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Research Articles: Therapeutics, Targets, and Development
Fatty acid synthase inhibition results in a magnetic resonance–detectable drop in phosphocholine
James Ross, Amer M. Najjar, Madhuri Sankaranarayanapillai, William P. Tong, Kumaralal Kaluarachchi and Sabrina M. Ronen
James Ross
Amer M. Najjar
Madhuri Sankaranarayanapillai
William P. Tong
Kumaralal Kaluarachchi
Sabrina M. Ronen
DOI: 10.1158/1535-7163.MCT-08-0015 Published August 2008
Expression of fatty acid synthase (FASN), the key enzyme in de novo synthesis of long-chain fatty acids, is normally low but increases in cancer. Consequently, FASN is a novel target for cancer therapy. However, because FASN inhibitors can lead to tumor stasis rather than shrinkage, noninvasive methods for assessing FASN inhibition are needed. To this end, we combined 1H, 31P, and 13C magnetic resonance spectroscopy (MRS) (a) to monitor the metabolic consequences of FASN inhibition and (b) to identify MRS-detectable metabolic biomarkers of response. Treatment of PC-3 cells with the FASN inhibitor Orlistat for up to 48 h resulted in inhibition of FASN activity by 70%, correlating with 74% inhibition of fatty acid synthesis. Furthermore, we have determined that FASN inhibition results not only in lower phosphatidylcholine levels but also in a 59% drop in the phospholipid precursor phosphocholine (PCho). This drop resulted from inhibition in PCho synthesis as a result of a reduction in the cellular activity of its synthetic enzyme choline kinase. The drop in PCho levels following FASN inhibition was confirmed in SKOV-3 ovarian cancer cells treated with Orlistat and in MCF-7 breast cancer cells treated with Orlistat as well as cerulenin. Combining data from all treated cells, the drop in PCho significantly correlated with the drop in de novo synthesized fatty acid levels, identifying PCho as a potential noninvasive MRS-detectable biomarker of FASN inhibition in vivo. [Mol Cancer Ther 2008;7(8):2556–65]
fatty acid synthase
magnetic resonance spectroscopy
choline metabolism
choline kinase
Fatty acid synthase (FASN) is the key enzyme in the de novo synthetic pathway of long-chain fatty acids (1). In most normal cells, FASN expression is low and fatty acids are obtained from the diet (2). However, FASN expression is significantly increased in a wide variety of human cancers including prostate, breast, colon, and ovarian cancer (3–7). Furthermore, this overexpression is associated with poor prognosis, particularly in the case of breast and prostate cancer (4, 8).
In light of these observations, FASN has been proposed as a novel target for cancer therapy (9–11). Indeed, recent studies show that inhibition of FASN by pharmacologic (e.g., Orlistat, cerulenin, C75, and EGCG) or small interfering RNA treatments result in cell cycle arrest and apoptosis of transformed cells in vitro, whereas in vivo studies show that treatment with FASN inhibitors results in inhibition of tumor growth (12–17). Importantly, normal epithelial cells are not affected by FASN inhibition (9, 12). Consequently, the use of FASN inhibitors, as well as inhibitors of other enzymes involved in fatty acid synthesis (18–20), presents a promising therapeutic approach.
However, because response to FASN inhibitors can result in tumor stasis rather than tumor shrinkage, conventional imaging methods may not be adequate to rapidly assess therapeutic response. Consequently, additional noninvasive methods for monitoring inhibition of fatty acid synthesis are needed. Furthermore, whereas the direct consequences of FASN inhibition on fatty acid synthesis and the subsequent modulation of membrane phosphatidylcholine (PtdCho) levels have been investigated in detail (12, 13, 21), additional studies are required to assess further effects of FASN inhibitors on other aspects of cellular metabolism.
Magnetic resonance spectroscopy (MRS) is a noninvasive, nondestructive method that can provide longitudinal information regarding tumor metabolism as well as its modulation following treatment. It has been used previously to monitor choline phospholipid metabolism, glucose metabolism, and cellular energy levels as well as response to chemotherapeutic agents and therapies targeted to specific oncogenic pathways (22–28). As such, MRS can therefore provide a method for both investigating the overall metabolic consequences of FASN inhibition and noninvasively assessing the molecular action of FASN inhibitors in vivo.
The goal of the work described here was therefore twofold: (a) using MRS, to simultaneously determine the effect of FASN inhibitors on fatty acid synthesis and on other metabolic pathways, and (b) using the MRS findings, to identify a MRS-based, noninvasive, metabolic biomarker of FASN inhibition that, in the long term, could be used to monitor FASN inhibition in vivo. To this end, we have used 1H, 31P, and 13C MRS to investigate the metabolic consequences of FASN inhibition. Our observations are in line with previous reports showing that, in addition to inhibiting de novo synthesis of fatty acid, FASN inhibition also leads to a drop in membrane PtdCho levels. However, we also show, to our knowledge for the first time, that FASN inhibition also results in a drop in de novo synthesis of the PtdCho precursor phosphocholine (PCho) and a drop in cellular PCho levels. Importantly, the drop in PCho was correlated with the drop in de novo synthesized fatty acid levels, identifying PCho as a potential MRS-based metabolic biomarker of FASN inhibition.
Cell Culture and FASN Inhibition
PC-3 human prostate, MCF-7 human breast, and SKOV-3 human ovarian cancer cells were routinely cultured in DMEM/F-12 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and 100 units/mL penicillin-100 μg/mL streptomycin, 0.25 μg/mL amphotericin (Life Technologies), and 2 mmol/L l-glutamine (Cellgro) at 37°C in 5% CO2. For all FASN inhibition studies, fetal bovine serum was lowered to 5% (to limit the amount of available extracellular fatty acids) and glucose in the medium was reduced by half to 8.76 mmol/L (financial reasons). To inhibit FASN, PC-3 cells were incubated for 24 and 48 h with 30 μmol/L Orlistat (treated) or with carrier DMSO at 0.5% (v/v; control cells). MCF-7 and SKOV-3 cells were incubated for 48 h with 30 μmol/L Orlistat (treated) or with DMSO at 0.5% (v/v; control cells). MCF-7 cells were also treated with 30 μmol/L cerulenin (treated) or with DMSO at 0.5% (v/v; control cells).
FASN Activity Assay
FASN activity was determined as described previously (29). Briefly, ∼5 × 106 cells were trypsinized, washed in PBS, and frozen at −80°C. Cells were resuspended in 1 mL lysis buffer containing 1 mmol/L EDTA, 150 mmol/L NaCl, 100 μg/mL phenylmethylsulfonyl fluoride, and 50 mmol/L Tris-HCl (pH 7.3), subjected to vortex for 30 s at 0°C, and disrupted at 0°C by ultrasonication for 10 periods of 1 s. The lysates were centrifuged (16,000 × g, 15 min) and the supernatant was stored at −80°C and assayed within 1 week. A sample was taken to measure protein content (Bio-Rad DC protein assay, Bio-Rad Laboratories). Lyophilized supernatant (∼5 μg) was added to a mixture of 200 mmol/L potassium phosphate buffer (pH 6.6), 1 mmol/L DTT, 1 mmol/L EDTA, 0.24 mmol/L NADPH, and 30 μmol/L acetyl-CoA in 0.2 mL reaction volume. After monitoring at 340 nm (Beckman Coulter DU800 UV/visible spectrophotometer) at room temperature for 3 min to measure background NADPH oxidation, 50 μmol/L malonyl-CoA was added and the reaction mixture was assayed for 10 min. Data were analyzed using Beckman Coulter DU800 System and Applications Software.
Flow cytometry was used to determine the effect of FASN inhibition on cell cycle as follows. PC-3 and MCF-7 trypsinized cells (∼2 × 106) were fixed in 95% ice-cold ethanol and then stained with a PBS solution containing 40 μg/mL propidium iodide (Sigma-Aldrich) and 100 μg/mL RNase A (Sigma-Aldrich). Cells were analyzed using a BD FACSCalibur (Becton Dickinson) instrument using a 488 nm excitation wavelength. Cells of uniform width were gated and propidium iodide intensities were plotted and analyzed using ModFit LT (version 3.1) software (Topsham). In the case of PC-3 cells, the integration values of the two major G1 populations [diploid (2n) and aneuploid (2.8n)] within the culture were summed to derive the final cell cycle distribution results. The values for their corresponding G2 (4n and 5.6n) and S phase populations were also combined.
Apoptosis Assay
PC-3 and MCF-7 cells (1 × 105) were plated in 4 mm2 wells, incubated overnight, and treated with 30 μmol/L Orlistat for 3, 24, and 48 h. At each time point, the medium was removed and transferred to a 1.5 mL tube to preserve any floating cells. The cell monolayers were then washed once with PBS, trypsinized, resuspended in 0.5 mL PBS, and transferred to the corresponding media tubes. The cell suspensions were then centrifuged at 1,000 × g for 5 min, washed once with PBS, and lysed using 100 μL lysis buffer [50 mmol/L HEPES (pH 7.4), 0.1% CHAPS, 5 mmol/L DTT, 0.1 mmol/L EDTA]. The lysates were centrifuged at 10,000 × g for 10 min and stored at -20°C. Caspase-3 activity was measured by incubating 10 μL of each lysate with 30 μmol/L Ac-DEVD-AMC substrate (Biomol) in assay buffer [50 mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 0.1% CHAPS, 10 mmol/L DTT, 1 mmol/L EDTA, 10% glycerol] in a 100 μL total volume for 1 h at 37°C. Cleavage of the substrate by caspase-3 was measured using a Safire plate reader (Tecan Group) at excitation and emission wavelengths of 360 and 460 nm, respectively.
Cell Extraction, MRS Acquisition and Analysis
For MRS studies, glucose in the medium (8.76 mmol/L) was replaced by equal concentrations of [1-13C]glucose and unlabeled glucose (to monitor fatty acid synthesis and glycolysis) and choline in the medium was replaced with [1,2-13C]choline at its normal concentration of 64.1 μmol/L (to monitor choline metabolism). PC-3, MCF-7, or SKOV-3 cells (logarithmic growth) were cultured with Orlistat (treated) or DMSO (control) for 24 and/or 48 h, replenishing medium every 24 h. Spent medium was saved. To monitor cell metabolism, ∼5 × 107 cells were extracted using the dual-phase extraction method (25, 30). Briefly, cells were rinsed with ice-cold saline, fixed in 10 mL ice-cold methanol, and scraped from the culture flask surface. Alternatively, cells were trypsinized and centrifuged and the cell pellet was combined with 10 mL ice-cold methanol (results from both methods were indistinguishable). Following vortexing, 10 mL ice-cold chloroform was added followed by 10 mL ice-cold de-ionized water. After phase separation and solvent removal, cellular proteins were collected and stored at −80°C until measurement (see below). To acquire 1H and 13C spectra, the aqueous fraction was reconstituted in 500 μL deuterium oxide (aqueous phase) and the lipid fraction was reconstituted in deuterated chloroform (lipid phase). To acquire aqueous 31P MR measurements, 25 μL EDTA in deuterium oxide was further added to a final concentration of 5 mmol/L. To acquire lipid 31P MR measurements, deuterated chloroform was evaporated and the precipitate was resuspended in 500 μL of a 2:1 mixture of deuterated chloroform and 60 mmol/L methanolic EDTA adjusted to pH 7.3 with CsOH. MR spectra were acquired on an Avance DRX500 Bruker spectrometer (Bruker Biospin). 1H spectra: 20 ppm spectral width, 30° flip angle, and 5 s repetition time with water suppression. 13C spectra: 240 ppm spectral width, 30° flip angle, and 3.5 s repetition time with broadband proton decoupling. 31P spectra: 60 ppm (aqueous 31P) or 35 ppm (lipid 31P) spectral width, 30° flip angle, 4.5 second repetition time, and broadband proton decoupling.
