorescent protein (GFP), the transfection rate could be evaluated directly under a fluorescence microscope. Following transfection, the fluorescence images demonstrated that the percentage of GFP-positive cells was >90% (Fig. 5A, B, D and E). RT-qPCR was performed to further examine CtBP2 and TGIF mRNA expression in the ECA109 cells transfected with the vectors. In comparison with the negative control (NC), the CtBP2 and TGIF mRNA expression levels were significantly increased or decreased (P<0.05) following transfection in the ECA109 cells (Fig. 5C and F). In addition, western blot analysis was performed to examine the CtBP2 and TGIF expression levels in the ECA109 cells transfected with the vectors. As was anticipated, the CtBP2 and TGIF expression levels increased or decreased following transfection in ECA109 cells, as compared with the NC. Of note, TGIF expression was robustly and simultaneously increased when CtBP2 expression was significantly upregulated in the cells transfected with LV-CtBP2 and simultaneously decreased when CtBP2 expression was significantly downregulated in the ECA109 cells transfected with sh-CtBP2 (Fig. 5G and H).
Overexpression or knockdown of CtBP2 and TGIF in ECA109 cells. (A and D) ECA109 cells were observed under a fluorescence and phase contrast microscope following transfection. The recombinant lentiviral vectors contained the gene encoding the green fluorescent protein. Scale bar, 500 µm. (B and E) statistical analysis of percentage of positive cells in panels A and D. (C and F) mRNA expression levels of CtBP2 and TGIF in ECA109 cells following transfection was examined using RT-qPCR. (G and H) Expression levels of CtBP2 and TGIF in ECA109 cells following transfection were examined using western blot analysis. Data are presented as the mean ± SD, and the Student's t-test and one-way ANOVA were performed. *P<0.05, **P<0.01 and ***P<0.001. NC, negative control; CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor; RT-qPCR, reverse transcription quantitative PCR; LV, overexpression of CtBP2 and TGIF; Sh, knockdown of CtBP2 and TGIF.
XAV939 is an inhibitor of the Wnt signaling pathway (38), and TGIF plays a role in tumorigenesis through Wnt signaling (33). Therefore, it was hypothesized that CtBP2 may interact with TGIF to promote the proliferation and migration of ECA109 cells through the Wnt/β-catenin signaling pathway. First, the effects of XAV939 on cell viability and β-catenin expression were examined in the ECA109 cells. The results of CCK-8 assay revealed that cell viability was significantly decreased (P<0.05) in the ECA109 cells treated with XAV939 (10 and 100 nM, and 1, 10 and 100 µM) for 48 h (Fig. 6A). The expression of β-catenin was also significantly downregulated (P<0.001) in the ECA109 cells treated with XAV939 (10 nM, 100 nM, 1, 10 and 100 µM) for 48 h (Fig. 6B). Therefore, the optimal concentration of XAV939 that was used in subsequent experiments was 10 µM.
Effects of XAV939 on the viability and the expression of β-catenin in ECA109 cells. (A) Viability of ECA109 cells treated with various concentrations of XAV939 was examined using CCK-8 assay. (B) Expression of β-catenin in ECA109 cells treated with various concentrations of XAV939 was examined using western blot analysis. Data are presented as the mean ± SD, and the Student's t-test and one-way ANOVA were performed. *P<0.05, **P<0.01 and ***P<0.001 vs. blank.
Furthermore, CCK-8 assay was performed in order to measure the viability of the ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 24, 48 and 72 h. In comparison with the negative control (LV-NC), cell viability was markedly increased in the LV-CtBP2-transfected group; however, it was markedly decreased (P<0.05) in the XAV939 group, compared with the LV-NC-transfected group (Fig. 7A). These results indicated that XAV939 inhibited the CtBP2-mediated viability of ECA109 cells.
Signaling pathway involved in the biological activity of CtBP2 in ECA109 cells. (A) Viability of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 24, 48 and 72 h, was measured using CCK-8 assay. (B) The proliferation of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 48 h was measured using EdU staining. Scale bar, 50 µm. (C) Statistical analysis of EdU staining results. (D) The migration of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 48 h was measured using wound healing assay. Scale bar, 100 µm. (E) Statistical analysis of the wound healing assay results. (F) The migration and invasion of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 48 h was measured using Transwell assay. Scale bar, 100 µm. (G) Statistical analysis of the Transwell assay results. Data are presented as the mean ± SD, and the Student's t-test and one-way ANOVA were performed. *P<0.05, **P<0.01 and ***P<0.001. CtBP2, C-terminal-binding protein 2; LV, lentiviral; EdU, 5-ethynyl-2′-deoxyuridine.
EdU staining was then employed to examine the effects of CtBP2 and XAV939 in ECA109 cells. The results demonstrated that the number of EdU-positive cells was significantly decreased (P<0.05) in the LV-CtBP2 + XAV939 group, compared with the LV-CtBP2-transfected group (Fig. 7B and C). This demonstrated that XAV939 inhibited the CtBP2-mediated proliferation of ECA109 cells. The results of wound healing and Transwell assays also revealed that XAV939 inhibited the CtBP2-mediated migration and invasion of ECA109 cells (Fig. 7D-G). These results indicate that CtBP2 exerts its biological activity through the Wnt/β-catenin pathway in ECA109 cells.
The extremely high incidence of EC in certain regions of China has prompted a number of researchers to investigate the disease pathogenesis and develop effective treatment strategies for this disease in China. Due to comprehensive tumorigenesis and the development of EC involving complex regulation of oncogenes and tumor suppressor genes, the in-depth elucidation of the pathogenesis of EC at the molecular level in order to identify an ideal molecular target or effective therapeutic drugs for EC is a promising therapeutic approach (10,39). In the present study, the proteins interacting with CtBP2 were identified and the mechanisms of the biological activity of CtBP2 in ESCC were investigated.
CtBP2, a transcriptional co-repressor, acts as a bridge molecule between DNA-binding proteins and transcriptional repressors, in order to inhibit gene transcription by specifically binding to DNA-binding proteins and transcriptional repressors (12). Therefore, proteins interacting with CtBP2 were first determined in order to investigate the underlying mechanisms of CtBP2 biological activity in ESCC. The STRING database has been used for the detection of known protein-protein interactions and the prediction of protein-protein interactions (40). The results obtained from the STRING database indicated that there was an interaction between CtBP2 and TGIF and the subsequently performed Co-IP analysis indicated that these two proteins co-localized in the nucleus.
