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Glucose restriction decreases telomerase activity and enhances its inhibitor response on breast cancer cells: possible extra-telomerase role of BIBR 1532
Cancer Cell International volume 14, Article number: 60 (2014)
Considerable progress has been made to understand the association between lifestyle and diet in cancer initiation and promotion. Because excessive glucose consumption is a key metabolic hallmark of cancer cells, glucose restriction (GR) decreases the proliferation, and promotes the differentiation and transformation of cancer cells to quiescent cells. The immortality of cancerous cells is largely assured by telomerase, which is an interesting target for inhibition by BIBR 1532. In this study, we investigated the effect of GR on telomerase activity and on the efficacy of its inhibition by BIBR 1532.
Breast cancer MDA-MB 231 and MCF-7 cells were cultured in DMEM (Dulbecco’s modified eagle’s media) with 0, 1 or 4.5 g/l of glucose. The telomerase activity was measured via quantitative Real-Time PCR, and the two telomerase subunits were semi-quantified by RT-PCR. Proliferation test and mitochondrial metabolism were assessed via tetrazolium salt reduction and cell counts; apoptosis was assessed via caspase-3 quantification and flow cytometry.
A decrease in the telomerase activity of more than 75% was associated with a significant reduction in the mRNA expression of its catalytic subunit hTERT (Reverse Transcriptase) and a decrease in the mitochondrial metabolism by more than 80% under restricted glucose conditions. In addition, GR increased the effect of BIBR 1532. Glucose deprivation induces apoptosis via BIBR 1532-mediated telomerase inhibition in triple negative breast cancer cells, as assessed by caspase-3 measurements and Annexin analysis.
Taken together, our results suggest that the effect of BIBR 1532 is potentiated by GR to induce triple negative breast cancer cell death.
Caloric restriction (CR) or reducing the total caloric intake by 20 to 40% without limiting essential vitamins and minerals has long been considered as a means to prevent and cure cancer and to extend life span [1–3]. Recently, a potential role of CR in the treatment and intervention of age-related diseases, including cardiovascular disease, diabetes, hypertension, atherosclerosis and cancer, has been investigated [4, 5].
Cancer is a complex disease associated with multiple DNA mutations that lead to abnormal cell proliferation, immortality and tissue invasion at distant sites . The strong relationship between nutrition and cancer, as also demonstrated by several epidemiological studies [7, 8], has been extrapolated by some clinicians into complete abstinence from food. In fact, ten oncologists did allow their patients who suffered from breast, esophageal, prostate and lung cancer to fast from 48 to 140 hours pre-chemotherapy and 5 to 56 hours post chemotherapy . Patients who received chemotherapy with water-only fasting reported a reduction in side effects and also showed a better response to treatment than the control group. This latter observation contradicts the dietary recommendation of the American Cancer Society that advises patients to increase their protein and calorie intake before chemotherapy administration.
Cell division is potentially a risky process, and cell division is limited in normal tissues. Telomerase is up-regulated in 85% of malignant tumors. Therefore, they escape senescence to have unlimited cell division at the earlier stage of tumorogenesis . Telomerase is the key enzyme responsible for the immortalization of cancerous cells .
Telomerase is a ribonucleoprotein complex enzyme that consists of two major subunits, the human Telomerase Reverse Transcriptase (hTERT) and human RNA component (hTR), in addition to several accessory proteins that modulate the activity of this enzyme . Eighty five percent of malignant tumors escape senescence to have unlimited cell division following telomerase up-regulation during the early stages of tumorogenesis . The enzyme telomerase stabilizes the length of telomeres, the end of linear chromosomes, via the de novo addition of telomeric repeats, which have been lost during incomplete DNA replication, telomere degradation or oxidative stress. The telomerase-induced telomere maintenance and reparation is believed to play a critical role in senescence, aging and cancer . Human telomerase consists of two major subunits, the human Telomerase Reverse Transcriptase (hTERT) and human RNA component (hTR), in addition to several accessory proteins that modulate the activity of this enzyme .
An increasing body of literature has demonstrated that several metabolic changes occur during cancer evolution . On the molecular level, cancer cell proliferation is tightly related to cell metabolism . In multicellular organisms, cell proliferation is controlled, and any aberrant individual cell proliferation is prevented when nutrients exceed the levels needed to support cell division. In contrast, cancer cells overcome this growth factor control via several DNA mutations that alter the receptor-initiated signaling pathway [16, 17]. This altered metabolism of tumor cells is exhibited by an increased glucose uptake and elevated aerobic glycolysis, which was first recognized by Warburg 70 years ago . Unsurprisingly, abnormal glucose metabolism has become the target of several studies because of its uniqueness in cancer cells and its possible role in preventing and curing cancer. However, GR is a metabolic stressor to many signaling pathways, including the anti-apoptotic pathway IGF1R-IR/PI3K/Akt/mTOR . Glycolytic enzymes are up-regulated following the activation of this pathway. In addition to glycolysis activation, c-Myc and SP1, which are transcription factors induced by a high glucose level, cooperate to activate the transcription of the catalytic subunit of telomerase (hTERT)  The activation of AMP-activated protein kinase by GR down-regulates mTOR, glycolysis activity, and cell proliferation [21–23]. Furthermore, glucose withdrawal increases ketone bodies in the blood circulation. These ketone bodies can reportedly help to prevent and treat cancer by protecting mitochondria from inflammation and ROS (Reactive Oxygen Species) . Finally, Li and colleagues  demonstrated that GR impairs precancerous cell growth via the epigenetic control of the telomerase subunit (hTERT) and p16 expression.
GR is an attractive modality to explore because it has been shown to induce changes in molecular pathways, especially those altered in cancer. Altering these pathways could make cells more susceptible to treatment with the telomerase inhibitor BIBR 1532. This drug is a potent and selective inhibitor of two telomerase units; it interferes with the action of telomerase .
The primary objective of this article was to investigate the effect of GR on the telomerase activity and on its inhibition by BIBR1532 in two breast cancer cell lines, the highly invasive and metastatic triple-negative (ER-, PR-, Her2-) MDA-MB 231 and the less invasive Estrogen-dependent (ER+) MCF-7 cells.
Effect of glucose restriction (GR) on telomerase activity
The effect of GR on the telomerase activity was first assessed using the MDA-MB 231 and MCF-7 breast cancer cell lines. Figure 1 shows that decreasing the glucose concentration in culture media led to a partial telomerase activity inhibition at 1 and 0 g/l. Importantly, the cells incubated with 0 g/l of glucose were cultured in 10% of FBS that contains 0.65 g/l of glucose. This concentration was measured for each test in a medical laboratory and mimics physiological glycaemia in fasting situations. MDA-MB 231 reduced their telomerase activity in response to GR by more than 50% (P < 0.05) and 80% (P < 0.01) at 1 g/l and 0 g/l of glucose, respectively. This same pattern was evident in MCF-7 cells, as indicated in Figure 1b. The telomerase activity decreased by 40% and 75% in cells incubated in DMEM containing 1 g/l (P < 0.05) and 0 g/l glucose (P < 0.01), respectively.
Glucose restriction but not BIBR 1532 impaired hTERT expression
Because the hTR component is constitutively expressed in all cells and hTERT is up-regulated in most tumor cells, we investigated the ability of GR to modulate the expression of hTERT in human breast cancer cell lines after 48 h of treatment with 10 μM BIBR1532. The RT-PCR method was used to examine the expression of both hTR and hTERT mRNA using GAPDH as an internal control. Importantly, BIBR 1532 inhibits the protein, but not the mRNA level of hTERT; it inhibits the mechanism of action of the enzyme.The relatively high telomerase activity at high glucose concentrations shown in Figure 1 was confirmed by high mRNA expression (Figure 2). However, we could not demonstrate any changes in the expression of hTERT and hTR mRNA in cells grown in 1 and 0 g/l of glucose; hTERT was expressed at 55% (P = 0.0248) and 59% (P = 0.033) at 1 and 0 g/l of glucose, respectively, in MDA-MB 231, while hTERT was expressed at 41% (P = 0.021) and 37% (P = 0.0178) in 0 g/L and 1 g/L, respectively, in MCF-7 cells, as shown in Figure 2c. As expected, BIBR 1532 did not affect the telomerase transcription because it acts directly on the protein.
Glucose restriction decreased cell proliferation and enhanced the effect mediated by BIBR 1532
The effect of GR on the MDA-MB 231 cell count and proliferation was only significant at 0 g/l of glucose compared to 4.5 g/l of glucose, as shown in Figure 3a. Although cell proliferation at 1 g/l of glucose seems to be lower compared to cells grown in 4.5 g/l of glucose, the results did not reach significance (P = 0.08). In contrast, the proliferation of cells in 0 g/l of glucose was very slow, and this difference was statistically very significant compared to those incubated in 1 and 4.5 g/l of glucose (P < 0.001). The effect of the telomerase inhibitor BIBR 1532 was very pronounced for cells grown in 0 g/l of glucose. By the end of week three, the number of cells incubated in 0 g/l of glucose and 2.5 μM BIBR 1532 was almost zero (Figure 3a). Contrary to this result, BIBR 1532 seemed to have an effect in weeks one and two for cells grown in 1 and 4.5 g/l of glucose. This trend disappeared at week three, and the level of cell proliferation was similar to that of the control.The effect of GR was more pronounced on the MCF-7 cell line. As shown in Figure 3b, reducing the glucose concentration in the media significantly decreased the cell proliferation over time. The proliferation of cells grown without glucose reduced by 30% from the first week (P = 0.057), and this reduction was enhanced at the second week, when only less than 40% of cells remained alive (P = 0.0001). The MCF-7 cell count was not extended into the third week because all of the cells grown in DMEM containing 0 g/L of glucose had died. Cells incubated in 1 g/L of glucose exhibited reduced proliferation by 20% at the second week (P = 0.0139). After 15 days, 2.5 μM BIBR 1532 could reduce cell proliferation by 35% only when cells were incubated in physiological glucose concentrations (P = 0.0051).