Data were analyzed using Bruker Topspin 2.0.a software. Changes in metabolite levels were determined relative to matched controls. Absolute metabolite quantification was obtained by determining the appropriate peak area, normalizing to an external reference (tetramethylsilane, 1H MRS; deuterated chloroform, 13C MRS; methylenediphosphonic acid, 31P MRS), correcting for saturation and nuclear Overhauser effects and normalizing to cellular protein. In quantifying the product of de novo fatty acid synthesis (13C MR spectra), only the methylene carbons at 29 to 30 ppm were used and it was further assumed that fatty acids observed represented only palmitate. From 1H MR spectra of lipid extracts, only methylene protons at 1.2 to 1.3 ppm were used to quantify the total pool of fatty acid and all fatty acids were assumed to be palmitate.
To quantify the cellular proteins that precipitated during metabolite extraction, the pellet was resuspended in 1 mL of 1 mol/L NaOH by incubating for 1 h at 50°C. A sample was then taken for measurement of protein content (Bio-Rad assay, Bio-Rad Laboratories).
Choline Kinase Assay
Choline kinase (ChoK) activity was determined as described (31). Following incubation with DMSO or Orlistat for 48 h, ∼15 × 106 PC-3 cells were trypsinized, washed in ice-cold PBS, and resuspended in 400 μL Tris-HCl (pH 8.0) containing 10 mmol/L DTT and 1 mmol/L EDTA in deuterium oxide. Cells were homogenized for 30 s at 0°C and were disrupted at 0°C by ultrasonication for 10 periods of 1 s. Cell lysates were centrifuged (16,000 × g, 30 min) and the particle-free supernatant transferred to a NMR tube. Initial PCho levels were measured by 1H MRS as described above. ChoK activity was assayed by monitoring temporal accumulation of PCho by MRS following addition of choline chloride, ATP, and Mg2+ in Tris-HCl buffer (final concentrations: 5 mmol/L choline chloride, 10 mmol/L ATP, 10 mmol/L MgCl2). ChoK activity was determined from a straight-line fit to plots of PCho as nmol/mg protein versus time following addition of all substrates.
The effect of Orlistat on ChoK activity was also monitored after only 1 h of incubation. Cells were incubated for 47 h with DMSO alone and then Orlistat was added during the last hour of incubation, creating a treated group. Cells were then lysed and ChoK activity was assayed as before. To detect any direct interaction between Orlistat and ChoK, cells were incubated for 47 h with DMSO alone. Following extraction, half the cell lysates were exposed to Orlistat for 1 h at room temperature, creating a treated group, and ChoK activity was assayed as before.
Quantitative PCR Analysis
PC-3 cells (2.5 × 105) were seeded in 10 cm2 dishes for 24 h before treating with 30 μmol/L Orlistat. RNA from treated and control cells was extracted cells using TRIZOL (Invitrogen) and standardized to a concentration of 50 ng/mL. cDNA was produced using a Transcriptor First-Strand synthesis kit (Roche) according to the manufacturer's instruction. The expression of PCho cytidylyltransferase (CCT) and ChoK following treatment was measured by quantitative PCR using an ABI 7500 instrument (Applied Biosystems). PCR primers were designed based on GenBank sequences NM_005017 (CCT; forward 5′-TGTTCAGCCAAGGTCAATGCAAGG-3′ and reverse 5′-TTCTCGTTCATCACCGTGAAGCCT-3′) and NM_001277 (ChoK; forward 5′-TATCTTGTTGCTGGAAGGCCGAGA-3′ and reverse 5′-TGGGCGTAGTCCATGTACCCAAAT-3′). Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous standard and amplified using primers 5′-CCATGGAGAAGGCTGGGG-3′ (forward) and 5′-CAAAGTTGTCATGGATGACC-3′ (reverse). Expression levels of CCT and ChoK were first normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase levels (ΔCt) and subsequently to the untreated control of each group (ΔΔCt). The results were expressed as percentages of relevant control using the equation 2ΔΔCt.
PC-3 and MCF-7 cells were treated with DMSO or Orlistat (30 μmol/L) for 3, 24, and 48 h and then lysed using cell lysis buffer containing 1% NP40, 1% SDS, and 1 μL/mL protease inhibitor cocktail set III (Calbiochem). Lysates were centrifuged at 12,000 rpm for 10 min at 4°C, the protein supernatant was collected, and total protein concentrations were determined using Bio-Rad DC protein assay reagents (Bio-Rad Laboratories). Proteins were separated by SDS-PAGE using 10% gels and transferred electrophoretically to 0.45 μm nitrocellulose membranes. Membranes were blocked in blocking buffer containing 5% nonfat dry milk in TBS (pH 7.6) and 0.1% Tween 20 and incubated overnight at 4°C with primary antibodies as follows: Akt (1:1,000; Cell Signaling Technology), phospho-Akt (P-Akt, 1:1,000; Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (1:5,000; Stressgen). This was followed by 1-h incubation with horseradish peroxidase–conjugated secondary anti-rabbit (Cell Signaling Technology) and anti-mouse (Cell Signaling Technology) antibodies at dilutions of 1:1,000 and 1:2,000, respectively. Membranes were washed with enhanced chemiluminescence reagents (LumiGLO & Peroxide, Cell Signaling Technology) for 1 min and exposed to Hyperfilm (Amersham Biosciences), which was developed on a Konica SRX-101 automatic developer (Konica). The intensity of individual bands of interest was quantified using Image J software (NIH) and normalized to the intensity of glyceraldehyde-3-phosphate dehydrogenase (loading control) and Akt and P-Akt protein expressions compared between DMSO- and Orlistat-treated groups.
Unless otherwise stated, experiments were repeated three to five times. Results are expressed as mean ± SD. Two-tailed unpaired Student's t tests were done to assess the statistical significance of results. P < 0.05 was considered significant.
Orlistat Treatment Leads to Inhibition of Fatty Acid Synthesis, Reduced Fatty Acid Levels, Inhibition in Cell Proliferation, and Lower Nucleotide Triphosphate Levels
First, we investigated in detail the effect of the FASN inhibitor Orlistat on PC-3 prostate cancer cells (known to overexpress FASN; ref. 7). Cells were treated for 24 and 48 h with 30 μmol/L (saturating dose; ref. 12) of Orlistat. As expected, this resulted in inhibition of FASN activity by 51 ± 10% (P < 0.05) and 70 ± 21% (P < 0.05) at 24 and 48 h, respectively, when compared with controls (Fig. 1A ). Figure 1B illustrates 13C MRS spectra obtained from the lipid fraction of control and treated PC-3 cells grown in the presence of medium containing 13C-labeled choline and glucose. From these data, it was possible to monitor fatty acids synthesized de novo from the 13C-labeled glucose present in the growth medium. As expected, fatty acid synthesis was inhibited following Orlistat treatment. Because cellular protein content (1.9 ± 0.2 × 10-7 mg/cell) was unaffected by Orlistat treatment, metabolite levels, determined by MRS, were normalized to protein content. Control cells synthesized 30 ± 12 nmol/mg protein of de novo 13C-labeled fatty acids during 24 h and 56 ± 18 nmol/mg protein during 48 h. In cells treated with Orlistat, de novo 13C-labeled fatty acid synthesis decreased significantly by 45 ± 21% to 15 ± 2 nmol/mg protein (P < 0.05) at 24 h and by 74 ± 12% to 13 ± 4 nmol/mg protein (P < 0.02) at 48 h. It is noteworthy that the measured decrease in de novo fatty acid synthesis correlated with the measured decrease in FASN activity following 24 and 48 h of Orlistat treatment (R2 = 0.99, P < 0.05). Figure 1C illustrates the 1H MRS spectra of the same cellular extracts monitoring the total fatty acid pool. Assuming that on average all fatty acid are 16 carbons in length, the cellular fatty acid pool was estimated as 523 ± 147 nmol/mg protein in control PC-3 cells. Following Orlistat treatment, and consistent with the inhibition of de novo fatty acid synthesis, total cellular fatty acid levels dropped slightly to 91 ± 31% following 24 h and significantly to 70 ± 17% (P < 0.04) following 48 h of FASN inhibition.
Inhibition of FASN causes a drop in fatty acid synthesis and eventually a drop in total fatty acid levels. A, % (relative to control) FASN activity (black columns), de novo fatty acid synthesis (white columns), and total fatty acid levels (striped columns) of PC-3 prostate cancer cells following treatment with Orlistat for the indicated times. Bars, SD. *, P < 0.05, statistically significant difference from its control (two-tailed unpaired t test). B, representative 13C MR spectra of lipid-phase extracts of PC-3 cells showing the effect of 48 h Orlistat treatment on de novo fatty acid synthesis. All peaks shown represent fatty acids, but only the peak labeled "FA" was used for quantitative analysis. C, representative 1H MR spectra of the lipid-phase extracts of PC-3 cells showing effect of 48 h Orlistat treatment on cellular fatty acid levels.
Orlistat treatment also led to cell cycle arrest in PC-3 cells (Fig. 2A ). Cell fractions in the G1 phase increased in association with length of treatment. A matched concomitant decrease in the S and G2-M phase fractions was also observed in both cell lines. Cell cycle arrest was associated with inhibition of cell proliferation. Thus, the number of Orlistat-treated PC-3 cells remained essentially unchanged at 111 ± 17% at 24 h and 83 ± 11% at 48 h compared with cell numbers before treatment. In contrast, untreated control cell numbers increased to 149 ± 38% (P < 0.05 relative to treated cells) and 206 ± 62% (P < 0.005 relative to treated cells) at 24 and 48 h, respectively. Apoptosis was not evident during the course of Orlistat treatment. Specifically, no significant increase in apoptotic cells was observed in treated cells, where the maximum percentage of the apoptotic cell fraction (sub-G1) did not exceed 0.9 ± 0.3% in treated PC-3 cells. Furthermore, an increase in caspase-3 activity was not detected at 3, 24, and 48 h post-treatment in contrast to the significant increase detected in a positive control, staurosporine-treated Jurkat cells (data not shown).
FASN inhibition results in cell cycle arrest without the induction of apoptosis in PC-3 (A) and MCF-7 (B) cells. Left, representative histograms showing cell cycle distribution of cells treated with Orlistat (30 μmol/L) for 24 and 48 h. The PC-3 cell line exhibits two major G1 populations, diploid (2n) and aneuploid (2.8n), and two corresponding G2-M peaks. Right, cell cycle phase distributions shown in histograms were quantified using ModFit LT and the results are illustrated in the bar graphs. Each value was determined from the average of three analyses. Bars, SD.