TGIF, a transcriptional repressor, is involved in a number of cellular signal transduction pathways, particularly the TGF-β pathway (41). As an interaction between CtBP2 and TGIF was observed, the expression of TGIF and the correlation between CtBP2 and TGIF in ESCC was then examined. A previous study by the authors revealed that the expression of CtBP2 was significantly increased in ESCC tissues and was positively associated with the histological grade of the tumor (22). As was anticipated, the expression levels of CtBP2 and TGIF were significantly simultaneously increased in ESCC tissues and cells. GEPIA was used to further analyze the correlation between the expression of CtBP2 and TGIF in ESCC. There was a direct correlation between CtBP2 and TGIF expression in ESCC tissues, with a correlation coefficient of R=0.45. These results indicated that the interaction of CtBP2 with TGIF plays a crucial role in ESCC.
The 5-year survival rate of patients with EC has been reported to be ~10%, and the recurrence and mortality rates remain high (8). Therefore, there is an urgent need for the identification of novel prognostic biomarkers for ESCC. In the present stuyd, Kaplan-Meier analysis was performed to examine the association between CtBP2 or TGIF expression and the survival of patients with ESCC. The results indicated that both CtBP2 and TGIF expression levels were significantly associated with metastasis and survival. Patients with a low expression of CtBP2 or TGIF had a longer cumulative survival. Therefore, CtBP2 and TGIF expression may serve as prognostic indicators of the clinical outcome of patients with ESCC.
XAV939 is an inhibitor of the Wnt signaling pathway (38), and high levels of TGIF are associated with high levels of the Wnt signaling pathway and a poor survival rate (33). Wnt exerts its effects through three signaling pathways, the most classic being the Wnt/β-catenin signaling pathway, mediated by β-catenin (42). It has been reported that the abnormal activation of the Wnt/β-catenin signaling pathway is involved in a number of diseases, including cancer, genetic diseases and organ fibrosis, as well as in the occurrence of EMT (43). Therefore, it was hypothesized that CtBP2, as a transcriptional co-repressor, may interact with TGIF and participate in the development of ESCC through the Wnt/β-catenin signaling pathway. TGIF expression was robustly increased or decreased simultaneously when CtBP2 expression was significantly upregulated or downregulated, and vice versa. The results of CCK-8, EdU staining and Transwell assays indicated that CtBP2 promoted the proliferation, migration and invasion of ECA109 cells through the Wnt/β-catenin pathway. In future studies, the authors aim to perform TCF reporter assay, in order to further verify the signaling pathway involved.
In conclusion, the present study demonstrates the existence of an interaction between CtBP2 and TGIF expression in ESCC, and these two proteins were co-localized in the nucleus. The CtBP2 and TGIF expression levels were robustly increased simultaneously in ESCC tissues and cell lines, and their expression was significantly associated with metastasis and survival. CtBP2 interacted with TGIF and promoted the progression of ESCC through the Wnt/β-catenin pathway.
The present study was supported by the China Postdoctoral Science Foundation (2018M632337), Natural Science Foundation of Shanghai (21ZR1449800), and the Natural Science Research Project of Nantong Science and Technology Bureau (XG202006-4).
QJ, MJ and HS contributed to the conception and design of the study. QJ and HS provided administrative support. QJ, MJ, WH, QY, ZL and HS provided all the study materials or patient clinicopathological data and samples. QJ, MJ, WH, QY and HS performed data collection and assembly. QJ, MJ, WH and HS contributed to data analysis and interpretation. QJ and HS confirmed the authenticity of all the raw data. QJ, MJ and HS wrote the manuscript. All authors have read and approved the final manuscript.
Patient written informed consent was obtained before the study, according to the guidelines of the Ethics Committee of the Affiliated Hospital of Nantong University, and ethics approval has been also provided from the respective ethics committee (2015 L132).
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Help! Do I Have Bed Bugs?
(honolulu, hi, usa)
i have a question. I am not sure if what I have is bed bugs. the bugs I have been seeing are very very small insects. It is almost impossible to see unless your really looking, and they are a tannish in color. I have been waking up with bites. I have about 20 on me right now, some old and some new.
The insects seem to be in my bedroom and in my living room. could these be bed bugs or something else?
Editor Comment: How to Tell if You Have Bed Bugs
Seeing insects and having bites, but not being certain that they are bed bugs can be a frustrating and confusing experience. Bed bugs are small, reddish-brown insects that are about the size of an apple seed, and their bites can cause red, itchy welts on the skin. However, there are other common insects, such as mosquitoes, fleas, and spiders, that can also bite and cause similar symptoms.
To determine if the insects are bed bugs, it's important to take a close look at the bugs themselves. Bed bugs are typically found in the seams of mattresses and box springs, in cracks and crevices in bed frames and headboards, and in other areas where people sleep. They are most active at night, and their bites are often found in a line or cluster on the skin.
If you suspect that you may have bed bugs, it's important to contact a professional exterminator to conduct an inspection. They have the specialized tools and knowledge to identify bed bugs and to determine if an infestation is present. An exterminator can also provide you with a plan of action to eliminate the infestation.
In case the bites are not caused by bed bugs it's important to take appropriate measure to deal with the insects that are causing the bites. Identifying the insect and understanding their behavior can help you to take the appropriate steps to get rid of them.
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Twenty years after his father mysteriously disappeared with the Roman Ninth Legion in rural Caledonia, a Roman soldier ventures into the treacherous territory. His goal: to recover Rome's golden eagle emblem and redeem his family's name. Set in 140 A.D., the tale follows determined centurion Marcus Aquila and his slave Esca--a native of Britain whom he's not certain he can trust--as they journey into the remote lands occupied by savage warrior tribes that live to slay Romans. Channing Tatum, Jamie Bell, Donald Sutherland and Mark Strong star in this first-rate historical epic. Directed by Kevin Macdonald; screenplay by Jeremy Brock. Based on the novel The Eagle of the Ninth by Rosemary Sutcliff.