Regulation of mitochondrial metabolism by glucose restriction and BIBR 1532
Water-soluble tetrazolium salt WST-1 was used to assess the cell viability. This test reflects not only the cell viability but also the metabolism of mitochondria, as reported by several works [27, 28]. Figure 4a shows that GR apparently decreased the succinate reductase activity in MDA-MB 231 cells by 55% and by more than 5-fold for cells grown in 1 g/l and 0 g/l of glucose, respectively. In MCF-7 cells, GR reduced the mitochondrial activity by 35% and by more than 80% in cells grown in 1 g/l and 0/gl glucose, respectively (P = 0.0009). This decrease was not caused by cell death because healthy quasi-confluent cells were detected in all wells using microscopic examination, bromophenol blue staining, and cell counts. This experiment was extended to evaluate the effect of BIBR 1532 in the GR conditions. As shown in Figure 5a, BIBR 1532 significantly reduced succinate reductase activity in MDA-MB 231 cells grown in 1 g/L and 4.5 g/L glucose. This effect was potentiated at a high glucose level (4.5 g/L), where 10 μM BIBR 1532 could reduce WST-1 transformation by 35%. However, this effect was absent when cells were grown in 0 g/l glucose.As shown in Figure 5b, higher concentrations of BIBR 1532 are required to elicit the same effect observed in MDA-MB 231 cells. This telomerase inhibitor could reduce WST-1 transformation by 25% starting from 50 μM, and this reduction became more significant when the BIBR 1532 concentration was increased to 100 μM (P = 0.0011). A high concentration of BIBR 1532 (100 μM) is required to inhibit the mitochondrial metabolism by ~25% in cells incubated in 1 g/L of glucose (P = 0.029). This modulator effect was absent in cells grown without glucose.Telomerase-negative human osteosarcoma Saos-2 cells have previously been studied to examine the role of telomerase in mitochondrial metabolism modulation. The Saos2 cell line was cultured in different glucose concentrations and then treated with 10 and 50 μM BIBR 1532 over 48 h. The results demonstrated that GR displayed the same profile as those exhibited by the two breast cancer cell lines. Reducing the glucose concentration in the media decreases the WST-1 transformation by 40% and more than 80% for cells grown in 1 g/L and 0 g/L glucose, respectively (Figure 5c). As shown in Figure 5d, BIBR 1532 exhibits the same effect in Saos-2 cells grown in 1 and 4.5 g/L glucose (P = 0.017 and P = 0.0032, respectively).
hTERT mRNA silencing did not affect mitochondrial metabolism
To further validate the results concerning the modulation of mitochondrial metabolism by BIBR 1532, we treated MDA-MB 231 and MCF-7 cells grown at different glucose concentrations with TERT siRNA. Surprisingly, Figure 6(a, b) shows that 20 nM TERT siRNA did not affect the formazan dye formation compared to the control at the three different glucose concentrations; however, a decreased tendency was evident only in cells incubated in 4.5 g/l glucose. The transfection efficiency was confirmed by using electrophoresis and cell death control, which is a blend of highly potent siRNAs targeting ubiquitously expressed human genes indispensable for cell survival (Figure 6) and hTERT gene expression. This apparently discrepant result could be explained by a high residual telomerase activity even after partial hTERT mRAN silencing. Furthermore, hTERT siRNA likely did not elicit an effect similar to that of BIBR 1532 due to the insufficient incubation time to deplete hTERT.
Glucose deprivation induced apoptosis mediated by BIBR 1532
To demonstrate whether BIBR 1532 induced apoptosis in MDA-MB 231 and MCF-7 cells, the expression of caspase-3 was assessed via ELISA (Invitrogen).
Although apoptotic signals are believed to be mediated by a hierarchy of caspase activation, only two pathways of caspase activation have been described. Nevertheless, caspase-3 is a common downstream enzyme activated in apoptosis that is induced by most stimuli.
The level of caspase-3 was measured in cells grown at different concentrations of glucose in the presence and absence of a high concentration of BIBR 1532 (50 μM).Figure 7 demonstrates that 50 μM BIBR 1532 did not affect MDA-MB 231 apoptosis except in cells grown in 0 g/l of glucose. Caspase-3 was not expressed in MCF-7 cells, and this result confirmed several reports that indicated caspase-3 deficiency in this cell line.
Caspase-3 expression was not detected in the Saos-2 cell line incubated at different glucose concentrations or treated with BIBR 1532. (Data not shown).
The results obtained from this experiment suggest that caspase-3 is involved in the induction of apoptosis in BIBR 1532-treated MDA-MB 231 cells that are deprived of glucose. Apoptosis induction was also investigated via annexin V staining. Figure 8 and Tables 1 and 2 shows same results displayed in Figure 7. The percentage of annexin V-positive cells significantly increased in treated cells with BIBR 1532 without glucose compared to the control cells. These data suggest that the growth inhibitory effect of BIBR 1532 on MDA-MB 231 cells is partly due to its effect on apoptosis induction under restricted glucose conditions. In the MCF-7 cell line, high concentrations of BIBR 1532 did not elicit apoptosis as observed in MDA-MB 231, unless cells incubated in high glucose concentration (4.5 g/L) are treated with 50 μM BIBR 1532, as shown in Figure 8b and Tables 1 and 2.
Due to its major role in cell immortalization, telomerase has become an attractive target for selective cancer therapy. Several approaches have been studied to decrease or completely inhibit the activity of telomerase. Among these efforts, we mention the direct and indirect inhibition of telomerase, immunotherapy using hTERT as a tumor-associated antigen, and gene therapy with a promoter-driven suicide promoter . Because its expression is mainly restricted to tumor cells and it is the rate-limiting subunit in telomerase activity, hTERT has become the main target of inhibition to stop tumor growth. However, these efforts directly face a major obstacle described by the lag phase between the initiation of telomerase inhibition and the impact on the proliferation capacity [29, 30]. Therefore, tumor cells with long telomeres will continue to grow upon telomerase inhibition until substantial telomere erosion has occurred. This limitation negatively impacts the application of this inhibition in patients with a high tumor mass. Out of several telomerase inhibitors, two molecules have been extensively studied: BIBR 1532 (2-((E)-3-naphtalen-2-yl-but-2-enolylamino)-benzoic acid) and Costunolide. The beneficial effect of caloric restriction, especially GR, on life span extension and cancer prevention and treatment has been very well documented. However, cell metabolic changes during this stress, especially on the enzymes activities, are not completely understood .
In the present study, we investigated the effects of BIBR 1532 on cell proliferation, mitochondrial metabolism, and apoptosis in MDA-MB 231 and MCF-7 cells under restricted glucose conditions. Furthermore, the role of GR on the telomerase activity and cell division rate was assessed for extended periods.
Our in vitro study showed that cell glucose deprivation dramatically decreases the telomerase mRNA expression and activity and cell proliferation. The important role of telomerase in cancer cell immortalization agrees with previous studies that reported a correlation between caloric restriction and cancer prevention and treatment. The cells incubated in 0 g/l of glucose were actually exposed to 0.65 g/l from FBS, which perfectly matches the physiological glycaemia in fasting state, whereas 1 g/l glucose mimics a postprandial state and 4.5 g/l mimics non-controlled glycaemia in diabetic patients or constant high glucose consumption. The increased telomerase activity in the presence of high glucose concentrations could at least partly explain the association between diabetes and cancer predisposition [31, 32].
Despite the similar hTERT expression in cells grown in 1 and 0 g/l of glucose, a complementary experiment involving the cell count showed different results. In fact, cells grown in media containing 4.5 and 1 g/l of glucose were relatively similar, with minor significant differences in the cell number compared to cells grown in 0 g/l of glucose.
To confirm the role of telomerase on mitochondrial metabolism, a WST-1 assay was performed on a cell line negative for human telomerase (Saos-2). The same pattern in the mitochondrial metabolism that was observed for MDA-MB 231 and MCF-7 cells was found in the osteosarcoma cell line Saos-2. The same experiments were extended to investigate the effect of BIBR 1532 under restricted glucose conditions. We began by examining the effect of this inhibitor on cell proliferation. The MDA-MB 231 cell number reduced to almost zero when the cells were treated with BIBR 1532. These results could be explained by an increased vulnerability of telomerase to its inhibitor in response to GR.
Mitochondria are important organelles specified in energy production and cellular calcium homeostasis, and the capacity to release apoptotic proteins (members of the Bcl-2 family) is of important consideration . Moreover, mitochondria are considered to be a major intracellular source of ROS. Succinate dehydrogenase (SDH), also known as quinone oxidoreductase, is a key regulator of metabolism and aerobic energy production. It couples the oxidation of succinate in the citric acid cycle to the reduction of ubiquinone in the electron transport chain, allowing cells to rely more on mitochondrial oxidative phosphorylation (OXPHOS) than aerobic glycolysis. Thus, this enzyme could reflect the activity of the mitochondrial respiratory chain . Cell oxidative stress is produced with increased OXPHOS. These ROS have been implicated as second messengers in multiple signaling pathways, especially in apoptosis, as indicated by many reports . We suggest that cells become more dependent on the Warburg effect than on OXPHOS after reducing the dehydrogenase activity via BIBR 1532 in 1 and 4.5 g/L of glucose. Aerobic glycolysis allows cells to rapidly multiply and contributes to ROS detoxification because carbons are diverted into the pentose phosphate pathway (PPP). This mechanism helps cancer cells survive under oxidative stress .
In MDA-MB 231, a high BIBR 1532 concentration did not stimulate apoptosis, as assessed by caspase-3 measurement, except for cells grown in 0 g/l of glucose independently of SDH activity modulation. This result could be explained by the extra-telomeric function of telomerase, i.e. its anti-apoptotic role . The remaining telomerase activity (~20%) could sustain the proliferative capacity of cells grown in low glucose. The additive reduction activity of telomerase enhanced by BIBR 1532 treatment rendered cells more susceptible to apoptotic signals triggered by this inhibitor and was sufficient to induce caspase-3 expression in MDA-MB 231 cells. The absence of apoptosis mediated by the telomerase inhibition in MCF-7 cells is likely due to the up-regulation of hTERT by the estrogen receptor . Further studies should be conducted to examine the effect of BIBR 1532-mediated telomerase inhibition in the MCF-7 cell line.