Levels of the high-energy metabolites phosphocreatine and nucleotide triphosphates (predominantly consisting of ATP) were determined from 31P MR spectra of the aqueous fraction of control PC-3 extracts at values measuring 16 ± 7 and 57 ± 22 nmol/mg protein, respectively. Phosphocreatine levels decreased significantly following 24 h of Orlistat treatment to below detection level, where they remained through 48 h of FASN inhibition. Nucleotide triphosphate levels also decreased following treatment, with their levels falling significantly by 34 ± 23% (P < 0.03) and 59 ± 10% (P < 0.002) at 24 and 48 h, respectively (Fig. 3A ).
Inhibition of FASN causes a drop in PCho synthesis leading to a decrease in total PCho levels, which is correlated with a drop in fatty acid synthesis. A, representative 31P MR spectra of the aqueous extracts of PC-3 cells showing the effect of 48 h of Orlistat treatment on cellular PCho levels and cellular energetics. Inset, % (relative to control) cellular PCho levels of PC-3 cells following treatment with Orlistat for the times indicated. B, representative 13C MR spectra of the aqueous extracts of PC-3 cells following Orlistat treatment for 48 h on de novo PCho synthesis. Inset, % (relative to control) de novo PCho synthesis in PC-3 cells following Orlistat treatment for the indicated times. Bars, SD. *, P < 0.05. C, PCho levels as a function of de novo fatty acid synthesis based on experiments summarized in Table 1 and indicating a correlation between FASN activity as indicated by de novo fatty acid synthesis and PCho levels (R2 = 0.73; P < 0.03).
Orlistat Treatment Leads to a Drop in PCho Levels
Levels of the membrane lipid PtdCho were determined from the 31P MR spectra of the lipid fraction of cell extracts (Fig. 4A ). Changes in the levels of this phospholipid were below detection level during 24 h of Orlistat treatment, with values at 110 ± 44% (P = 0.74) relative to control. However, following 48 h of treatment, PtdCho dropped by 31 ± 4% (P < 0.0005) from 115 ± 31 to 80 ± 24 nmol/mg protein consistent with previous reports (12, 13, 21). 13C MRS studies (Fig. 4B) indicated that this was due to a reduction in de novo phospholipid synthesis. Control cells synthesized 21 ± 6 and 61 ± 12 nmol/mg protein PtdCho during 24 and 48 h, respectively. Following Orlistat treatment for 24 h, PtdCho synthesis decreased by 54 ± 24% to 10 ± 4 nmol/mg protein (P < 0.03) and by 60 ± 9% to 25 ± 9 nmol/mg protein (P < 0.004) following 48 h of treatment.
Inhibition of FASN causes a drop in the synthesis and total levels of membrane phospholipids. A, representative 31P MR spectra of PC-3 lipid extracts following 48 h of treatment with Orlistat. a, cardiolipin; b, plasmologen-PtdEtn; c, PtdEtn; d, PtdS; e, sphingomyelin; f, phosphatidylinositol; g, plasmologen-PtdCho. Inset, % (relative to control) cellular PtdCho levels of PC-3 cells following treatment with Orlistat for the times indicated. B, representative 13C MR spectra of lipid extracts following 48 h of Orlistat treatment; peaks at 63 to 64 ppm have not been unequivocally identified. Inset, % (relative to control) de novo PtdCho synthesis in PC-3 cells following Orlistat treatment for the indicated times.
In addition, and somewhat unexpectedly, 31P MRS spectra of the aqueous fraction of cell extracts indicated that levels of total PCho, the precursor of PtdCho, were also affected by Orlistat treatment (Fig. 3A). PCho dropped significantly by 41 ± 24% from an average 182 ± 20 to 107 ± 44 nmol/mg protein following 24 h of Orlistat treatment (P < 0.02) and by 60 ± 21% from 199 ± 63 to 70 ± 26 nmol/mg protein (P < 0.02) following 48 h of treatment. The total choline–containing metabolite signal detected by 1H MRS (data not shown) also dropped by 33 ± 27% (P = 0.05) and by 47 ± 32% (P = 0.06) at 24 and 48 h, respectively, further confirming the effect of FASN inhibition on PCho levels. 13C MRS studies were used to study PCho synthesis simultaneously with fatty acid synthesis and Fig. 3B illustrates the 13C spectra of control and Orlistat-treated cells. These studies indicated that the drop in PCho levels was due to its reduced synthesis. Control cells synthesized 83 ± 13 nmol/mg protein of de novo PCho during 24 h and 131 ± 27 nmol/mg protein during 48 h. Synthesis decreased significantly on treatment with Orlistat by 39 ± 12% to 47 ± 11 and 59 ± 23% to 37 ± 15 nmol/mg protein after 24 and 48 h, respectively. Importantly, total as well as de novo PCho levels correlated with FASN activity (R2 = 0.99, P < 0.05).
Drop in PCho Is Associated with a Drop in Cellular ChoK Activity
The data above indicate that following 48 h of Orlistat treatment the drop in total PCho levels (129 ± 89 nmol/mg protein) was comparable with the drop in PCho synthesized de novo (94 ± 42 nmol/mg protein). This indicated that the drop in PCho levels was due to reduced PCho synthesis rather than increased utilization. To confirm this hypothesis, ChoK activity was assayed in control and treated PC-3 cells using MRS as described previously (31). The rate of PCho synthesis, a measure of cellular ChoK activity, was 34 ± 6 nmol/mg protein h in control cells. Following FASN inhibition with Orlistat for 48 h, this rate dropped significantly to 21 ± 7 nmol/mg protein h (P < 0.05; Fig. 5A ).
FASN inhibition is associated with a drop in ChoK activity (A) but not in long-term inhibition of PI3K signaling (B). A, 1H MR spectrum of cell lysates showing endogenous PCho and added Cho. Inset, representative plot of PCho synthesized by ChoK extracted from control cells and 48 h Orlistat-treated cells. Lines represent the straight-line fits to the data used to obtain rates of PCho synthesis (R2 = 0.89, control; R2 = 0.96, Orlistat) indicating inhibition of ChoK following Orlistat treatment. B, Western blot analysis of P-Akt, Akt, and glyceraldehyde-3-phosphate dehydrogenase (loading control) expression in control and treated PC-3 cells following 3, 24, and 48 h of Orlistat treatment.
We questioned how quickly Orlistat affects choline metabolism and, to this end, also monitored ChoK activity following 1 h of Orlistat treatment. At that time point, FASN inhibition resulted in a drop in ChoK activity by 49 ± 10% (P < 0.05) relative to controls. This inhibition was comparable with that observed following 48 h of treatment and indicated that the effect of FASN inhibition on choline metabolism occurs rapidly and is sustained during treatment.
To rule out any direct inhibitory interaction between Orlistat and ChoK, Orlistat was also added directly to the extracted cell lysates. Incubation of cell lysates with 30 μmol/L Orlistat for 1 h did not alter the activity of ChoK, which remained at 110 ± 36% (P = 0.67) of control levels.
To determine the reasons behind the drop in cellular ChoK activity, we monitored enzyme expression. Quantitative PCR analysis showed that ChoK expression was unchanged at 24 h with gene expression at 106 ± 4% relative to control. However, 48 h of Orlistat treatment resulted in a drop in ChoK expression to 60 ± 28% (P < 0.05) relative to controls. The expression of CCT was not significantly altered during the same time, with quantitative PCR data showing CCT expression at 95 ± 9% and 99 ± 10% at 24 and 48 h, respectively. Thus, changes in ChoK expression can explain the drop in PCho levels following 48 h of treatment but not the drop in ChoK activity and PCho levels observed at the earlier time points following FASN inhibition.
Phosphatidylinositol 3-kinase (PI3K) signaling has been linked to FASN expression in clinical samples (11, 32, 33); recently, a bidirectional link between FASN and PI3K signaling has been shown, with PI3K controlling FASN expression and FASN inhibition resulting in a reduction in PI3K signaling downstream of HER-2/neu (11, 34, 35). We have shown previously that inhibition of the PI3K pathway results in a drop in PCho levels (36), and ChoK activity can be controlled by PI3K signaling (37). We therefore questioned whether PI3K signaling provided the mechanistic link between fatty acid and choline metabolism and could explain the drop in ChoK activity and PCho levels following FASN inhibition, particularly at the earlier time points. To answer this question, we monitored the effect of Orlistat on P-Akt levels in PC-3. A slight drop in P-Akt levels was observed following 3 h of Orlistat treatment in PC-3 cells (Fig. 5B), indicating some inhibition of PI3K signaling. However, 24 or 48 h of exposure to Orlistat did not result in a significant reduction in P-Akt levels.
Drop in PCho Is a General Observation following FASN Inhibition
To confirm the generality of our observations, we extended our studies of Orlistat to another two cell lines: MCF-7 human breast cancer and SKOV-3 human ovarian cancer cells. We further extended our studies to a second FASN inhibitor, cerulenin. Our findings are summarized in Table 1 . MCF-7 cells were treated with 30 μmol/L Orlistat for 48 h. This lead to a drop in cell numbers to 57 ± 14% compared with controls, associated with cell cycle arrest and no significant apoptosis, as in the case of PC-3 cells (Fig. 2B). As expected, FASN inhibition resulted in a drop in de novo fatty acid synthesis by 46 ± 9% in MCF-7 cells. Importantly, however, the inhibition of FASN activity was associated, as in the case of PC-3 cells, with a drop in de novo synthesized PCho levels by 63 ± 12% resulting in a drop in total PCho levels by 38 ± 6%. Levels of total choline also dropped by 49 ± 15% in MCF-7 cells. The effect of Orlistat treatment on cellular energetics was less substantial than in PC-3 cells, with nucleotide triphosphate levels falling by 28 ± 32%. Western blot analysis of P-Akt levels in MCF-7 cells yielded similar results to PC-3 cells, with no significant inhibition of PI3K signaling observed following 48 h of Orlistat treatment.
Summary of common affects of FASN inhibition on PC-3, MCF-7, and SKOV-3 cancer cells, expressed as % of control
Treatment of SKOV-3 cells with Orlistat also lead to inhibition of PCho synthesis and a reduction in PCho levels (Table 1). Specifically, FASN inhibition resulted in a drop in SKOV-3 cell number to 79 ± 7% relative to control associated with a drop in de novo fatty acid synthesis by 37 ± 7%, a drop in de novo synthesized PCho levels by 31 ± 21%, and a drop in total PCho levels by 56 ± 14%.
MCF-7 cells were also treated with 30 μmol/L cerulenin for 48 h (Table 1). The effect on cell proliferation was negligible, with cell numbers remaining at 99 ± 17% of control. Nonetheless, treatment with cerulenin was associated with a reduction in de novo fatty acid synthesis by 40 ± 6%. Furthermore, as in the case of Orlistat, the inhibition of FASN activity was associated with a drop in de novo synthesized PCho levels by 21 ± 12%; importantly, total PCho levels dropped by 32 ± 14% in cerulenin-treated MCF-7 cells, whereas total choline dropped by 48 ± 4%. Cerulenin treatment had no noticeable effects on cellular energetics, with nucleotide triphosphate levels remaining at 97 ± 32%.