The National Association of Pension Funds (NAPF) is warning that the EU's assessment of Solvency II-type rules for pension funds are "seriously flawed".
According to NAPF, they don't provide an adequate test of regulations that could damage UK pensions and businesses for decades.
In particular, the Association believes that the Holistic Balance Sheet (HSB) doesn't take adequate account of the complex structures of many large, multinational companies and their pension schemes.
NAPF added that the HBS fails to provide an accurate valuation of an employer's support of a pension, particularly in complex groups and in schemes with a degree of government support.
The comments come in a response to the European Insurance and Occupational Pensions Authority's Quantitative Impact Study, which it testing the proposals of the European Commission.
NAPF has also criticised the short eight-week timeframe given for providing feedback on the Solvency II-based regulation – many NAPF members said they didn't have time to respond.


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text:
/*
 *    THIS FILE IS GENERATED. DO NOT MODIFY.
 *
 *    SOURCE TEMPLATE: trait.c.h
 *    SOURCE PROTO: weave/trait/heartbeat/liveness_trait.proto
 *
 */
#ifndef _WEAVE_TRAIT_HEARTBEAT__LIVENESS_TRAIT_C_H_
#define _WEAVE_TRAIT_HEARTBEAT__LIVENESS_TRAIT_C_H_

    //
    // Enums
    //

    // LivenessDeviceStatus
    typedef enum
    {
    LIVENESS_DEVICE_STATUS_ONLINE = 1,
    LIVENESS_DEVICE_STATUS_UNREACHABLE = 2,
    LIVENESS_DEVICE_STATUS_UNINITIALIZED = 3,
    LIVENESS_DEVICE_STATUS_REBOOTING = 4,
    LIVENESS_DEVICE_STATUS_UPGRADING = 5,
    LIVENESS_DEVICE_STATUS_SCHEDULED_DOWN = 6,
    } schema_weave_heartbeat_liveness_trait_liveness_device_status_t;