Our results again suggest the importance of GR to inhibit telomerase action; an additive effect between GR and telomerase inhibition by BIBR 1532 is also evident in apoptosis initiation in the triple-negative cell line MDA-MB 231.
This report demonstrated that the telomerase inhibitor BIBR 1532 exhibits an apoptotic effect via telomerase inhibition that is potentiated by restricted glucose conditions.
Materials and methods
Cell culture and glucose restriction
The established human triple-negative breast carcinoma cells (MDA-MB 231), human-estrogen positive breast carcinoma cells (MCF-7) and human negative telomerase osteosarcoma cells (saos-2) were purchased from ATCC (American Type Culture Collection). All MDA-MB 231, MCF-7, and Saos-2 cells were cultured in DMEM (Dulbecco’s modified eagle’s media, Sigma-Aldrich) at different glucose concentrations (0, 1 and 4.5 g/l) and supplemented with 10% FBS (fetal bovine serum, Sigma-Aldrich, Taufkirchen, Germany), 1% Pen-Strep (penicillin-streptomycin, Sigma-Aldrich, Taufkirchen, Germany) and 2 mM glutamine (Sigma-Aldrich, Taufkirchen, Germany) and maintained in a humidified atmosphere of 5% CO2 at 37°C. The cells were pre-incubated for five days in the appropriate media, and treatment occurred when cells reached 80% confluence.
MDA-MB 231 AND MCF-7 cells were seeded at very low densities in 25 cm2 flasks and grown in DMEM culture media containing 10% FBS and 1% Pen/Strep at different glucose concentrations with or without 2.5 μM of BIBR 1532. The medium was replaced every two days, and fresh telomerase inhibitor was added each time. At the end of each week, the cells were split and counted at least three times using bromophenol blue and a hemocytometer. Figures 3a and b show the effect of GR and treatment with 2.5 μM BIBR 1532 on MDA-MB 231 and MCF-7 cells by calculating the obtained cell number after each count on those seeded.
Cell viability and mitochondrial metabolism assay
The cell proliferation was evaluated using WST-1 (Takara Bio Inc, Ostu, Shiga, Japan), a tetrazoluim salt, cleaved into formazan by succinate-tetrazolium reductase, which exists only in the mitochondrial respiratory chain and is active only in viable cells. The production of formazan is directly related to the cell number due to the active mitochondrial metabolism and to its absorbance, which was measured using an ELISA reader at 450 nm. In our study, cells grown in different glucose concentrations were seeded in 96-well plates at a density of 104 cells per well and allowed to adhere for 24 hours, and the assay was performed following the manufacturer’s instructions.
Telomerase activity assay
MDA-MB 231 and MCF-7 extracts, from 1.5 million cells for each condition, were prepared according to the manufacturer’s protocol using a Quantitative Telomerase Detection Kit (Allied Biotech, Inc Vallego, California, USA). Briefly, the cell pellets were washed twice with cold PBS, and the cells were then lysed with an appropriate volume of the provided lysis buffer. After 30 minutes of incubation on ice, the suspension was centrifuged for 30 min at 4°C and 12000 × g. The supernatant was then aliquoted to further assess the telomerase activity and protein concentration.
The telomerase activity was measured by Real-Time Polymerase Chain Reaction (PCR) (Applied Biosystem, USA), which is based on the ability of telomerase in the cell extracts to synthesize telomeric repeats onto an oligonucleotide substrate, and the resultant extended products are subsequently amplified by PCR. The generated PCR products were then visualized using the highly sensitive DNA fluorochrome SYBR Green. The PCR products were detected following the binding of SYBR Green dye to double-strand DNA.
Detection of hTERT and hTR gene expression
Total RNA from MDA-MB 231 and MCF-7 cells was extracted using THE QIAamp RNA extraction Kit (Qiagen Inc., Valencia, CA, USA). Complementary DNA (cDNA) was synthesized from 0.1 to 2.5 μg of total RNA in a 20-μl reaction using the Omniscript Reverse Transcription Kit (Qiagen Inc., Valencia, CA, USA). The expression of hTERT and GAPDH was assessed using the multiplex hTERT/GADPH PCR technique and specific primers. The primers were defined as follows: hTERT: 5′-CGGAAGAGTGTCTGGAGCAA-3′, 5′-GGATGAAGCGGAGTCTGGA-3′; hTR: 5′-GCGGAAGACAGTGGTGAACT-3′, 5′-AGCTGGAGTAGTCGCTCTGC-3′; GAPDH (Glyceraldehyde 3-phosphate dehydrogenase): 5′-TGGGATGGACTGTGGTCATGAG-3′, 5′-ACTGGCGTCTTCACCACCATGG3-′, which was used as the internal control. PCR amplification was performed using Dream Taq Green PCR 2× Master Mix (Fermentas, USA), and the reactions were subjected to 40 PCR cycles (BioRad, Germany) of 94˚C for 30 seconds, 60˚C for 30 seconds, and 70˚C for 30 seconds followed by 7 minutes of extension at 72˚C. The PCR products were separated via 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining using the UVP BioDoc system (UVP, England).
The protein concentration was measured with the Bradford method according to the manufacturer’s recommendations using an acidic solution of Coomassie Blue G-250 dye, which is a reagent in the Bio-Rad Protein Assay, and BSA, which served as the standard. Serial dilutions of BSA at known concentrations were prepared. Five milliliters of Coomassie Blue solution (5× diluted) were added to the diluted proteins. The absorbance of the colored solution was measured with a spectrophotometer at 595 nm.
Caspase-3 apoptosis assay
The caspase-3 level was determined using a caspase-3 (active) human ELISA Kit from Invitrogen. Briefly, MDA-MB 23, MCF-7 and saos-2 cells were seeded in 6-well plates in DMEM at different levels of GR and treated with 50 μM BIBR 1532. After 48 hours of treatment, the cells were collected in cold phosphate buffered saline 1 × (PBS) and lysed with cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF and protease inhibitor cocktail). The cell extracts were then used to determine the concentration of caspase-3. The ELISA method was based on the fixation of cleaved caspase-3 at Asp 175/Ser 176 between two antibodies. This enzyme was detected after a detection antibody bound to the second antibody, followed by adding a substrate solution that was acted upon by the bound enzyme to produce color. The O.D. (optical density) of this colored product was measured at 450 nm with an ELISA Reader (Thermo Scientific, USA), and this O.D. is directly proportional to the concentration of human active caspase-3 present in the sample.
Annexin V-FITC apoptosis assay
The FITC annexin V apoptosis detection kit (Abcam) was used following the manufacturer’s instructions. MDA-MB 231 and MCF-7 cells grown in different glucose concentrations were plated at a density of 2 × 105 cells/well on six-well plates. After 48 h of treatment with 50 μM BIBR 1532, the cells were trypsinized and collected in ice cold PBS. The cells were washed twice with cold PBS and re-suspended in 500 μL binding buffer containing 5 μL Annexin V-FITC. The signals were detected using a FACScan flow cytometer (MACSquant, Germany) with excitation and emission settings of 452 nm.
TERT siRNA transfection
MDA-MB 231 and MCF-7 cells were maintained in DMEM at different concentrations of glucose and plated in 96-well plates at a density of 2 × 104 cells per well. Prior to seeding, 5 μL of specific siRNA, Hi-perfect (Qiagen) and serum-free media were added into each well and incubated for 10 minutes at ambient temperature. One hundred microliters of cell suspension were added to each well to initiate a treatment of 72 h. Two siRNAs were used in this assay; the first was TERT3, which targets the mRNA of the sub-catalytic unit of telomerase and the second was Death, which targets expressed human genes that are indispensable for cell survival. This siRNA was used as a positive control because it induces cell death that was visible by light Microscopy. The siRNA sequences were designed with the RNAi Human/Mouse Starter Kit (Qiagen Inc., Valencia, CA, USA). For hTERT, the siRNA sense sequence was 5′-GGAGCAAGUUGCAAAGCAUTT-3′ and the anti-sense was 5′-AUGCUUUGCAACUUGCUCCAG-3′, the target sequence was 5′-CTGGAGCAAGTTGCAAAGCAT-3′. Transfection monitoring was assured by the cell death control siRNA, which is a blend of siRNAs that target human genes essential for cell survival.
The statistical analysis was performed with t-test using the online statistical software Graph-Pad Quickcalcs (http://www.graphpad.com/quickcalcs/ttest1.cfm).
This project was approved by the Ethical Committee of the Faculty of Medicine of Saint-Joseph University, number 53/2012.
Human telomerase reverse transcriptase
Human template RNA
Reactive oxygen species
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Jiang W, Zhu Z, Thompson HJ: Dietary energy restriction modulates the activity of AMP-activated protein kinase, Akt, and mammalian target of rapamycin in mammary carcinomas, mammary gland, and liver. Cancer Res. 2008, 68: 5492-5499. 10.1158/0008-5472.CAN-07-6721.
Pollak M: Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer. 2008, 8: 915-928. 10.1038/nrc2536.
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Pascolo E, Wenz C, Lingner J, Hauel N, Priepke H, Kauffmann I, Garin-Chesa P, Rettig WJ, Damm K, Schnapp A: Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate. J Biol Chem. 2002, 277: 15566-15572. 10.1074/jbc.M201266200.
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Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang J, Shen M, Bellinger G, Sasaki AT, Locasale JW, Auld DS, Thomas CJ, Vander Heiden MG, Cantley LC: Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science. 2011, 334: 1278-1283. 10.1126/science.1211485.
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We are very thankful to Dr. Rabih Talhouk at the American University in Beirut for providing the MDA-MB 231 and MCF-7 breast cancer cell lines.
We state here that none of our authors has financial or other competing interests that might be construed as influencing the results or interpretation of our study.
LW carried out the cell culture, the telomerase activity measurement, and the mitochondrial metabolism experiments and drafted the paper. NA, IR and RS contributed to the design of the study, drafted the paper and contributed to the discussion. RS performed the ELISAs experiments. CK performed the flow cytometry experiments. GH proposed the original idea of this study, designed the experiments and drafted the paper. All authors read and approved the final manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Wardi, L., Alaaeddine, N., Raad, I. et al. Glucose restriction decreases telomerase activity and enhances its inhibitor response on breast cancer cells: possible extra-telomerase role of BIBR 1532. Cancer Cell Int 14, 60 (2014). https://doi.org/10.1186/1475-2867-14-60