Drop in PCho Levels Correlates with the Drop in Fatty Acid Synthesis following FASN Inhibition
Finally, we wanted to confirm that PCho levels were correlated with FASN activity. As mentioned above, FASN activity tightly correlates with de novo synthesis of fatty acid. We therefore used changes in de novo synthesis of fatty acid as an indicator of FASN activity in the exact same experiment in which PCho levels were measured. Our findings are summarized in Fig. 3C in which we combined the data obtained from the different experiments summarized in Table 1. We find that the drop in PCho levels significantly correlates with the drop in de novo fatty acid synthesis (R2 = 0.73; P < 0.03), validating PCho as an indicator of FASN inhibition.
FASN is overexpressed in several cancers, and inhibition of FASN leads to cell cycle arrest, apoptosis, and inhibition of tumor growth, presenting a novel therapeutic approach for cancer treatment (9–17). Here, we monitored the metabolic consequences of FASN inhibition with the goal of determining the effects of this inhibition on cellular metabolism and identifying MR-based metabolic biomarkers of the molecular action of FASN inhibitors. We used 1H, 31P, and 13C MRS, combined with 13C-labeled glucose and choline, to assess fatty acid synthesis, choline metabolism, and cellular energetics. First, using Orlistat, we determined in detail the MRS-detectable consequence of FASN inhibition in PC-3 cells. We then confirmed the generality of our findings by investigating the effect of Orlistat on two other cell lines and the effect of an additional FASN inhibitor.
As expected, FASN inhibition in all three cell lines resulted in inhibition of de novo fatty acid synthesis and a drop in total fatty acid levels, which was generally associated with inhibition in cell proliferation. These findings are consistent with previous reports (12, 13, 15–17, 38–41). Our MRS data also showed that FASN inhibition affects cellular energetics, with levels of phosphocreatine (when detectable) and nucleotide triphosphates generally decreasing with FASN inhibition. However, this effect was not always large enough to be deemed a reliable indicator of FASN activity.
Because this study used MRS, where the metabolic fate of different precursors can be monitored simultaneously, it was possible to monitor choline metabolism at the same time as fatty acid synthesis. Choline is the precursor of the main membrane phospholipid PtdCho in the Kennedy (CDP-choline) pathway. A drop in PtdCho levels has been reported previously (12, 13, 21) and others have shown a link between fatty acid synthesis and the Kennedy pathway through transcriptional and posttranscriptional control of the rate limiting enzyme CCT (42). Our studies also showed a drop in PtdCho levels. However, in this study, we observed, to our knowledge for the first time, that inhibition of fatty acid synthesis also results in inhibition of the first step in this pathway, namely synthesis of PCho by ChoK. As a result, PCho levels were lower following treatment; importantly, PCho levels correlate with fatty acid synthesis levels. Thus, we propose that a reduction in levels of the MRS-detectable metabolite PCho can be used as a noninvasive diagnostic indicator of FASN inhibition in vivo.
Regarding the mechanism for the observed drop in PCho, we hypothesized that FASN inhibition and inhibition of fatty acid synthesis lead to upstream inhibition of the first enzyme in the Kennedy pathway, namely ChoK. To test this hypothesis, we monitored the cellular activity of ChoK and our data confirm that cellular ChoK activity is indeed significantly reduced following FASN inhibition. This modulation of ChoK activity results, at least in part, from the reduction in ChoK expression observed following 48 h of Orlistat treatment. However, the fact that the activity of ChoK was reduced before this time point (within 1 h of treatment) indicates that other factors are also involved in modulating ChoK activity following FASN inhibition. We thus considered the possibility that cellular ChoK activity was also modulated by inhibition of PI3K signaling, reported to occur downstream of FASN inhibition (11, 35, 37). In line with previous reports, our data indicated that PI3K signaling was indeed reduced following FASN inhibition. However, this effect was only observed following FASN inhibition for a relatively short time (3 h) and was not sustained for 24 or 48 h. Our data therefore indicate that most likely multiple translational and posttranslational factors are involved in modulating ChoK activity following FASN inhibition. It is possible that modulation of ChoK activity could be mediated by PI3K shortly after FASN inhibition, whereas the effects of longer-term treatment could be mediated by altered ChoK expression.
In conclusion, this study highlights the value of MRS for simultaneously monitoring the effect of one inhibitor on several metabolic pathways. Furthermore, our data in three different cell lines and using two FASN inhibitors indicate that a drop in PCho levels is correlated with inhibition of fatty acid synthesis following FASN inhibition. MRS is a noninvasive method that can be used in patients in vivo to monitor PCho either by monitoring the phosphomonester peak in the 31P spectrum or, more commonly, by monitoring the total choline signal in the 1H spectrum. Consequently, we propose that this previously unreported metabolic change could serve as a noninvasive in vivo MRS metabolic pharmacodynamic biomarkers of FASN inhibition.
We thank Wendy Schober (The University of Texas M. D. Anderson Cancer Center Core Facility) for help in flow cytometry analysis.
Grant support: National Cancer Institute Cancer Center support grant CA016672 for the support of Core NMR Facility.
Note: J. Ross and A.M. Najjar contributed equally.
Accepted May 11, 2008.
Received January 7, 2008.
Revision received April 11, 2008.
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Mol Cancer Ther August 1 2008 (7) (8) 2556-2565; DOI: 10.1158/1535-7163.MCT-08-0015
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Startups Money Venture Capital Analysis
Deal Roundup: Startup funding remains sluggish in the first week of 2020
Photo Credit: VCCircle
Shashank Didmishe	3 Jan, 2020
The first week of 2020 saw lukewarm deal activity in the technology startup space. In all, six startups raised $20 million, which was lower than the already low $61 million scooped up by five startups in the previous week.
Targetone Innovations, which owns the automobile servicing platform GoMechanic, raised the highest funds among the six entities, from venture capital firms Chiratae Venture and Sequoia Capital India. It was also the only company whose funding round was above Series A. Three of the remaining rounds were seed while two were Series A.
Two entities, technology startup Buildpan and agri-tech firm Shivrai Tech, did not disclose the amount of funds raised.
The week was devoid of mergers and acquisitions and fund closure. In the initial public offering (IPO) space, cloud-based communications services provider Route Mobile received approval from the Securities and Exchange Board of India for its proposed Rs 600 crore listing.
Top funding deals
GoMechanic: The Targetone Innovations-owned automobile servicing platform raised $14.7 million in a Series B investment round led by returning venture capital firms Chiratae Ventures and Sequoia Capital India. Orios Venture Partners also participated in the round. The investment will be used to support its expansion into 10 cities by the end of 2020, in its EV thrust, brand promotion and in streamlining spare parts procurement.
WealthBucket: The fintech platform owned by Wealth Bucket Capital Investment raised $3 million in a seed round from private equity firm NorthStar and some HNIs (high net worth individuals). The company will use the funds to increase its staff strength to 100 from the current 45 and expand its operations. Along with this, the funds will be used to get technology to simplify the know your customer (KYC) and investment process. The company also plans to extend the platform to handle the investment portfolio of an entire family through one account.
Quicko: The online tax solutions provider owned by Quicko Infosoft, raised $300,000 in a seed round from Zerodha-owned Rainmatter Capital, an incubator which invests in fintech startups. Founded by Vishvajit Sonagara, Quicko provides income tax filing services to resident and non-resident taxpayers. Salaried individuals can upload Form 16, review and quickly file returns. The platform also helps stock traders figure the right tax forms, the applicability of tax audit, due dates, and file tax returns including profit and loss statement and balance sheet. The platform claims to have on-boarded more than 50,000 taxpayers.
Leverage Edu: The Leverage Ed-tech-owned platform, which owns the higher education counselling platform, raised $1.5 million in a funding round led by existing investors venture capital firm DSG Consumer Partners and early-stage venture fund Blume Ventures. Other investors which participated in the round include Saama Capital founder Ash Lilani and fintech firm PayU CEO Amrish Rau. With this round, the Delhi-based startup has so far raised $3 million.
Shivrai Technologies: The Pune-based company, which owns and operates an agriculture ERP (enterprise resource planning) platform FarmERP, raised an undisclosed amount in the Series-A investment round from Singapore-based software firm Technogen IT Services.
FarmERP, which has a customer base in 25 countries, will employ artificial intelligence, machine learning, computer vision and blockchain technologies to help users mitigate climate risks in agriculture and achieve sustainability.
Buildpan: The Indore headquartered company, which helps developers to test, integrate and deploy apps, raised an undisclosed amount in seed round from a group of HNI (High Networth Individuals), including Yusho Kawata, partner at Elrise Corporation Inc Japan, angel investor Sunil Kumar Singhvi and a US based HNI. The platform detects crashes, bugs and device compatibility issues in apps using artificial intelligence and machine learning to detect errors.
Route Mobile: The omnichannel cloud communications service provider received approval from markets regulator SEBI (Securities and Exchange Board of India) for its proposed Rs 600 crore IPO (initial public offering). Through the issue, the company will raise Rs 240 crore by issuing fresh shares. Promoters Y Sandipkumar Gupta and Rajdipkumar Gupta will receive about Rs 360 crore through an OFS (offer-for-sale). Additionally, Route Mobile has kept the open the option of a pre-IPO placement of up to Rs 100 crore. In case the company chooses to use the option, the IPO size will be reduced by that amount.
goMechanic Targetone Innovations Chiratae Ventures Sequoia Capital WealthBucket NorthStar Route Mobile Shivrai Technologies Technogen IT Services IPO Quicko Infosoft Zerodha Rainmatter Capital Leverage Edu Leverage Ed-tech DSG Consumer Partners Blume Ventures