#endif // _WEAVE_TRAIT_HEARTBEAT__LIVENESS_TRAIT_C_H_

redpajama_set_name: RedPajamaGithub


text:
Kosher Pickle
Should a Jewish nursing home be required to welcome Gentiles?
By Erick Trickey
Jim Campbell
In its fight against housing discrimination, the Metropolitan Strategy Group usually takes on bigots and predators: apartment managers who treat white applicants better than black ones, lenders who jack up interest rates on the poor.
Then there's the lawsuit against the Jewish nursing home.
In fall 1998, Carmela DiCicco tried to get her 95-year-old Catholic mother into the Montefiore Home in Beachwood. But the nursing home, which admits only Jews and their spouses, turned her away. DiCicco went to Metro Strategy, which complained to the Ohio Civil Rights Commission, then sued.
It may be the most ironic lawsuit at Cleveland's federal courthouse. An organization created to protect minorities' right to live where they want is trying to force a century-old Jewish institution to admit Gentiles. And the directors of Montefiore, members of one of history's most persecuted religious minorities, are accused of religious discrimination.
DiCicco and her siblings "desperately wanted [their] mother to live in a well-run nursing home that was close to them, so they could visit her," says fair-housing attorney Ed Kramer. But when Montefiore turned Lena Amantea away for being Catholic, her children cared for her themselves until she died in January 2000.
The accusation that Montefiore discriminated against Amantea seems to unnerve local Jews. Leaders of the Jewish Community Federation and Anti-Defamation League declined comment on the suit. Rabbi Daniel Schur of the Heights Jewish Center, who supervises Montefiore's kosher kitchens, says this isn't a case of discrimination. "It's not an apartment building where people live, and this color lives [here, and] this color not. We're talking about health."
An illness recently sent Schur's son to a hospital. "He's now in Montefiore for rehab. His whole spirit is different. It's extremely therapeutic to be amongst your own."
Montefiore, founded by a Jewish fraternal order in 1882, celebrates only Jewish holidays, observes the Sabbath, offers Holocaust discussions and Torah lessons, and hosts Jewish weddings and funerals.
Montefiore also has a synagogue. That, its directors argue, makes it exempt from fair-housing laws, which include an exception for nonprofit groups connected to a religious organization.
The OCRC agreed. Last year, it refused to order Montefiore to admit Gentiles. And since the federal and state laws are almost the same, the odds favor Montefiore in the pending federal case, too. "We're very confident we're going to win this thing," says Howard Landau, a spokesman for Montefiore who declined further comment.
Metro Strategy may soon give up on its effort to integrate the home. Kramer says the two sides are discussing a settlement which would allow Montefiore to remain all-Jewish if it strengthens its religious ties to clearly comply with the law.
Still, other nursing homes with religious affiliations admit everyone. "Part of our social teaching is to take care of people in need, no matter what," says Margot Klima, spokeswoman for Catholic Charities, which runs St. Augustine Manor in Cleveland.
But Reverend Marvin McMickle, civil-rights leader and pastor of the Antioch Baptist Church, sees nothing wrong with Montefiore's all-Jewish policy. "A people who had a history of discrimination have creatively found ways to serve themselves. They couldn't get into hotels, so they got into the hotel business. They couldn't get into a recreational facility, so they started their own facility. I would rather commend them for [that] than badger them for the very same reason."
Discrimination has encouraged Jewish separateness, agrees Peter Haas, professor of Jewish Studies at Case Western Reserve University. Still, he finds Montefiore's policy "a bit surprising" and believes the home should admit Gentiles.
That would be fine with Maxine Gannis, a seven-year resident of Montefiore. Although she's happy to be in a Jewish home, she says if it admitted non-Jews, "It wouldn't matter to me."


text:
Murray Leah (Author)
Edge-of-your-seat military romantic suspense...
Ex-Navy pilot Will Fletcher has returned home to the fishing village of Looe, Cornwall, and bought a pub. As far as he's concerned, he's done with the military, with trouble, and with women. All he's interested in is living a quiet life in the country with his dogs and his boat.
Desperate to escape a traumatic past, local vet Jo Williams has fled her native New Zealand for Looe. She's finding her new home rather lonely, but a relationship is the last thing she wants. After all, her stalker ex-boyfriend Jack is the reason she ran in the first place.
Yet when Jo meets Will the chemistry between them is undeniable, and it is clear that fate has her own plans for them. Unfortunately, so does Jack-and his include murder.
Reader Review
"Great romantic suspense! I was enthralled from the beginning. A veterinarian on the run and a pub owner full of angst and secrets. Grab a beverage of your choice, a sandwich and snacks because you will not want to break for food once you start. Extremely cohesive plot, fascinating characters, drama galore and steamy scenes that actually help move the plot along. This is the first book I have read for this author but it will not be the last. I highly recommend this book." JC, Amazon Reviewer
Author note
Ebb Tide is a standalone military romantic suspense that is the first in a series of books that are set in Cornwall but feature different heroes and heroines. The second book in the series will be out soon.
Bluewave Press Limited
5.5 X 8.5 X 0.58 inches | 0.72 pounds
Romance - Military
past staff: Cameron's Picks! VIEW LIST (61 BOOKS)


Our clients typically ask us: how do I find and own my business story? And a follow up question, what are the secret ingredients to make a good business story. With this in mind, I'd like to focus this episode of Visual Storytelling Today show on these fundamental questions I am sure any business leader can relate to. About our guest I have the great pleasure to speak today with Diana Yazidjian, a business storytelling expert with DFY consulting based in Montreal, Canada. Diana has extensive experience in marketing and technology helping start-ups, Fortune 500, retailers, and municipalities. She provides strategic and hands-on training on social media, brand storytelling, stand-out content and thinking like the customer. What you will learn: What makes an effective business story? How does the end-to-end process of creating an effective Visual Storytelling program look like? Where the future of business storytelling is headed? Great industry examples and actionable tips to get you started Read our episode's blog post on VSI Blog. This podcast is brought to you by the Visual Storytelling Institute (VSI) from Miami, FL. Ready to become a pro visual storyteller? We help you rise above the communication noise with personalized visual storytelling workshops, so you can connect better with your audience, empower their lives and grow faster your company. Schedule an appointment!
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Showing below 14 results starting with #1.
Create the page "Custom Celebration VI" on this wiki!
[[Category:Custom Celebration VI]] ...m and an Exo-Squad Jump Trooper jetpack. It was cheesy and such a terrible custom by today's standards, but it was fun.
[[Category:Customizer Interviews]][[Category:Custom Celebration VI]] ... I had a Storm Shadow figure that no one else had at the time. I made some custom nunchucks for him and after that started making more ninja weapons.