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SafeCare BC Hosts National Alliance Meeting: Memorandum of Understanding to Guide Future Collaboration
September 21, 2016 | News
Leadership is a key element of this year’s Be Care Aware campaign and this weekend, SafeCare BC took it to the national level as host of the National Alliance for Safety and Health in Healthcare (NASHH) annual meeting.
NASHH includes representatives of health care safety associations from Alberta, Saskatchewan, Ontario, Quebec, New Brunswick and Nova Scotia. This year’s meeting included the participation of WorkSafe BC, SafeWork Manitoba, Canadian Association for Long Term Care and the Saskatchewan Workers Compensation Board. Among other things, the Alliance focused on long term care injury rates across Canada, violence prevention, accreditation, certification and health human resources.
The Alliance’s work will be guided by a Memorandum of Understanding (MOU) to raise awareness of high injury rates in long term care and share best practices to address this growing national challenge. Watch for a launch of the Alliance’s web-site in the coming weeks and more updates in the year ahead. Click here for a link to the MOU.

domain: health


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October 26, 20175:02 AM ET
Heard on Morning Edition
President Trump declared a public health emergency to deal with the opioid epidemic Thursday, freeing up some resources for treatment. More than 140 Americans die every day from an opioid overdose, according to the Centers for Disease Control and Prevention.
“We are currently dealing with the worst drug crisis in American history,” Trump said, adding, “it’s just been so long in the making. Addressing it will require all of our effort.”
“We can be the generation that ends the opioid epidemic,” he said.
Trump also directed agency and department heads to use all appropriate emergency authorities to reduce the number of deaths caused by the opioid crisis.
Trump spoke personally about his brother Fred who struggled with alcoholism and died in his 40s. Because of him, Trump said, he had never tried alcohol or cigarettes. “He would tell me ‘don’t drink, don’t drink,’ ” Trump said. “He had a very, very, very tough life because of alcohol.”
The administration will also launch an ad campaign so that young people can see “the devastation and ruination [drugs cause] people and people’s lives.”
Dr. Andrew Kolodny, co-director of the Opioid Policy Research Collaboration at Brandeis University’s Heller School, calls the announcement “very disappointing.” Without funding for new addiction treatment, he says, declaring a public health emergency isn’t enough. “This is not a plan,” he says. “The administration still has no plan” for dealing with opioids, he says.

domain: news


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For computer gaming, I have a mac. Does anybody know of good hunting games available for it? There is a freemium option in the app store for Deer Hunter Reloaded.
There seems to be a pretty realistic hunting game out there for the PC called "The Hunter." Are there any games like that available for Mac or even PS3 that you guys know of? Thanks.

domain: entertainment


Jeff Sides caught shots from all 17 3200s at the Hoka One One Dash for Doobie!
Live Twitter updates from Dash for Doobie!
Two loaded fields are set to take place tomorrow at the Dash for Doobie!
Meet records look to be broken this weekend at Dash For Doobie, though we wonder if one of the records is possibly unbreakable.
Heat sheets are out for the Hoka One One Dash For Doobie!
The 10th Annual Hoka One One Dash for Doobie looks to be the best one yet!


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Significant Figures Chemistry Worksheets Math Classroom Decimals.
Zeros that in front of nonzero are not. zeros at the end of the number and to the right of a decimal are significant. zeros Significant figures in chemistry this is a series of lectures in videos covering chemistry topics taught in high schools. review the rules of significant figures and apply the rules in calculations.
Fun engaging students review significant figure rules scientific method lesson plans chemistry teacher prep. Significant figures practice worksheet answer key persuasive writing prompts word problem equations phonics worksheets. Dimensional analysis worksheet stem sheets chemistry worksheets relationship. Scientific notation chart publishing teaching chemistry math methods. Significant figures worksheet chemistry lovely practice practices worksheets persuasive writing prompts. Scientific notation worksheet chemistry worksheets. Profile.