redpajama_set_name: RedPajamaCommonCrawl


Get a slice of Highland life on this day trip to western Scotland, taking in one of its most scenic lochs, the old capital of Stirling, and the Trossachs: land of William Wallace and Rob Roy.
Departing Edinburgh, we travel westwards for our first stop of the day at the bonnie banks of Loch Lomond; one of the prettiest and largest lochs in the whole of Scotland. Here you have the option of an hour-long cruise with spectacular views of the loch's many islands and surrounding mountains. Alternatively, take a stroll through the picturesque lochside village of Luss; with its quaint houses, cafes and stunning views across the Loch's western shoreline.
We continue through the Trossachs National Park; known as 'the Highlands in Miniature' for its abundance of lochs, mountains and forests. It's also famous as the home of the legendary outlaw, Rob Roy MacGregor. We will spend time exploring the area before stopping for lunch in a small village immersed in its beautiful surroundings.
After lunch, we make our way through the town of Callander, past the medieval Doune Castle before making our final stop of the day at Stirling Castle. This is one of Scotland's most historically significant fortresses, sitting high on volcanic rock and offering wonderful views of the surrounding landscape. You will have time to visit the castle and learn of the exploits of key historical figures including William Wallace, Robert the Bruce and Mary Queen of Scots, before hitting the road again, back to Edinburgh.
This tour may occasionally run in reverse, beginning in Stirling and ending at Loch Lomond.
We thoroughly enjoyed our tour today with Gary! He shared with us his in-depth knowledge of the different areas interspersed with humour and music which which made it even more enjoyable!
Nice tour. Unfortunately, there is a substantial amount of driving time and too little time at Sterling Castle (only 1.5 hours — not nearly enough). Our guide, Martin, was very good.