At my sons' pediatric check-up, the doctor asked if we had any "Mr. Yuk" stickers. I told her that we didn't and she brought us a few to place around the house. When I looked at those stickers, memories of my childhood came flooding back. I immediately recalled the old "Mr. Yuk" commercial from the 70s. That commercial scared the crap out of me when I was a kid. The ominous "Mr. Yuk is mean. Mr. Yuk is green" followed by diabolical laughter was enough to keep me out of the medicine cabinet. "Mr. Yuk" was a bad dude and I didn't want any part of him.
This walk down memory lane prompted me to do a YouTube search to find the commercial. I was pleased to find it and I'd like to share it with you. All of you 70s kids will be able to relate.
I showed the commercial to my kids and explained to them why they should not touch items with "Mr. Yuk" stickers. They didn't seem as scared of "Mr. Yuk" as I was. Maybe it takes more than a green dude to frighten this generation.
My wife, KayEm, and I were frightened a few years ago when my son, N, swallowed some of the oil I use to lubricate my clippers. KayEm freaked out when she noticed him walking out of our bathroom holding the bottle.
"Oh my, God," she said. "He drank some of that oil." I tried to play it cool, but I was scared because I took drug counseling course that taught us how oils coat the lungs and cuts off breathing. We weren't sure if he had ingested any or not, but we weren't about to take any chances. K called Poison Control to find out what we needed to do. They gave us detailed instructions and we followed them to the letter. We are thankful that he only swallowed a small amount and we were able to flush his system before any serious damage occurred. But the situation could have been much worse if we didn't have access to the Poison Control Center.
I implore each of you to make sure that you have the Poison Control number in an accessible place. Post it on the refrigerator, in the bathroom and any other location that houses hazardous chemicals. For those who don't have the phone number, it is (800) 222-1212.


text:
Paul Patterson
Sidney Cooper Mobiles: Paul Patterson and CCCU Contemporary Music Ensemble
Free`
Sidney Cooper Gallery
Canterbury, United Kingdom
St.Peter's Street
CT1 2BQ
Canterbury West or Canterbury East
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This concert is centred on Canterbury Christ Church University's visiting professor Paul Patterson's work 'Tate Modern Mobiles'.
Contemporary Music Ensemble (dir. Lauren Redhead):
Kelly Butler (voice, piano), Trixie Collins (voice, guitar), Jack Dorman (voice, bass guitar), Diane Dunn (voice, percussion), Hannah Firmin (voice, clarinet), Sinead Kearns (voice, flute), Nicola Lamonte (voice, glockenspiel), Poppy Stanley-Gilbert (voice, viola)
Scratch Duet:
Jason Kavanagh (guitar), Ben Rocca (piano)
This concert is centred on Canterbury Christ Church University's visiting professor of composition Paul Patterson's work Tate Modern Mobiles. These pieces, performed this evening for mixed ensemble and fixed media playback, act both as short stand-alone compositions and as picture-frames for the pieces in this concert. The music employs open notation, often specifying playing technique rather than pitch or rhythm, and allows the musicians a great degree of creative expression in their interpretation. The five Tate Modern Mobiles in this case frame works for spoken and sung voice by undergraduate composers at CCCU and improvised music performed by two members of the Canterbury Christ Church University Scratch Orchestra.
Paul Patterson will introduce the concert.
Paul Patterson is one of the most versatile, successful and internationally-respected British composers of his generation. All of the major UK orchestras, and in numerous countries abroad, have performed his works, as have eminent ensembles and international soloists, and he has a substantial discography. He has been the featured composer at many festivals both in the UK and abroad, and has also been BBC Composer of the Week. Music-lovers worldwide enjoy his music as it never alienates - rather, creates an enriching experience that draws both audiences and performers in.
His substantial oeuvre, which includes several prestigious commissions, is stylistically varied; he is known for his sympathetic and often humorous writing for instruments, making a popular and successful name for himself in the choral, brass, organ, orchestral, concerti and children's music. His composition style of challenging but idiomatic writing has resulted in many of his solo works have been chosen as set pieces for international competitions in Europe, Australia, Israel, Thailand and the USA.
Patterson's increasing international reputation as one of the foremost contemporary composers for the harp began with the popularity of Spiders. More works are being commissioned currently, and he has been invited to be the featured composer at the International Harp Congress in Hong Kong in 2017, his 70th birthday year.
Over the course of his career, Patterson has made significant contributions to many of the country's leading musical institutions. At the Royal Academy of Music he initiated a pioneering series of annual Composer Festivals – an idea swiftly emulated elsewhere. He is a fellow of many of the UK's leading music colleges and has received several awards in recognition of his services to music.
To book your place at the concert, please visit our website: http://www.canterbury.ac.uk/arts-and-culture/event-details.aspx?instance=49008
Twitter @Sidney_cooper01
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This page was added 10th March 2016, 15:46 by @user137158