domain: chemistry


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- Gender and Relationships»
- Relationship Problems & Advice
Jealousy or envy, whatever you call it, is powerfully detrimental to your well being
How to control jealousy and not let it control you
“Look at me, look at me!!!” shouts the five year old girl as she swings higher than your head on the swing set.
You think about when you were that age and doing good to get four feet off the ground without having a panic attack that you were going to fall out and crack your head open.
Your friend posts pictures of backstage passes to a Lady Gaga concert on her Facebook page along with her picture hugging the dame and playing air guitar with the bassist as she boasts about her rockin' night with the crew. Instead of getting thousands of posts about how wonderful it is that she got to see a singing legend, the comments section is empty and while you know you should tell her how great it is, you stay silent too.
Your ex shows up at a function he knows you will be attending and brings his new girl friend. They hang all over each other. She is gorgeous and a good thirty pounds slimmer than you and younger too with breast cleavage big enough to hide a wallet in.
Logically you know it makes no sense to be jealous of any of the above and if you are like most people you are embarrassed to admit that you feel diminished by other people’s accomplishments, especially the five year old on the swing.
It's just petty to be that concerned over what other people do and feel jealous that you can never do the same things, but yet, you still feel that way, so what's up with that?
You want to take the high road and be glad for others accomplishments, but instead you find yourself feeling a bit hurt and slighted and evaluating yourself and wondering what it is you are doing wrong to not have the kind of life that others have.
It is hard to take the high road when you feel people are purposefully trying to make you look bad and themselves look good though.
Jealousy, like anger stirs up a flood of biochemical reactions in the brain that we have to struggle to suppress and if we only suppress them and fail to deal with why we have them in the first place, they will resurface even stronger the next time we feel like someone is trying to boost themselves up while using us as a springboard.
A 2009 article in Psychology Today actually suggested that jealousy was a survival mechanism and not merely a flaw in character.
Richard Smith, professor of psychology at the University of Kentucky states that we feel jealous when we feel that someone is trying to take away something that is rightfully ours. Of course he feels that jealousy is only felt when it involves a love interest, but those of us who have felt jealous, know that we can feel that way over job privileges, siblings, even Christmas gifts and birthday parties.
If your brother gets a remote race car for his birthday and a party at the skating center and you get a new shirt and a cupcake with a candle in it, you will probably not only feel slighted, but have pangs of jealousy toward your brother even though he did not intentionally do anything to hurt you.
You can be jealous of someone who has talents you want, or friends you desire to have yourself. There are numerous stories of women trying to steal other people's babies because they are so jealous that someone else has a child and they cannot have one.
Wars start over jealousy, when one nation seems to have more than any other and does not want to share because they think they deserved what they got and so others with nothing did not deserve to have any better.
People who go "postal" and shoot up schools and work places often do so because they are jealous that others are given more, while they are made to feel unworthy and have had things taken away from them by the very same people who feel justified to make themselves look better while ignoring the needs of any other.
The dictionary defines jealousy as resentment against a rival or a person; particularly one enjoying success and having an advantage over us. The survival instinct tells us that we must level the playing field and shift the advantage back toward ourselves or we will cease to exist and our competition will take over and push us out.
Jealously usually leads to resentment and if not dealt with, anger and attack on the person we feel is taking away something that is rightfully ours. This is when it becomes a problem that we need to control.
So, how does knowing jealousy is a defense mechanism designed to help us survive, give us insight into how to control it?.
First of all you have to determine whether what you are feeling is envy or jealousy as they are not quite the same and need to be dealt with a little differently.
Technically envy is worse than jealousy when it involves feelings of inferiority and wanting to retaliate. Envy is often seen as bitterness and longing to have what someone else has whether you deserve to have it or not.
Jealousy is wanting to keep what you already have and prevent it from being taken away by a rival. If you have ever had a specific talent in a group that no one else has and are highly valued for that talent and then a new person comes in who is even more talented and is now asked to do all those things you did in the past and enjoyed doing, you are going to be hard pressed not to feel jealous.
You may even feel that you are being pushed out of the group and are no longer wanted, which can be devastating to your well being, but there are some steps you can take to counter these feelings of jealousy to varying degrees of success.
1. When you are feeling your self esteem slipping away, write down a list of all the good things about yourself.
If you are kind and gentle, meek and mild mannered, always willing to help someone in need, these are the best characteristics to focus on.
Try to avoid superficial things, like, I’m pretty and smart, and thin and have lots of friends. These things tend to fit in that envy category and instead of helping you overcome your jealousy, it will only make it worse.
2. Remind yourself of what is really important to you. Do you really want to be rich and famous and talented, or are you okay with being generous, always there when your friends need you, willing to try your hand at anything even though it might not look as good or turn out as well as someone with more talent.
3. How badly do you need the attention of others to feel good about yourself? This is a big one for me. I don’t like getting praise, but I love it when people appreciate the things I do for them and say a simple, “Thank you, that was nice.”
If people stop acknowledging me and take me for granted or worse yet abuse me and take what they can get without ever showing gratitude, then I tend to get upset with them, but instead of being rude and wanting to strike out at them, I remind myself to use their behaviors to remind me not to do the same thing to others and indeed, to go out of my way to thank someone for a kindness they have done and let them know how much I appreciate them and everything they do.
This way you turn a potential retaliation desire into a desire to do unto others as you would like done unto you, rather than do unto others what was actually done to you and caused you distress.
This not only builds self esteem, but boosts the esteem of others. The big thing is to say thank you genuinely and not just to make yourself look like the boss thanking his or her loyal followers who dutifully serve their wonderful leader.
4. Recognize why you feel so upset. Technically the person that steals your thunder and takes away the thing that is so special to you is a thief and deserves to be punished or at least prevented from doing more harm. This is where that survival instinct kicks in.
In times like this, just ask yourself, “Did I really want to be in the spotlight or was I happier in the background?” and “Am I so desperate that I need someone to praise me to feel good about myself or can I look at everything I have accomplished in life and be happy that I had the opportunity and ability to do those things, even if no one else really noticed but me?”
Sure, the majority of us would like to have money and lots of friends, and a nice new home, well behaved children and the physical health to do anything we desire, but just because we don't have those things and someone else keeps flaunting those things in our faces, does not mean we would be happier if we were the flaunters and they were us!
Chances are they have lower self esteem than we do and they need all those things to make them feel good about who they are.
If you can get that kind of thinking into your head and feel sorry for people who try to make you look small, rather than envy them and want to retaliate, you can begin to overcome jealousy and take power over your own emotions.
If someone close to you really cuts you deep and does nothing but brag on how wonderful they are and how great their life is compared to others, feel free to gently remind them that it is great that they have all these nice things, but that some people do not and this is not because they did not work hard enough or desire to have nice things and just sat around being lazy and making unwise choices. Encourage them to be sensitive toward others and share their talents rather than brag about them.
If someone has a $100,000 Mercedes, rather than brag about how fast and dependably built it is, why not offer to take the woman down the street to the grocery store rather than speed past her as she waits in the rain at the bus stop? This would be more impressive to most people and would definitely set a better example than being prideful.
If you have not had a vacation in five years, you drive a beat up old car and can barely pay the rent each month and your friend comes back from the nail salon and tells you she has just booked a cruise to the Bahamas and she’s taking her new man, a wealthy doctor with his own yacht with her and too bad you don’t have any money and can’t do anything fun, rather than get jealous or envious or pray for a hurricane to blow her and lover boy out of the ocean, focus on something good in your life or even volunteer to help someone less fortunate than you.
That way instead of feeling left behind, you can feel lifted up and truly blessed to have the wonderful life you have.
When you feel jealous, look for the good in yourself and the things that really matter to you. Acknowledge that it hurts to see other people get the attention and recognition you want, and hurts even more when people purposefully push you away in order to take everything good for themselves and deny you a shot at it, but understand this is their character flaw and not yours. Try to have sympathy for their insecurities rather than being insecure yourself and wanting to strike back at them.
Perhaps the best way not to feel jealous is to not be prideful or boastful yourself. If you are humble and thankful you will rarely give people a reason to be jealous of you and will rarely be jealous of someone else.
It is only when we start to feel like no one likes us and no one cares and that we are not as good as other people that the jealousy really starts to take an ugly turn and drag us down a path we really know we shouldn't go.
If people are not praising you enough for your liking, then start praising them more. If you feel like they are trying to out compete you, stop competing with them. Let them compete against themselves. That way they can be their own burden and not yours.
If someone is trying to run faster and further than you, drop back and offer to help a slower runner along with words of encouragement. If you are the slowest runner, enjoy the scenery and know that you are inspiring a passerby to get out and run as they think, "well, if that fat old lady/man can run, why can't I?"
Find the humor in all things. If you can laugh at yourself and focus on your own improvements, then you won't feel so competitive or left behind.
If someone is trying to steal your spouse, ask your spouse how they feel about the person and if they seem attracted to all the attention, ask them would they prefer to go be with that person over you. If they say no, then tell them that they need to set boundaries with that person and that you will gladly give them all the attention they want and more. Chances are that is all it takes to break the other person's hold over your significant other.
You have the power to prevent jealousy from making you a bad person and retaliating and acting the same way the person you are jealous of is acting, but it does take some work to get to that point and having the support of friends and family who love you for who you are makes it a lot easier to get there!

domain: psychology


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A national atheist group has joined the effort to convince President Barack Obama to designate the Islamic State's persecution of Christians and other minority groups in the Middle East a "genocide."
The Center for Inquiry, a national nonprofit atheist group, joined the International Religious Freedom Roundtable in asking the Obama administration to properly label the atrocities committed by the Islamic State.
Michael De Dora, director of public policy at the Center for Inquiry, told The Christian Post on Thursday that there are several reasons why his group has chosen to support the International Religious Freedom Roundtable.
"As a organization driven by humanist values, CFI believes that the right to freedom of conscience is a fundamental and universal liberty — including the rights to freedom of religion, belief, thought, and expression. We strive to protect these rights for all individuals and groups, especially those facing threats or violence," De Dora, who also serves as the United Nations representative for his group, told CP.
"We have been distressed to read reports of ISIS' systematic destruction of ancient religious minority communities in Iraq and Syria, and believe the international community must come together in a spirit of goodwill — regardless of theological differences — and strongly engage in efforts to defeat ISIS, and aid their victims," De Dora added.
The public policy director went on to argue that the Islamic State's crimes clearly fit the United Nations' definition of genocide.
"[...] the simple fact is that many, if not most, of ISIS's crimes amount to genocide as defined by the UN. Convention on the Prevention and Punishment of the Crime of Genocide. ISIS's campaign against Christians, Yazidis, Shia Muslims, Shia Turkmen, and Shabak — including persecution, enslavement, and outright extermination — clearly fits the UN definition," De Dora said, adding that "even ISIS's crimes which are not necessarily aimed at outright extermination — including intimidation, oppression, and coercion — cause grave harm to religious minorities and are clearly intended to effectively destroy their communities. This would also qualify as genocide under the UN's definition."
The International Religious Freedom Roundtable describes itself as an "informal, non-governmental group of religious leaders and human rights activists" that includes over 100 non-governmental organizations, religious leaders, and human rights activists.
The group sent a February 17 letter to President Obama explaining why the U.S. government should label ISIS's actions as a genocide, pointing to the Islamic State's "reign of terror and destruction" against "religious minority communities in Iraq and Syria," including Christians, Yazidis, and the Shabak people.
"We believe that without a formal declaration the U.S. Government has abdicated its duty and vow to confront the most heinous of human rights violations, namely genocide," the letter, a copy of which was obtained by The Christian Post, reads.
Several prominent religious leaders and advocacy groups have signed the letter, including Tony Perkins, president of the Family Research Council, Russell Moore, president of the Southern Baptist Ethics and Religious Liberty Commission, and Thomas Farr, director of Georgetown University's Religious Freedom Project, among others.
The letter goes on to reference numerous instances in which the Islamic State has admitted to its desire to wipe out minority religious populations, as seen through features in its Dabiq propaganda magazine.
In the magazine's October 2014 issue, the terrorist group shows a photo of an Islamic State black flag flying over the Vatican with the threat: "We will conquer your Rome, break your crosses, and enslave your women, by the permission of Allah, the Exalted. This is His promise to us; He is glorified and He does not fail in His promise."
Along with human rights groups and religious leaders, political groups have also called on the Obama administration to use stronger language when discussing the Islamic State's destruction.
In March 2015, the United Nations released a report suggesting that the International Criminal Court should prosecute the Islamic State for trying to wipe out the minority Yazidi population.
"Clearly international war crimes and crimes against humanity and possibly genocide appear to have been committed during this conflict. The genocide part relates particularly to the Yazidis," Hanny Megally, chief of the Asia, Pacific, Middle East and North Africa branch of the U.N. Human Rights Office, said in a statement announcing the report, according to Reuters.
Earlier this month, the European Union passed a resolution that also called on the International Criminal Court to prosecute Islamic State militants for acts of genocide.
"[ISIS] is committing genocide against Christians and Yazidis, and other religious and ethnic minorities, who do not agree with the so-called ISIS/Daesh interpretation of Islam," the resolution reads.
The Obama administration has sought to defend its refusal to label ISIS actions as a genocide, with White House Press Secretary Josh Earnest saying earlier this month that such a label would pose legal issues.
"There are lawyers considering whether or not that term can be properly applied in this scenario. What is clear and what is undeniable and what the president has now said twice in the last 24 hours is that we know that there are religious minorities in Iraq and in Syria, including Christians, that are being targeted by ISIL terrorists because of their religion and that attack on religious minorities is an attack on all people of faith and it is important for all of us to stand up and speak out about it," Earnest said during a Feb. 4 press conference.
"This is an open question and one that continues to be considered by administration lawyers," Earnest added.