Steve Nash takes in the trade talk and returns a measured response, but Suns coach Alvin Gentry isn't … He stepped in to end Nash's interview and then he made out trade speculators to be ridiculous. "Steve's not going to be traded, that I can tell you," Gentry said. "If he's getting traded, I'm going along with him, OK? Have you looked at the way he's playing? He's playing at a real high level. Why would we trade him? I don't understand that. That's nothing that's been discussed here. It's not going to happen."
HoopsVibe's Very Quick Call: Enough was enough –at least for Phoenix Suns Coach Alvin Gentry.
This morning, Gentry emphatically shot down rumors the Suns were trading superstar point guard and franchise face Steve Nash.
According to Gentry, Nash isn't available. He's too valuable to the Suns and their 'Seven Seconds Or Less' system.
Of course, Gentry has a point. Nash has two years and $22 million left on his contract, so the Suns ultimately control his fate.
However, Nash, a two-time MVP, has leverage. Fans sympathize with him because Phoenix owner Robert Sarver is more concerned with turning a profit than winning a championship.
Fans would understand if he asked for a trade. Sarver would likely accommodate. And Gentry's opinion, frankly, wouldn't matter.


COVENTRY City's Player of the Season Gary Breen faces a hernia operation.
Breen was forced to pull out of the Republic of Ireland's World Cup qualifying game against Portugal at Lansdowne Road on Saturday and will also miss Wednesday's game against Estonia.
He will now go under the surgeon's knife to get the problem sorted out as quickly as possible.
Breen has not played since suffering the injury in the 3-2 defeat against Aston Villa on May 5 that relegated City.


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A comic served up fresh, despite Tony controlling the elements while Wes made a witch's brew.
I don't care if I'm not supposed to talk about it, the hidden comic made this 10 times better than it already was.
I think we have found a game winning formula here.
What happens to him in a ball pit?
Apparently, he's his team's secret weapon…victory through creepout!
They're not ready yet to do it without gloves.
It was TMI…but I guess you can't live in a bubble your whole life…mmmmm giant bubble.
Alas, our love can not be. The referee limits our time together for 6 seconds, and threatens to separate us forever for "delaying the game".
If that's wrong, who wants to be right?
I was almost expecting the hidden comic to be an animated gif similar to the one from the ASP Short 'Mugger'.


A few years ago, as a project for my photography course, we had to register the festival known as, "El Señor de los Milagros" (The Lord of Miracles). It was a traditional event brought to Chile by their Peruvian residents that celebrate "Cristo de Pachacamilla" (Christ of Pachamilla).
The image of Christ is carried on the back of several devotees. I'm not sure the exact weight of it, but I could tell that it was very heavy. This festival is done every year on October 18 and 19, reuniting the Peruvian immigrants in Santiago that pay their homage to the image.
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A quick, delicious, and hearty salad; perfect for any occasion! 5 Minute Bean and Chickpea Salad is a BBQ, picnic, and potluck favourite!
Dear Reader, if there's one thing I have learned more than anything over the past five years, it's that no matter how prepared I think am, there's always going to be hitch. You see, I've never been diagnosed with OCD, but I've been accused of being OCD many, many, many times. And, I don't object, because if I'm to tell the truth here, I think I just might be.
In a previous life, when I was married, we were young and inexperienced, so we didn't often do much that required excess preparation or planning. We were simple, stay-at-home folk, with a new baby and bills. I'm sure some of you can relate. Well, fast forward a few years and the marriage has dissolved, the baby has grown into a beautiful and talented young woman, and my adult life finds me with more time on my hands.
I'm a planner. I plan to the point of talking myself into something and talking myself right out of it again. I plan excessively! But, in contrast to that, I'm also quite impulsive. So, it's always an internal battle. It drives John.e crazy, and I know this, because he has politely told me so on several occasions.
But, in my defense, Dear Reader, I tend to over-plan, over-prepare, and over-organize, because he doesn't do any of those things. John.e is more of a spontaneous person when it comes to doing something. Let me give you an example. Picture it: it's Saturday evening; let's say it's around 5 pm. "Let's go to a movie tonight," he says. Fear and panic immediately stricken me and I scan my thoughts for good excuses.
You see, it's not because I don't want to go to a movie, it's because I now don't have time to think about it or plan it. What will we see? Should we have purchased tickets ahead of time? What will I wear? What theatre will we go to? Will we drive or will we take the subway? Did I have dinner plans already and will popcorn fit into my calorie count for the day? (That one is kind of a big deal!) Is it opening weekend? Will it be too crowded? What's the weather like outside and do I have a jacket to suit my outfit?
When we spend a long period of time at the cottage, and not just a quick weekend, I like to take advantage of the outdoor grill so that I can add grilling recipes to Lord Byron's Kitchen. That also takes planning. So, I prepare a list of things I'll need based on each recipe and how I would like to style the photos for the post. What plates would I like to use? Do I need props? Should I bring utensils or use the utensils at the cottage? I will even think about if I need any special tools, such as a good knife, aluminum foil, a cheese board, or baking dishes. Two years ago, I even brought along baking sheets and a muffin tin!
With all of that planning, it's no wonder that sometimes my soul searches for something quick and easy; something that takes no planning at all. My 5 Minute Bean and Chickpea Salad solves all of those pesky planning issues, really. It's probably the easiest recipe you'll fine at Lord Byron's Kitchen!
So, if you're like me, and you like to plan everything, keep this 5 Minute Bean and Chickpea Salad recipe on hand for those terrible moments when life throws you a curve ball, and planning ahead is just not an option. Besides, the salad is delicious, so make it as a part of your meal plans too!
In a large mixing or salad bowl, whisk together the garlic, olive oil, vinegar, salt, lemon juice, and black pepper.
Add in the beans, chickpeas, and parsley. Toss to coat.
Refrigerate for two hours for best results.
You are most certainly not alone, Ella. And, thank you for your lovely comments. Have a great trip! Sounds wonderful!!


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We also supply high quality LED controls from the leading manufacturers Sunricher & LTech.
The controls that we offer start from the simple single colour LED dimmers to full LED DMX control and the new WiFi Smartphone and Iphone LED lighting control App's (App's are free to download from our site).
We are constantly adding to our selection of LED lighting control, lighting and other equipment! So please come back and visit us every so often to see what we have added.
Please checkout our range of Sunricher LED controllers!


Welcome to the Utah Viola Society!
We are a fun-loving group of alto-clef reading violists who get together regularly to celebrate all things Viola! We are interested in promoting your recital, helping find teachers for students, providing masterclass opportunities with world renown guest-artists, sponsoring guest recitals, providing information about all things VIOLA! If you are interested in joining us in our alto clef adventures, please sign up for our newsletter or subscribe to our blog. You'll stay on top of all things Viola in the Beehive State.
Join us on September 29 for Viola Day 2018! Our special guest will be Brant Bayless, Principal Viola of the Utah Symphony.
1:15 Meet Your Maker: Try out all the violas!