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text:
Phillips, DiPisa & Associates Partners With O.E. Strategies
July 14, 2017 – Executive search firms are challenged with identifying the most qualified candidates for their clients' organizations.
Within the healthcare sector, recent and rapid transformations have made it imperative that leaders assuming a role within an organization in this field have the proper social, cultural and professional "fit."
It is also critical that healthcare groups provide a well-conceived onboarding experience to avoid the high costs of failure and to ensure the success of the executive.
Phillips, DiPisa & Associates, an executive search firm serving the healthcare sector, has entered into a partnership with O.E. Strategies to provide candidate executive assessments. The alliance was formed to ensure a seamless cultural fit between candidate and client, as well as a smooth onboarding process.
The complementary services that Phillips DiPisa and O.E. Strategies offer will address the fit and onboarding challenges, as well as other aspects of the search process. Employing its staff and consultant network of industrial/ organizational psychologists, O.E. Strategies will use scientifically-based leadership and evaluation protocols to help the recruiters assess their broad base of clients across the U.S. O.E. Strategies will also offer clients onboarding coaching services.
"Phillips, DiPisa has experience and a proven record of success with the ever evolving retained search process," said Daniel Phillips, the firm's president. "Our partnership with O.E. Strategies lets us add a very refined, specialized layer to that process, allowing us to thoroughly evaluate a candidates' appropriateness for placement and then to ensure that they can thrive in their new positions."
Why Onboarding Is Key to a Successful Recruiting Strategy
Effective onboarding can make or break an executive's success in a new role, yet less than a third of organizations actively support their executives to adapt to the cultural and political climate – which rank as the top reasons new leaders fail.
"Phillips DiPisa is known nationally for the breadth of their executive-level contacts and the multi-step process they use from search to hiring to post-hiring assessment," said Dr. Suzanne Miklos, president and founder of O.E. Strategies. "O.E. Strategies can complement their established efforts by assessing a candidate's leadership effectiveness, including strategic thinking ability, business acumen, work habits, interpersonal influence, character, motivation and potential for growth."
Michael Corey, a partner with Phillips DiPisa, said that by enhancing the onboarding process, clients and candidates have a greater chance of mutual success. "We're finding that the entities that are searching for a new direction in their executive leadership are themselves rapidly changing direction in the healthcare marketplace," he said. "The assessment process O.E. Strategies employs allows us to dig deeper and to find the nimble and creative executives the healthcare system requires."
The Onboarding Process
According to a recent CareerBuilder survey, over a third of employers (36 percent) lack a structured onboarding process, and a significant number are reporting costly consequences. Forty-one percent of these employers said the lack of a structured onboarding process has had a negative impact on their company, including lower productivity (16 percent), greater inefficiencies (14 percent), higher employee turnover (12 percent), lower employee morale (11 percent), lower level of employee engagement (10 percent), lower confidence among employees (10 percent), lack of trust within the organization (seven percent) and missed revenue targets (six percent).
"While onboarding is a critical component in setting new employees up for success from Day One, this study shows some companies are neglecting fundamentals in the onboarding process – and running into serious consequences that can impact the bottom line," said Rosemary Haefner, chief human resources officer at CareerBuilder.
Off-Boarded Employees
In a recent Mullin International online poll, off-boarded employees have been shown to have increasing influence over prospective employees' decisions to either approach or avoid a company. In fact, more than four out of five respondents said they were increasingly concerned about exiting employees' impressions. Effective outplacement can minimize the negative effects of downsizing on company reputations.
The benefits of using a deliberate, thought-out process are substantial. Employers using a structured onboarding program reported that employees are more engaged (49 percent) and have more confidence. The CareerBuilder report also found that successful onboarding leads to employees having greater trust in the organization (45 percent), giving workers great efficiencies (44 percent), higher productivity (42 percent), higher morale (38 percent) as well as lower turnover.
"Too many businesses believe they are successfully bringing newly hired executives into the fold, when in reality they are simply covering administrative basics," said Egon Zehnder partner Mark Byford. "That level of onboarding is outdated and largely ignores the most challenging obstacles for new leaders – unfamiliar cultural norms and expectations."
The consequences of improper onboarding, he added, include financial repercussions and talent retention issues. "Organizations must focus on getting integration right upfront to ensure new leaders succeed in today's high-stakes environment," said Mr. Byford.
Contributed by Scott A. Scanlon, Editor-in-Chief; Dale M. Zupsansky, Managing Editor; Stephen Sawicki, Managing Editor; and Will Schatz, Managing Editor – Hunt Scanlon Media
Check Out These Related Articles On Search Firms Expanding Their Offerings:
Morgan Samuels Acquires Human Capital Consulting Firm Greenwood / Asher Forms Alliance With Devine Group Caldwell Partners Latest Recruiter to Offer Assessment Services Analytic and Assessment Tools Gain Traction Among Headhunters Addison Group Latest Firm to Gain New Offerings By Acquisition Big Data & Analytics Sector Turning Into Big Business for Recruiters
Sterling Martin Associates Seeks New Leaders for Denver-Based Non-Profits
Human Capital Group Taps CIO for Dean Foods
Hunt Club and Parentaly Partner to Help Companies Adopt Equitable Parental Leave Programs
Mogul Forms Alliance with Brimstone
Coulter Partners Forms Alliance with Differing Minds