From The Washington Times: http://www.washingtontimes.com/news/2016/may/15/tom-gresham-why-hillary-clinton-cant-ban-ar-15-rif/
She should learn something about the guns and the fear she clings to
Sunday, May 15, 2016
At every opportunity, Hillary Clinton attacks, denigrates and marginalizes roughly a third of the American population — the 100 million who own firearms — with her promises to “do something” about this country’s “gun culture,” to reverse the Supreme Court’s decision in the Heller case that individual citizens have the right to own guns, to remove the protection from junk lawsuits Congress gave the firearms industry when it was faced with an organized assault (30-plus lawsuits) designed to put all gun companies out of business, and to bring back and expand the Clinton ban on the most popular firearms in America — semi-automatic rifles, best exemplified by the AR-15.
The problem is that neither she, nor Congress, can ban this ubiquitous rifle, which is owned and used by millions of Americans for hunting, competition and recreational shooting.
Before getting into the “why” she can’t ban these rifles, it’s critical to review exactly what the AR-15 model is, as well as what it is not.
The largely-ignorant media still willfully misinforms the public by calling this rifle an “assault weapon.” Duped by the gun-ban industry into repeating a term with no definition, but which was created with the express intent of confusing and misleading the public into banning this gun, the media becomes part of the gun-ban movement each time it repeats the scary-but-meaningless term “assault weapon.”
Josh Sugarman, executive director and founder of the Violence Policy Center, a tiny gun-ban group, is credited with creating the term as part of the effort to ban yet another slice of firearms, when he famously said, “Assault weapons’ menacing looks, coupled with the public’s confusion over fully-automatic machine guns versus semi-automatic assault weapons — anything that looks like a machine gun is assumed to be a machine gun — can only increase the chance of public support for restrictions on these weapons.”
Note the admission that these rifles are not machine guns. Ask almost anyone who is not firearms knowledgeable, however, what guns the now-defunct Clinton Gun Ban (the so-called “assault weapon” ban) of 1994 banned, and the response likely will be “machine guns.”
So, what was included? Basic, auto-loading (also called semi-automatic) rifles. They fire only once with each pull of the trigger. President Teddy Roosevelt hunted with a semi-automatic rifle. My first deer hunt, in 1962, was with a semi-automatic rifle, when I was 11 years old. There is nothing new, nor sinister, about self-loading rifles. What was new, however, was the interest in the semi-auto version of the M-16 rifle used by the military, and the opportunity to confuse the public because of these similar looks.
Continue reading at: http://www.washingtontimes.com/news/2016/may/15/tom-gresham-why-hillary-clinton-cant-ban-ar-15-rif/


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Handmade Upcycled Sari Rag Wreath
A beautiful rag wreath made from sari offcuts. Guaranteed to add colour to any festive occasion! The wreaths all use upcycled sari waste and hand cut by underprivileged women in India who have been trained in tailoring.
A sustainable way to decorate your home and welcome the festive season!
Each one is completely unique. Please see images to select your preferred colour way.
Size 35 x 35cm (14 x 14")
Share this Product
A pretty alternative to a traditional wreath.
The wreath is absolutely gorgeous - so different and can be used for Christmas with the hanger that it came with or if you want to, kept up permanently 💕
We are so thrilled with our recycled wreath it ticks all the boxes, gorgeous, reusable and helping others and looks absolutely perfect on the front door.
Thank you so much! So delighted that you're thrilled with the wreath and that it looks good on your door. A very merry Christmas to you :)
I ordered a new wreath for my front door as my normal Christmas wreath was falling to bits! I chose a wreath with lots of greens in it to match our front door - and am delighted with it. It hangs beautifully on the hanger that was sent from PriPri so it was super easy to put up and it looks beautiful. It makes me think of the amazing women in India who we are helping when we shop at PriPri and makes me feel that I am shopping responsibly. Thank you!
I am absolutely thrilled with my sari wreath. It is beautifully made, and the colours are stunning. The mix of fabrics is wonderful. Delivery was really prompt, and all packed really carefully with no waste. The wreath came with a really clever door hanger too, which I was not expecting. An item of beauty for years to come.

domain: design


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- Wegmans said its scan app will be discontinued due to high losses.
- The app, which is used with self-checkout, was introduced in 2020.
- Sam's Club, Walmart, and Tops have similar scanning apps still in use.
Wegmans told customers on Monday that they will no longer be allowed to use the Scan app, which allows shoppers to scan groceries as they go using a phone and pay at self-checkout.
The Scan app was hurting business, the company said in a September 12 email to customers, writing "unfortunately, the losses we are experiencing this program prevent us from continuing to make it available in its current state." Users will lose access on Sunday, September 18, president and CEO Colleen Wegman said in an email.
The Scan app was first introduced early on in the pandemic as a contactless checkout option and was available in nearly all of the chain's 107 locations across the eastern US. Wegmans offered customers a $5 digital coupon to adopt the app.
Shoplifting at self-checkout isn't unique to Wegmans, despite technological attempts to curb it, according to Loss Prevention Media. Some methods, like neglecting to scan some items, or inputting expensive items as cheaper ones, are ubiquitous on social media.
Self-checkout and handheld checkout devices are known to have greater losses for retailers, though those may be made up for in labor savings. Some major grocery retailers still have the option. Grocery giant Walmart has self-checkout, along with Scan and Go for Walmart+ members.
Sam's Club, owned by Walmart, also has Scan and Go. Some Costco locations have self-checkout, and grocery chain Tops told Buffalo News that it has no plans to discontinue its similar Shop and Scan program.
Wegmans said in a statement it will "continue to introduce new digital solutions to streamline your shopping experience for the future," but didn't offer any details on exactly what or when those might be.
Do you have a story to share about a retail or restaurant chain? Email this reporter at firstname.lastname@example.org.

domain: economics


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Book Club: Fever Pitch – Nick Hornby
'Fever Pitch' is widely regarded to be the best football book ever written. It's been made into two films, won the 1992 William Hill Sports Book of the Year award and inspired countless football fans around the UK to think more deeply about their football fandom. Its author, Nick Hornby, joins us in the studio for today's episode of the Book Club!
Nick sits down with Kate and Jim to remember the seismic impact of 'Fever Pitch', nearly thirty years after its initial publication. Nick's footballing obsession embedding itself within every aspect of his life and the book encapsulates the joy, frustration, hopelessness and deliriousness felt by a generation of football fans on the terraces every Saturday. From his deep love for Arsène Wenger to the state of the modern game, it's a fascinating chat with one of the forefathers of modern football writing!
You can purchase the latest edition of 'Fever Pitch' here.
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domain: literature


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Giving evidence to the Home Affairs committee in Parliament today, immigration minister Caroline Nokes suggested that employers may have to perform their own checks on EU citizens’ right to work in the UK post-Brexit, despite the fact that the Government’s proposed new ‘settled status’ scheme has not yet been fully rolled out.
Commenting, Stephen Doughty MP, member of the Home Affairs committee and leading supporter of a People’s Vote, said:
“The Government has treated the three million EU citizens living and working in the UK abysmally since the very start of the Brexit process. These people, who are our friends, our neighbours, our colleagues and in some cases our family, have had to put up with two years of uncertainty and worry about what their rights will be in the future, and based on today’s revelations, that uncertainty looks set to continue.
“Ministers have singularly failed to assuage the fears and concerns of EU citizens in the UK, and the proposed new ‘settled status’ scheme is already descending into a shambles – if you have an iPhone, for example, you can’t even complete the application, even though this problem was first raised months ago.
“And based on the evidence given today by Caroline Nokes, a minister responsible for immigration, employers and landlords may have no way of assessing whether individual EU citizens have the right to live and work in the UK post-Brexit. The warning signs are already there – with the Brexit clock ticking, the Government has not got a grip of this at all.
“People were promised an efficient new immigration system if they voted to leave the EU, but as with so much of Brexit, what’s being delivered doesn’t match up to what was promised – it’s a mess. That’s another reason why more and more people are calling for a People’s Vote on whatever emerges from the Brexit process – so that the people can have the final say.”

domain: sociology


Although most skin care companies will want to let you know how badly your skin can age if you don't take care of yourself - or in other words use their products. A Norwegian pharmacy has taken a more honest approach to its latest campaign which paints growing older in an entirely different light.
Their answer? a fascinating and quite poignant video titled 'A Lifetime in 60 Seconds' in which we see a frank and unaltered look at how our skin changes as we age from 0 - 100. The video was created in partnership with Vitus Apotek and ad agency T/A Pol and offers a refreshing take on showing the body just the way it is - no retouching involved. Check the video out below.
Here's a brief breakdown of the video:
Watch the full video here:


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ISU Softball Camps
Head Coach Cristal Brown
Cristal Brown was hired as the 8th head coach in Idaho State women's softball history. She comes to Pocatello after spending the last eight years as a Division III head coach at Whitworth.
She joined the Whitworth program in 2011 and after four seasons as head coach, Brown led the Pirates to three consecutive Northwest Conference regular season titles (2015-17). The 2015 season was the program's first-ever NWC regular season championship title as the team had an overall record of 31-12 and 21-7 in conference play which was a (then) school record for most victories in a season.
Following the 2016 NWC championship title, Brown led the program to their first NCAA Division III regional tournament appearance. The 2017 season would be the Pirates third consecutive NWC regular season championship, the first NWC tournament championship, and their second consecutive NCAA post-season tournament appearance. Whitworth went 2-2 in the NCAA regional tournament, earning the school's first NCAA tournament victories and an overall season record of 37-12. With this outstanding performance, Brown was selected the conference Coach of the Year in 2016 and 2017.
During the 2018 season, the Pirates finished third in the conference with a 24-18 overall record and went 17-11 in conference play. In 2019, Brown led the program to a strong second place finish going 31-11 overall and 23-5 in the NWC.
In addition to her coaching duties, Brown was also the faculty representative for the university's SAAC program which involves student athletes in NCAA legislation and also many community service events. She emphasizes academic excellence as the program earned top 10 GPA in the nation in 2015 and top three in 2016.
In her combined eight years at Whitworth, Brown led her team to seven consecutive winning seasons and an overall record of 221 wins and 124 losses.
Through her first seven years, Brown had 30 players named All-Northwest Conference and 15 players receive NFCA All-Region honors. In 2016 Whitworth posted the third-highest team GPA in all of NCAA DIII softball and had 14 players recognized as scholar-athletes by the NFCA. In 2015 Megan John became the first CoSIDA Division III softball Academic All-American in school history.
Before her exceptional career at Whitworth, Brown was the head coach at Kingsburg High School for four seasons (2008-2011) in Kingsburg, California. During her time there, the Vikings went 101-29, won four Central Sequoia League championships, won a Central Section (CIF) crown in 2008, finished as Central Section runner-ups in 2009, and was a three-time Central Sequoia League Coach of the Year honoree.
Her coaching experience includes two seasons as the assistant varsity coach at Valencia High School where they were ranked number one in the nation as well as a two year stint as an assistant coach with The Master's College helping them attain a top 25 national ranking.
Brown was raised in California and is a former small-college All-American. She was an intimidating left-handed leadoff batter who utilized her wide range of skills. She could hit for power and also had the ability to slap her way on base using her speed. She was the Western State Conference Junior College Player of the Year in 1998 at Bakersfield JC before earning Second Team NAIA All-America honors at Azusa Pacific in 2000. During her time at Azusa she set single season records for games played (68), at-bats (234) and stolen bases (40 out of 44). Brown helped lead the Cougars to a 53-15 record, a GSAC crown and a runner-up finish at the NAIA national championship.
She received her Bachelor of Arts in History in 2001 from The Master's College in Santa Clarita California and went on to receive her Masters of Arts in Coaching and Athletic Admin in 2011 from Concordia University in Irvine, California.


Here is an outfit for the Coachella Festival. It is fun, cool and everything spring.
AE Slouchy Jean Vest
AE Spring Graphic T-Shirt
AE Khaki Boyfit Bermuda Short
Asos Leather Cross Body Bag With Embroidery And Fringing
AEO Western Bootie
AEO Turqouise Studded Cuff
AEO Bracelet Watch
AEO Tropical Print Sunglasses
Courtesy of ASOS and American Eagle Outfitters
Have you ever wondered after looking at a tag on your sweater, what is Angora Rabbit Hair? Angora Rabbit Hair comes from a breed of rabbit that sprang up in Turkey. Angora Rabbits are known for their “long, soft wool” and were pets of “French royalty” during the “18th century.” Almost everyone say that Angora Rabbit Fur is “softer than cashmere.”
They can live to be 10 years old or more and are only fed commercial rabbit food because it meets all of their nutritional requirements. There are four different breeds of Angora Rabbit.
The English breed is tightly spun and is very popular for show because of its astonishingly luxuriant fur on the face and ears. They weigh anywhere between 5 and 7.5 pounds. They are groomed daily.
The French breed is 7.5 – 10.5 pounds and has fur that can be colored more because of the guard ration of the fur. They get groomed once a week.
The Satin Angora breed weighs anywhere between 6.5 – 9.5 and has a “shiny and satiny” feel to its fur. This breed’s fur can also be colored and is recommended more by spinners because of how easy it is to spin it.
The Giant Angora breed can weigh 10 to 20 or more pounds. It is the largest breed and the fur has to be trimmed off carefully unlike the other breeds that shed or molt. They get the angora carefully by using scissors in what is called “scissor harvesting.” The Angora Rabbits are not harmed in any way and they enjoy the constant care.
The Angora Rabbit wool can be dyed different colors and are delightful to spinners because the wool is strong and soft at the same time allowing the spinner to finish the process faster. It is mixed with other fabrics including, cashmere and sheep’s wool no more than at a 30% ratio because if an item was made out of 100% Angora Rabbit, it would be too hot and the texture would be too delicate to wear. Angora wool holds dye color permanently better than other wool fabrics.
Courtesy of Ramapo.edu and Homestead.org


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Author Reading with Antonya Nelson
Antonya Nelson, the author of eight books of fiction, will present a reading on Saturday, September 22, 2012 at
7:00 pm in the Canyon Community Center in Springdale.
Her works include Female Trouble and the novels Talking in Bed, Nobody’s Girl, and Living to Tell. Nelson’s work has appeared in the New Yorker, Esquire, Harpers, Redbook, and in many other magazines, as well as in anthologies such as Prize Stories, the O. Henry Awards, and Best American Short Stories. Her books have been New York Times Notable Books of 1992, 1996, 1998, and 2000. The New Yorker called her one of the “twenty young fiction writers for the new millennium.” She is also the recent recipient of the Rea Award for Short Fiction and is a recipient of an NEA Grant 2000-01 and a Guggenheim Fellowship.
|Support for this event was provided by the Utah Arts and Museums Program.|
|Support for this event provided by The National Endowment for the Arts|


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There are few things more refreshing than a cold beer on a hot day. Australia is home to some of the best breweries in the world, so it’s no surprise that there are plenty of great places to enjoy a pint.
Here are 11 of the best places to get beer in Australia:
1. The Local Taphouse, Sydney
With over 50 taps and 200 different bottles available, The Local Taphouse is a paradise for beer lovers. They offer something for everyone, with a focus on local and international craft beers.
2. The Cambridge Hotel, Newcastle
The Cambridge Hotel has been serving up great beer since 1885. They have an impressive selection of local and imported beers, as well as a range of ciders and wine.
3. The Scratch Bar, Brisbane
The Scratch Bar is a must-visit for any beer lover visiting Brisbane. This funky bar has an ever-changing selection of craft beers on tap, so there’s always something new to try. And if you can’t make up your mind, the knowledgeable staff are always happy to help you choose the perfect beer for your taste. Hungry? The Scratch Bar also serves up some mouth-watering pizzas that are perfect for sharing.
4. Little Creatures, Fremantle
Little Creatures is one of Australia’s most popular breweries, and their Fremantle brewery is the perfect place to enjoy their beer. They offer a range of different styles, including pale ales, IPAs, and stouts.
5. The Wheaty, Adelaide
The Wheaty is a pub devoted to all things beer. They have an ever-changing selection of 16 taps, as well as hundreds of different bottles. They also offer a range of beer-related events, such as meet the brewer nights and beer tastings.
6. The Public Bar, Melbourne
The Public Bar is one of Melbourne’s most popular bars, and for good reason. They offer a great selection of beers, as well as a range of other drinks and activities.
7. The Taphouse, Hobart
The taphouse is Tasmania’s leading destination for craft beer. They have 18 taps featuring a rotating selection of local and international beers. They also offer a range of beer-themed events, such as meet the brewer nights and tasting paddles.
8. Biero Bar, Melbourne
For a truly unique beer drinking experience, head to Biero Bar in Melbourne. This intimate bar specializes in Belgian-style beers and offers over 100 different varieties to choose from. So whether you’re a fan of fruity lambics or rich and malty dubbels, you’ll find a beer to suit your taste here. And if you get hungry, Biero Bar also serves up some delicious Belgian snacks like cured meats and cheeses.
9. The Brewhouse, Perth
If you’re looking for a relaxed place to enjoy a few beers with friends, The Brewhouse in Perth is the perfect spot. This laid-back bar has a huge range of local and international craft beers on tap, as well as an impressive bottle list. And if you get peckish, there’s a great selection of pub grub available too.
10. The Tavern, Hobart
The Tavern is a must-visit for any beer lover visiting Hobart. This historic pub dates back to 1824, and it’s the perfect place to enjoy a pint or two in front of an open fire. With a huge range of local and international beers on tap, as well as an impressive bottle list, you’ll be spoilt for choice here. And if you get peckish, the pub grub is worth trying.
11. The Hopscotch Bar, Darwin
The Hopscotch Bar is the perfect place to enjoy a beer or two in Darwin. With 24 taps pouring local and international craft beers, there’s something for everyone here. And if you get hungry, the bar also serves up some delicious pub grub like burgers and wings.
Looking for more places, check out Mornington pale.

domain: geography


Quality Switch recently opened the “Horace H. Sewell High Voltage Testing & Research Center” in 2016. This high voltage lab is dedicated to product development and R&D and includes a new 1.2 MV Impulse Generator test set from High Volt in Dresden, Germany. The lab is also equipped with a 700 kV hi-pot test transformer that is rated for continuous maximum applied voltages that was acquired from Phenix Technologies in Accident, Maryland. This allows us to measure partial discharge with our Omicron test set on test samples up to 700 kV and mimic the 1 hour test levels specified in the industry standards. At 700kV, the lab was found to have 2 pC or less of background partial discharge. Many of the test samples are tested in transformer oil, so the lab is equipped with GlobeCore oil processing equipment.
Additionally, Quality Switch can perform temperature tests on our switches with capabilities of 3500 amps continuous current application. The facilities are equipped with data collection methods for thermal tests, which include the functional life test that Quality Switch helped develop with the IEEE Transformers Committee. This life test to determine contact stability is now detailed in the IEEE guide C57.12.157-2015. Quality Switch also has capabilities to set up various mechanical endurance tests as well.