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Smell of Burning Plastic and Smoke Reported On the ISS; Space Station Aging Fast
The alarms on the International Space Station went off on Thursday after the smell of burning plastic and smoke was detected. The crew saw the smoke and reported it happened when the batteries of the ISS were being charged. The incident happened at the Russian-built Zvezda module which serves as living quarters.
The Russian space agency Roscosmos said the crew activated air filters to suck away the smoke and smell of plastic before retiring for the night after the air quality was restored to normal. A spacewalk already scheduled for Thursday is still billed to hold without any technical fears. The spacewalk will be to fully integrate the Russian-made Nauka science lab with the ISS; the ISS was slightly knocked out of position when the lab docked at the space outpost in July.
Russian authorities have expressed concerns that the ISS is getting old and should be replaced. In fact, the Russian government said in April that they would construct their own space station before 2030, but there has not been any further news on the proposed project. Russia said several failures have been occurring at the International Space Station due to outdated hardware and software. These have led to air leaks, misfiring engines, and cracks among other unexpected dysfunctions.
The ISS was constructed in 1998 as a joint project by the US, Russia, Japan, Canada, and other European partners. It was supposed to have a lifespan of 15 years, but it is now 23 years in orbiting operation and Roscosmos said it may not function beyond 2030 due to structural fatigue. Russia's Deputy Prime Minister Yuri Borisov said using the space station beyond that time could "lead to irreversible consequences – to catastrophe."
As at the moment, there are seven astronauts on the space station. Two from Russia, three from the United States, one from France, and one from Japan; Mark Vande Hei, Shane Kimbrough, and Megan McArthur from the US; Oleg Novitsky and Pyotr Dubrov from Russia; Akihiko Hoshide from Japan; and Thomas Pesquet from France. Novitsky and Dubrov are the ones to do the spacewalk required to integrate the Nauka science lab with the ISS.
JWST Discovers Carbon-Dioxide in Exoplanet Outside Our Solar System
Google Celebrates Stephen Hawking's Posthumous 80th Birthday with Captivating Doodle
Elon Musk's SpaceX Launches Four Astronauts on NASA's Behalf to the ISS
Meteorite Crashes through Roof and Lands on a Sleeping Woman's Pillow
All You Need to Know About Pool Chemicals
Unknown Space Junk Hits and Damages the International Space Station


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De Universiteit van Calcutta (Engels: University of Calcutta of Calcutta University; Bengaals: কলিকাতা বিশ্ববিদ্যালয়) is de oudste moderne universiteit van India. De universiteit, gelegen in Calcutta, wordt bestuurd door de overheid van de deelstaat West-Bengalen en kent diverse geaffilieerde onderzoeksinstituten.

De centrale campus (Ashutosh Shiksha Prangan) ligt aan College Street, maar er zijn ook campussen in Rajabazar (Rashbehari Shiksha Prangan), Ballygunge (Taraknath Palit Shiksha Prangan), Alipore (Sahid Khudiram Siksha Prangan), Hazra en Zuid-Sinthi.

Geschiedenis
De Universiteit van Calcutta is de oudste van de moderne universiteiten in India. De universiteit is in 1857 opgericht onder het bestuur van Charles Canning, de toenmalige gouverneur-generaal. Frederick John, de verantwoordelijke voor onderwijs bij het Britse bewind in India, had al het idee in Londen geprobeerd erdoor te krijgen om in Calcutta een universiteit op te richten naar Londens model. Hiermee wilde hij een geleerde bovenklasse creëren in India die de Britten zou helpen bij het besturen van India. In eerste instantie was hij echter niet succesvol hiervoor de benodigde steun te verkrijgen.

Later, in 1854, werd wel een voorstel geaccepteerd om zowel in Calcutta als in Bombay een universiteit op te richten, en de Calcutta University Act werd op 24 januari 1857 van kracht. Een senaat van 41 leden werd gevormd als beleidsmakend college van de universiteit.

De eerste kanselier en vicekanselier van de universiteit waren respectievelijk de toenmalig gouverneur-generaal Canning (van ambtswege) en de voorzitter van het hooggerechtshof James William Colvile. In maart 1857 werd het eerste toelatingsexamen afgenomen in het stadhuis van Calcutta onder 244 kandidaten. In 1858 werd naast de senaat ook een syndicaat ingesteld om mee te beslissen in het beleid van de universiteit.

De senaat hield in het begin haar bijeenkomsten in de raadskamer van het al eerder opgerichte Calcutta Medical College, terwijl het kantoor gevestigd werd in enkele gehuurde kamers aan Camac Street. De jaren erna zou het Writers' Building de ontmoetingsplek vormen voor de senaat en het syndicaat. Het zou tot 1862 duren voor de senaat besloot voor de universiteit een eigen gebouw te laten neerzetten. Vervolgens werd de Senate Hall gebouwd, die in 1873 werd geïnaugureerd.

In de loop der jaren werden vele instituten aan de universiteit geaffilieerd, zoals in 1857 het Nawab Singh Ahluwalia Government College in Kapurthala (Punjab), een van de eerste.

In 1858 studeerden Joddu Nath Bose en Bankim Chandra Chattopadhyay als eerste studenten af aan de universiteit, en in 1882 studeerden de eerste vrouwelijke studentes af: Kadambini Ganguly en Chandramukhi Basu.

In 1890 werd de eerste Indiase vicekanselier benoemd, Gooroodas Banerjee.

Calcutta
Calcutta

redpajama_set_name: RedPajamaWikipedia


textfiles.com has alexa rank of #62,463 in the world, with roughly 15404 daily unique visitors. This website has a Google PageRank of 5/10. It is a domain having .com extension and is hosted in United States. textfiles.com estimated worth is: $133,200.00 and have a daily income of around $185.00. This website is also listed on Dmoz. As no active threats were reported recently, textfiles.com is SAFE to browse.


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in: History of Morocco, Conflicts in 1911, Conflicts in 1912,
Wars involving France
Wars involving Morocco
French Morocco
France–Morocco relations
French conquest of Morocco
French artillery at Rabat in 1911
Franco-Moroccan conflicts
Larache expedition (1765)
Conquest of Algeria (1830–47)
Franco-Moroccan War (1844)
Bombardment of Salé (1851)
Conquest of Morocco (1911–12)
Zaian War (1914–21)
Rif War (1920–26)
Ifni War (1957–58)
The French conquest of Morocco took place in 1911 in the aftermath of the Agadir Crisis, when Moroccan forces besieged the French-occupied city of Fez. Approximately one month later, French forces brought the siege to an end. On 30 March 1912, Sultan Abdelhafid signed the Treaty of Fez, formally ceding Moroccan sovereignty to France, which established a protectorate. On 17 April 1912, Moroccan infantrymen mutinied in the French garrison in Fez. The Moroccans were unable to take the city and were defeated by a French relief force. In late May 1912, Moroccan forces unsuccessfully attacked the enhanced French garrison at Fez. The last aftermath of the conquest of Morocco occurred in September 1912 when French colonial forces under Colonel Mangin defeated Moroccan resistance at the Battle of Sidi Bou Othman.
France-Morocco relations
History of Morocco
Conflicts in 1911