When vacationing in Wellfleet, Massachusetts you'll surely check out popular attractions like the Cape Cod National Seashore, the Massachusetts Audubon Society Wildlife Sanctuary, and Wellfleet Harbor. You might also eat at some of the great restaurants in the downtown area or do some fishing on one of the town's kettle ponds and experience a quiet refrain from the ocean-side beaches, which you'll also likely visit.
While the mainstream, touristy characteristics of Wellfleet attract visitors every year, the area has some interesting stories that are worth looking into because they provide off-the-beaten-path locations to explore from your vacation rental. The following facts also present insight into how Wellfleet became what it is today, so read on if you're interested in learning more about this quirky Cape Cod town.
You're probably aware that bananas aren't native to the United States, as they are an imported tropical fruit. Did you know, however, that Captain Lorenzo Dow Baker, the man responsible for mass-scale banana importing in the country, was born and raised in Wellfleet?
In 1870, Baker took his ship to Venezuela, and on his way back to New York City, stopped in Jamaica to pick up a few bunches of bananas. Unfortunately, the produce spoiled by the time he reached the United States. Undeterred, Baker returned to Jamaica the following year and picked some green bananas, which ripened on his way home.
Baker earned a substantial profit from these bananas, as he could purchase a bunch for 25 cents and then sell it for $3, the equivalent of nearly $60 in 2018 prices. He would turn this practice into a very successful business where he'd make his fortune.
Eventually, Baker would form the Boston Fruit Company, an entity that has since become the United Fruit Company, a corporation that imports the Chiquita brand bananas you see in grocery stores today.
Baker's influence on Wellfleet didn't stop at bananas, as his sudden wealth and status led to the area becoming a summer hotspot for the rich and famous. In 1886, Baker, now known as The Banana King, opened the Chequesset Inn, a large hotel that sat on the old Mercantile Wharf and extended 400 feet into Wellfleet Harbor. Baker envisioned the resorts he had seen in his travels through Florida and the Caribbean when he built the inn, and made it an all-inclusive property that catered to high society.
The inn, a four-story-tall building with 62 rooms, collapsed into Wellfleet Harbor during an intense winter storm in 1934. There is a plaque near the spot the hotel stood, which is near Mayo Beach off Kendrick Avenue, and you can still see pieces of the pier in the water during low tide.
The Chequesset Inn is chiefly responsible for developing the early tourism industry in Wellfleet, as it gave people a reason to visit. As our next fact suggests, the industry has taken off exponentially since the late 1800s, to the point that Wellfleet is an entirely different place in the summer.
Wellfleet is a relatively small town, with an estimated population of about 3,500 people during the offseason. In the summer, however, the community swells to over 17,000 residents, many of whom own or rent Cape Cod vacation homes.
Although this temporary growth is an inconvenience for many locals, as it leads to lines and crowds everywhere, it is responsible for keeping the economy afloat. Cape Cod businesses rely on seasonal residents spending money in the area and, thus, the influx of visitors is essential for the survival of the town in its current form.
Marconi Beach is one of the more popular places to spend time in Wellfleet during the summer, but did you know that it got its name from world-famous Italian inventor and engineer Guglielmo Marconi? Marconi is known for his work with long-distance wireless telegraphs and credited with inventing our current system of using radio waves for transatlantic communication.
As the story goes, Marconi chose the area that is now Marconi Beach to set up Marconi Station in 1901, where he planned to make the world's first wireless transatlantic communication. He chose the beach because it is elevated without much vegetation, providing a clear shot across the ocean.
Marconi ultimately ended up sending the first communication to cross the Atlantic from Glace Bay, Nova Scotia. But on January 18, 1903, a message from President Theodore Roosevelt was sent to King Edward VII using his station in Wellfleet in what was the first transatlantic wireless communication to emanate from the United States. Pieces of the first wireless tower are still visible at the beach.
When you're in Wellfleet's downtown area, you're sure to hear the bells at the First Congregational Church on Main Street ring every half hour. This tradition occurs because the church is home to the only bell clock in the country that remains on ship's time.
On a ship, time is broken down into four-hour shifts that start both in the AM and PM at 12:00, 4:00, and 8:00. There is then one bell-ring for every half hour past the start of a shift. For example, the bell clock will ring once at 12:30, twice at 1:00, and three times at 1:30 for a shift starting at noon.
This quirk means that bell clock at this church in downtown Wellfleet rings all day and all night, but it'll never tell you the correct time unless you know how to decipher ship's time. When you head down to Main Street on your Wellfleet vacation, see if you can figure out what time it is just by listening to the bells.
If you spend any amount of time in Wellfleet in the coming months, take the time to explore the stories and locations behind these hidden facts. Some, like Marconi Beach and Town Clock, are easily accessible, while others, such as Captain Lorenzo Dow Baker's plaque, will take some digging to find. Wellfleet is a special place for those who live and spend their vacations there, and these hidden facts add to the allure of this quintessential Cape Cod town.


"With almost 30 years experience behind the turntables, DJ A to the L has been rocking both clubs and airwaves in the US and Europe since he was a teenage and brings a long established track record as a DJ on college, commercial, and underground Hiphop radio to The Ammo Dump on Beatminerz Radio. He has opened for and toured with various artists across multiple genres – from Jazzy Jay, Afrika Bambaataa, 50 Cent, KRS-One, Public Enemy, MC Lyte, Run DMC, The Beastie Boys, and Gang Starr; to Chocolate Puma, Diplo and Tiesto; to Justin Timberlake, Christina Aguilera, Destiny's Child and Britney Spears. He's also lectured at several universities on various Hiphop-related courses. As comfortable mixing Disco, Funk, House, RnB, and Reggae as he is with mixing Hiphop, there's no telling what direction an Ammo Dump mix might take over each two hour episode.


text:
Q: Struts2 isn't mapping correctly textfield from JSP to Action Class I have a problem with a Struts2 2.3.28 J2EE webapp deployed and running into Apache Tomcat 8. I have a JSP file in which a form is present and it contains a textfield with some name:
classificazioni.jsp:
<s:form action="editCategorie" >
<s:textfield label="Nome" name="namedCat" value="ciao"/>
<!--
    <s:textfield label="Nome" name="catBean.namedCat" value="ciao"/>
-->

...