We are currently seeking a highly motivated and talented engineer with experience in laboratory automation to support our genome mining efforts. As part of the Automation team at LifeMine, you will have the opportunity to work with an amazing group of scientists and life engineers to drive next evolution in drug discovery by incorporation automated technologies into biological and chemical workflows. You'll be responsible for successful development, implementation and validation of new automated protocols and act as the go-to person for automation and other technical supports. As we look toward expanding our automation capabilities, you will have the amazing opportunity to help design and build a state-of-the-art automation facilities to rapidly enable and accelerate LifeMine’s fungal, genomically-enabled drug discovery platform. We are looking for someone who is hands-on, passionate and with a strong willingness to learn and adapt in a dynamic environment.
- Work closely with scientists to develop and/or improve automated workflow utilizing various laboratory automation equipment with the key focus of increasing throughput, improving data quality, reducing costs and removing any operational bottlenecks.
- Program, optimize, validate, and implement new and/or improved methods on robotic liquid handling workstations and integrated laboratory automation systems and work cell.
- Design, implement and lead integration projects to enable LifeMine to grow at scale.
- Explore, design and prototype customized solutions and/or gadgets to meet automation needs.
- Establish and maintain automation operational protocols/documentations (i.e. SOP, scripts, backups, data analysis) and inventory of automation consumables.
- Work with Informatics and IT to establish and/or improve LifeMine's current LIMS and data analysis infrastructure.
- Develop customized scripts to enable lab connectivity and/or data analysis.
- Provide hands-on training to end users on various automation platforms and act as a go-to person for on-site support for all automation related issues.
- Collaborate with third-party vendors to evaluate, negotiate and implement new automation solutions.
- Proactively evaluate new technologies, analyze existing workflows, identify bottlenecks, and develop solutions to improve efficiency, throughput, cost, and quality.
- This position will collaborate with Microbiology, Next Generation Sequencing (NGS), Synthetic Biology, Cell Biology, Biochemistry, Data Science and Chemistry teams to design, implement and support high impact automated workflows.
- Other tasks as required or necessitated by the position.
- MS with 6+ years of experience or B.S. with 8+ years of experience in biomedical or biological engineering, chemical engineering or similar.
- Advanced knowledge with laboratory automation systems and experimental workflow development, utilizing robotic liquid handlers (Hamilton, Labcyte Echo, Agilent Bravo, Tecan, Dynamic Devices) and associated integrated accessories (Precise Arm, Liconic Incubators, plate washer and bulk dispensers, plates sealer/peelers etc...).
- Strong electrical and mechanical background to be able to assemble and troubleshoot automated system as needed.
- Knowledge of system design and rapid prototyping via Solidworks to generate customized solution from concepts to implementation.
- Experience with fully integrated automation solutions and software such as HighRes Cellario, Biosero Green Button Go, Thermo Momentum, PAA Overlord, Retisoft Genera, Transcriptic automation or similar.
- Knowledge with streamlining workflows and developing automated laboratory processes and instrumentation systems, including collecting and documenting requirements, developing concepts, design, testing/validation, implementing, and training.
- Familiarity with cloud architecture framework (AWS, GC, Serverless) and/or database (SQL, Postgres, noSQL) is a plus.
- Developing, optimizing and implementing integrated robotic systems for use in a high throughput drug discovery environment is a plus.
- Experience with project management and barcoded samples storage and tracking is a plus.
- Knowledge of standard molecular biology techniques such as nucleic acid isolation/purification, DNA assembly, transformation, CRISPR editing, NGS is a plus.
- Independent, service-oriented, willingness to learn and take on new challenges, and a strong passion to work in a dynamic team environment to improve patient outcomes and save lives.


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Recently, I addressed the question as to when a child is emancipated under the eyes of New Jersey law. As I indicated there, the New Jersey Supreme Court defines emancipation as "the act by which a parent relinquishes the right to custody and is relieved of the duty to support a child." Newburgh v. Arrigo, 88 N.J. 529 (1982). A related question also addressed by the Court in Newburgh is a parent’s obligation to contribute towards a child’s postgraduate education expenses.
The Supreme Court in Newburgh set forth a non-exhaustive list of factors for a court to consider in determining a parent’s obligation to contribute to such educational expenses. These factors were subsequently codified by statute at N.J.S.A. 2A:34-23(a) as follows:
1. Whether the parent, if still living with the child, would have contributed toward the costs of the requested higher education.
2. The effect of the background, values, and goals of the parent on the reasonableness of the expectation of the child for higher education.
3. The amount of the contribution sought by the child for the cost of higher education.
4. The ability of the parent to pay that cost.
5. The relationship of the requested contribution to the kind of school or course of study sought by the child.
6. The financial resources of both parties.
7. The commitment to and aptitude of the child for the requested education.
8. The financial resources of the child, including assets owned individually or held in custodianship or trust.
9. The ability of the child to earn income during the school year or vacation.
10. The availability of financial aid in the form of college grants and loans.
11. The child’s relationship to the paying parent, including mutual affection and shared goals as well as responsiveness to parental advice and guidance.
12. The relationship of the education requested to any prior training and to the overall long-range goals of the child.

domain: law


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Embellish your shoes !
For all the gold in the world ! Hand-sewn and made in France, the Chic Venitien is perfect to giv your outfil a unique look ! Protection of the shoe heel, confortable smooth and silent walking...
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domain: artistic


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Orange White Cat Personality
White cats were considered to be more shy lazy and calm while tortoiseshell cats were more likely depicted as more intolerant and more trainable.
Orange white cat personality. They typically have white faces chests and legs. The bicolor cat was also considered affectionate just as the white cat the black cat and the tricolor cat were also characterized as more shy lazy and less sociable. A study described by the University of California Berkeley discovered that.
People like the M sign of an orange cat which is visible in a cats forehead. In fact a survey conducted by the University of California Berkeley revealed that cat lovers were more likely to assign positive personality traits to orange cats than white or tortoiseshell cats. Calicos usually have black or orange foreheads ears and backs.
Given that your boy is a kitten hell probably be quite happy to play with another cat. Calicoes are usually about 25 to 75 white sporting large black and orange patches. The survey showed that the orange cat is considered friendly very affectionate and that cats of this color have more positive characteristics when compared to white cat for example.
Orange Cat Facts. None of the orange cats I know have felt the need to be the top cat though so hes probably a good match for your girl. The beautiful calico cats BREED are mostly.
Black and white orange and white brown and white and calico in terms of how the cats reacted to novel situations and handling by a stranger. Toby the first orange tabby cat owned by animal lover and activist Betty White Oscar and Miko two orange tabby cats owned by the late lead singer of Queen Freddie Mercury Morris the Cat the orange tabby cat from the 1970s television commercials for 9 Lives Cat Food. Orange tabby fur can be blended with white spots.
If youre not sure what colors can be found you should bring in a sample coat so you can see what you have in mind. They can also have yellow blue or greenish-black. Some people think that M represents the virgin while the Islamic people say that it means Mohammad.
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domain: pet


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The relationship comes with reached its second stage. The couple has worked check through the differences, but they nonetheless love one another and look connected to the other person. In this level, they may miss the feeling they felt in stage an individual, and they may even be tired of each other. During these stages, people tend to hop ship and locate new human relationships. Here are the stages of any relationship: The initiation, experimentation, and routine service. You might have a crush in your new spouse, but you aren’t sure how serious occur to be getting.
The first level is balance. This lasts for several years till one of the associates decides to finish the relationship. In this stage, the relationship remains stable, but the couple struggles to communicate. The other individual is keeping away from communication and eliminates the other person. This could lead to a power have difficulties or feeling of boredom, and the romance may even take danger of ending. Expansion requires switch, so both partners need to be pushed other than their ease zones.
The second stage is definitely stability. Get committed to one person and are not enthusiastic about dating someone else. You’ve decide to share your daily life with your partner. Your partner understands that you have work and other obligations, and that you do want to share your daily life with anyone else. During this level, you may even look hesitant to publish other people within your life. But , your partner has got understood your preferences, and they already have grown accustomed to your routine.
The third level is a transition stage. With this stage, weight loss stand to get apart from one another and are very likely to argue than communicate. You may even begin to question your relationship’s course. It is a a chance to make decisions about a future together. Once if you’re ready to consider your relationship to the next level, then you can definitely enjoy the associated with your life with all your new spouse.
The fourth stage is a stage of commitment. Both people within a relationship continue to be very suitable, but their requirements are very different. They could have related needs and preferences, nonetheless they may will vary goals. They might even have the same interests, while. This stage of the romantic relationship is the most hard, so it’s crucial to be patient. And remember, the most important thing is to have fun , and don’t forget might your partner for a date.
The fifth stage of a relationship is referred to as the stagnating stage. During this phase, the couple may communicate internally but not with each other. They can be mind-readers, yet this does not help to make communication likely. If you’re within a casual level, there’s no need to worry about the next thing. The relationship stage will remain stagnant for 2 years. During this period, the two of you can communicate with each other.
At this point, couples have got a lot of differences and often feel mad about the same details. During this stage, a couple is usually unsure of whether they can trust their partner, but they don’t let this quit them from trying to make their marriage work. Whenever they start questioning the direction of their relationship, they will find it hard to keep it going. Yet , this can be one of the most rewarding level for their romance. They’re likewise the ones who are prepared to give their very own hearts to the other.
The relationship is in the level stage. It could hard to become away from the other person in this stage. At the same period, the determination becomes good. A couple might have problems articulating their true feelings, nevertheless they’ll still try to make their romantic relationship work. In the end, this period is the most important stage of all. In case the two people in a relationship will be committed to the other person, it’s important that they communicate with each other.
With the second stage, a person commits to one person as well as the relationship will very likely be highly strong. From this stage, someone might be not able to be completely honest with them. The couple with the honeymoon period. Both of them have similar feelings. They might feel happy when they are with each other. Nevertheless the other person might be a little too sensitive and may not view the flaws. This is the first phase of any relationship.