I want to access this textfield value into the respective ActionClass, in which I provided the property's declaration and getter and setter:
EditCategorie.java:
private String namedCat;

public String getNamedCat() {
    return namedCat;
}

public void setNamedCat(String namedCat) {
    this.namedCat = namedCat;
}

/*
private CatBean catBean;

public CatBean getCatBean() {
    return catBean;
}

public void setCatBean(CatBean catBean) {
    this.catBean = catBean;
}
*/

public String execute()
{
    return SUCCESS;
}

public String display()
{
    System.out.println(getNamedCat());
    // System.out.println(getCatBean().getNamedCat());
    return NONE;
}

I know that this way the webapp should work correctly and print the namedCat textfield value when the display method is invoked. Here is my struts.xml interesting parts:
<interceptors>
    <interceptor-stack name="mystack">
        <interceptor-ref name="defaultStack" />
    </interceptor-stack>
</interceptors>

<action name="editCategorie" class="it.unisalento.se.actions.EditCategorie">
        <interceptor-ref name="mystack"/>
        <result name="success" type="tiles">classificazioni</result>
    </action>

    <action name="showCategorie" class="it.unisalento.se.actions.EditCategorie" method="display">
        <interceptor-ref name="mystack"/>
        <result name="none" type="tiles">classificazioni</result>
    </action>

I've just tried two different approaches, as you've noticed reading the code, but when I try to print the namedCat into EditCategorie's display method the console shows 'null', whilst with the Bean approach, despite I know Struts2 should automatically instantiate the bean class by itself with the correct filled attributes, this doesn't happen and obviously when I try to access to the namedCat property of the catBean instance I get a NullPointerException.
Eventually this is also the CatBean.java class:
public CatBean()
{

}

private String namedCat;

public String getNamedCat() {
    return namedCat;
}

public void setNamedCat(String namedCat) {
    this.namedCat = namedCat;
}

I have read many times here that the main Struts2 advantage is to avoid the use of direct Servlet's logic, and also I have read here that this two approaches should work. I know I could use also a ModelDriven approach (this works perfectly in other actions), but I have to deal with parametric textfields so it is not the case.

A: Struts 2 actions are instantiated per-request.
There is no data in the newly-instantiated action, hence nothing to display, when you hit the showCategorie URL. If you want to persist the value you'd need to keep it somewhere, e.g., database, session, application, etc.

redpajama_set_name: RedPajamaStackExchange


There are a number of important issues to bear in mind. The Chilean economy (along with other Latin American ones) has been hit by the decline in international prices for its major commodity. With the drop in copper prices from 2011, the Chilean economy grew at an average annual rate of less than 2 percent. Oftentimes an economic downturn alone would be enough to persuade former supporters to defect. In Chile, however, there were a variety of other factors at play. A series of corruption scandals, involved both the political right and the left, but impacted out-going President Michelle Bachelet given that her son and daughter-in-law have been implicated. Bachelet's approval rating plummeted to 25 percent as her critics characterized her as both incompetent and corrupt.
In addition, the Bachelet policy performance had little hope of satisfying the disparate demands of this deeply divided society. The administration carried out a series of reforms that for some did not go far enough fast enough, but for others went too far, too fast. There had been mass popular mobilization demanding reform of the country's highly unequal educational system since the mid-2000s. However, it was not until 2015 that Bachelet passed a law providing for free post-secondary education. However, widespread student demonstrations continued as protestors demanded deeper reforms that would exclude the participation of the private sector in education. At the same time, the increase in corporate taxation, needed to pay for the educational reform and other social programs, angered the private sector. Meanwhile, Bachelet's progressive social policies (legislation legalizing abortion under certain conditions and providing for gay civil unions) alienated socially conservative members of her own coalition, and galvanized the socially conservative political right. A profound sense of disillusionment with both the political process and the policy output, probably mostly among former left coalition (now known as the New Majority) supporters, may well have been at the root of the low voter turnout of only 48.5 percent.
In addition, the left-centre alliance that had ruled Chile for most of the years since the transition to civilian rule in 1989 was deeply divided by the 2017 election; by that time, it was faced with defections from both ends of the political spectrum. The socially conservative Christian Democrats, who had previously backed left-centre coalition candidates, fielded their own presidential candidate in 2017. In addition, a new political group involving left parties and social movements, the Frente Amplio, also ran its own presidential candidate, calling for a more substantive democracy, a new economic model involving a reduction in the concentration in economic wealth, and the establishment of social rights.
The political right, on the other hand, was strongly united this time around -something it had not been in the past. Given the country's sluggish economic growth, Piñera's criticism of the New Majority economic management resonated with a significant portion of the population. Finally, the political right had effectively used scare tactics, with Piñera comparing Guillier to Nicolas Maduro, the socialist leader of the collapsed Venezuelan economy.
Given all of this and the fact that 45 percent of the popular vote went to left candidate Guillier, it is premature to proclaim that Chile is "moving right" in terms of popular opposition to policies to mitigate socio-economic inequality. The country remains deeply polarized, with the recent election campaign probably serving to deepen that political polarization. Indeed, Sebastían Piñera promised to reverse the Bachelet's reforms aimed at reducing socio-economic inequality - reforms considered by many on the left to be far too timid. The pronouncements made by Chile's incoming President were more conciliatory on election day. Although Piñera declared that the country "needs agreements more than confrontations," and that "paths of the future unite us.", we need to remember that there are powerful interests backing the Chilean political right, and they will insist on their own policy agenda. Swings in electoral fortunes in Chile, and elsewhere in the region, do not necessarily signal any change in the basic constellation of political forces and their deep and abiding differences.