"Doug dug." by The Electric Toy Company is my current iOS commuting game of choice (previous title holders include: Bejeweled Blitz, Happy Street, Puzzle & Dragons and World's Biggest Picture Cross).
While Doug Dug initially feels like a quite shallow (and in some ways unfairly random) game, there are quite a lot of subtle design features that only make themselves apparent with extended play.
Doug Dug has two stated objectives: collect as much treasure as possible to gain a high score, and dig as deeply as possible (triggering the brilliant message "You have achieved a new low") without dying. There's no time limit or forced scrolling so the player is free to plan their moves (most of the time) without having to perform digital gymnastics.
Once the player learns the basic rules of how the game works, they can stop playing reactively, and start thinking about how to use the environment to achieve their immediate goals. Below I've listed some of the rules I've observed to give you a head start.
The treasure that Doug the dwarf seeks is encountered in several states.
Treasure that can be mined out of blocks is always visible and is instantly collected when a block is mined out by Doug. It takes the form of precious gold, so-so silver or shameful bronze ore (worth $300, $200 and $100 respectively) as well as coloured gems that become more valuable (up to $5,000 in my experience) the deeper one goes. If a block is destroyed by falling the treasure is destroyed with it. The easiest and stupidest way to die in Doug Dug is to tunnel horizontally through a cliff to reach a distant treasure, resulting in a cave-in.
Treasure dropped by killed enemies falls until it comes to rest on a flat surface. While this treasure only comes in gold/silver/bronze varieties, later enemies can drop many pieces. Loose treasure has some interesting properties: dragon's fire and lava particles are extinguished when they come into contact with it (so for instance, a dragon surrounded by loose nuggets is effectively harmless) and likewise rocks falling onto it are instantly destroyed. Unlike in Spelunky, loose treasure falling on enemies doesn't hurt them.
Background treasure is fixed into the back wall, and moves in slight parallax with the foreground scene. It's collected instantly on contact. It's worth noting that treasure (and items) within two spaces above Doug can be collected by swiping upwards, so sometimes it's possible to use air control to pluck background treasure even when there's no platform nearby.
Finally there are treasure chests, which can contain a random amount of treasure or which will spawn a random item (never the same as the one being carried by the player) or a lit bomb when opened.
A key thing to know if you're chasing a high score is that the game has a combo system: if three or more pieces of treasure are collected within about a second of each other, a Treasure Trove bonus of $1,000 x the number of pieces of treasure is awarded. This can often be exploited through careful route planning and 'pre-weakening' treasure-bearing blocks.
Collectible items that bestow temporary or permanent effects can sometimes be found in chests and crates, or just lying around in the world. Doug has a single inventory slot that can be used to store items for later activation. The last three items on the list are only found in crates and have a state purchase cost to access.
Gold Rush: Doug can dig faster for a limited time. Useful for out-running enemies or just to relieve the tedium of clearing a particularly dense expanse of rock.
Feather Fall: Doug can fall any distance without injury. Can be used mid-fall. In a game where lots of cheap deaths are caused by leaps of faith and collapsing floors, it's at least some solace.
Support: Makes a single block sized 'roof' that Doug can stand within to be safe from collapsing rocks. It can also be dropped on enemies.
Hard Hat: Essentially an extra life. Protects Doug from one instance of something that would kill him. Doesn't use an inventory slot. Useless against deep lava.
Jackhammer: Gold Rush on steroids. Doug is briefly invincible and constantly digs downward, although he can be steered to destroy blocks and enemies to his sides. Particularly useful for dealing with massive amounts of lava and trolls.
Invincibility: Doug becomes impervious to all hazards (including burning and crushing) for a few seconds. Also seems to speed up digging.
Rats: The weakest enemy, rats can move left or right and burrow through blocks in front of them. The can be killed by running into them or falling on them. Very often they commit suicide by digging out the support for blocks above them.
Bats: As in Spelunky, these are treacherous little swine. Bats fly in any direction until they hit a wall, and then turn 90 degrees. Doug can despatch them by 'digging' at them in any direction, but there are annoying edge cases where a bat will clip the player's hitbox off-centre and still kill them. As the game only tracks a few tiles above the top of the screen before garbage collecting, bats that fly off the top of the screen seldom return.
Dragons: Added in an update, dragons move similarly to rats, however they can randomly decide to stop walking and spew a stream of fire particles. (Their eyes flash momentarily to warn of this.) At close range their fire can destroy blocks and chests (and other enemies). While on the same level they can usually be easily dealt with, they can be extremely dangerous if Doug gets trapped downhill to one.
Mummies: Mummies are two squares tall and can be dealt with on open ground in the same way as rats and dragons. However, they're not affected by walls and falling rocks: if a mummy doesn't have enough room to stand on a platform, it will transform into a ghost and start slowly zig-zagging up the screen until it finds enough space to rematerialise. The strategy for dealing with them then becomes clearing enough space for them to spawn and chase Doug.
As with treasure, there's a combo system for making multiple kills – this only counts for kills Doug makes directly.
Lava: Fluid is simulated in the game as a collection of loosely jiggling particles which each have a heat/energy value (denoted by colour). Lava contained in a tight space will stay 'hot', whereas if its container is broken, it will slowly tumble downhill until it finds another container or else will slowly disperse. Falling rocks extinguish lava, so you can build stepping stones or bridges by engineering rockfalls above lava fields.
Dirt: The softest material that Doug can dig through is also the most dangerous. Dirt has very few 'health points', and can be weakened by too much weight, or sometimes just spontaneously, resulting in massive chain reaction collapses. When digging through dirt it's usually worth waiting and observing if surrounding pieces are cracking, and to ensure there's a clear escape route.
Falling: Doug can sometimes 'surf' on falling rocks, repeatedly landing on them as he falls, which cushions otherwise fatal long falls.
You can buy "Doug dug." on iOS and Android.


Today I thought that I would share some pics of places that we visited while on vacation.
One day we ended up at St. George Island. Its probably a good hour away from where we were staying. You have to drive across a long bridge to get there and when you finally arrive there are are homes and a few shops and a few restaurants. At the end of the island is the National Park that seemed to go on forever. The beach was covered in seashells which was beautiful!
The sand here was white and the water was so much better than where we were staying. It was a mixture of the greenish brown with some blue.
The neat thing about St. George Island were the sea turtles! Everywhere you looked you could find their nesting holes. It was pretty cool. We wished we would have been able to watch them hatch and scurry off to the ocean.
On another outing we drove about 25 minutes from Mexico Beach to Cape San Blas. You could actually see the island off in the distance from where we were staying. On one side of the island is the bay which had the exact same water we had. No bueno. It was as dark as rooter for a few days. Then on the other side you had massive sand dunes or rock walls. You had no idea what was on the other side of them.
Just over the dunes it was like I heard the angels singing! Okay, I am a little dramatic but it was sheer delight.
We had never seen such an amazing beach! Cape San Blas was GORGEOUS!
Oldest & Littliest goofing off the first day we visited. The beach had very few people.
It is so funny how different the water was here vs where we were staying.
I would love to rent a house on Cape San Blas.
Another area of the island all you see is a giant rock wall. Well I of course had to hop out to see what was on the other side. It was so pretty! Reminded me of some of the beaches we had seen in Maui.
There was path you could walk down to get to the beach. If you look in the distance you can see the giant rock wall.
Oldest & Littlest really enjoyed this beach. It was full of stumps and driftwood which made great for exploring.
Look at this awesome piece of drift wood!
This portion of the Florida Panhandle is perfect for those who like quiet remoteness with nothing but the beach. It is divine for sure and such a nice change of pace from all the congestion of the busier beaches. I could have staid forever.
The water cleared where we were staying just in time to enjoy our last day and we forever will be thankful for the memories we made.
They were fabulous places and we hated leaving. Wish we lived closer!
Beautiful Pamela I love those quieter beaches too. Looks like a wonderful vacation.
Nothing beats the quiet beaches Kris.Thanx for visiting friend!
What a beautiful place, Pamela! LOVE the shells on the beach. A perfect place to make memories before school begins!
It certainly was Yvonne! Memories the boys will always have. We hope to one day own a small place so that we can meet up with the grandbabies and keep the tradition going.
Your pictures are gorgeous! I have been to St. George's Island before but not Cape San Blas. They are very remote aren't they?
Yes, they are very remote Kelly. I did not really mind it but Mr. Front Porch and his daddy were not too fond of that aspect. They need things to do when they get bored that are not an hour away.
Looks like a wonderful place for a vacation, Pamela, and your photos are beautiful!
Thank you so much for your kind words of encouragement and prayers. I truly appreciate it!
Thanks for sharing Pamela...I haven't been to that area in years (more like 25 years).
I didn't know about St. George Island and will have to check it out. Is it dog friendly? Many places don't allow dogs on the beach.
Going to check out your other Florida links. We are in North Florida, and love to find new places to visit.
What a beautiful remote place. Everything about it is magical, sand, lots of shells, the blue ocean and sea turtles! Plus, it doesn't seem crowded, which for me would make it perfect. Happy weekend!


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Home Economy Navigating in Montpellier: Michael Delavos answers your questions on Mondays at 12:30pm.
Navigating in Montpellier: Michael Delavos answers your questions on Mondays at 12:30pm.
The day after the publication of our profile dedicated to Montpellier traffic, the Mayor and President of the city will live on Midilibre.fr and our Facebook page "Midi Libre Montpellier".
In our edition of this Sunday, December 12, dedicated to travel to Montpellier, Michael Delafoss, the city's mayor and president, said he "carries" his options. That is, its desire to develop alternatives to the car, to encourage motorists to change their habits and thus reduce traffic, and thus pollution, in the city. A policy he says is "proactive", and even if it has consequences for traffic, it is always more complex.
Since the subject is sensitive, Mr. Delafosse has agreed to answer questions from readers and netizens at Midi Libre, on Monday 13th December at 12:30pm. It was initially slated to be its Vice President for Mobility, Julie Frisch, who made an appointment with you but a personal handicap that prompted the mayor to take his place.
You can send your questions to him by writing to us via email at [email protected] The interview will be followed on our Facebook page.Midi Montpellier Freeor on Midilibre.fr
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Seven sick from Listeria in Canada
By Joseph James Whitworth contact
21-Jan-2016 - Last updated on 21-Jan-2016 at 17:18 GMT
Pre-packaged leafy greens and salad blends are being investigated as the source
Related tags: Listeria monocytogenes
Canada is investigating seven cases of Listeria monocytogenes with a link to pre-packaged leafy greens, salad blends and kits.
All cases have been hospitalized, and one person has died, however it is not clear if Listeria contributed to the cause of death.
The Public Health Agency of Canada (PHAC) said infections have been reported in five provinces: Ontario (3), Quebec (1), New Brunswick (1), Prince Edward Island (1), and Newfoundland and Labrador (1).
It said the risk is low, but given that Listeria can cause severe illness to some high-risk groups, Canadians should review and follow proper safe food handling practices in an effort to prevent illnesses.
Individuals became sick between September and early January 2016. The majority of cases (71%) are female, with an average age of 81 years.
In the milder form of the disease, symptoms can start the following day after eating a contaminated product. For the more serious form of the disease, the incubation period is generally much longer; on average about 21 days, but can be up to 70 days after exposure.
Link to leafy greens and salad
The source has not been confirmed but pre-packaged leafy greens, salad blends and salad kits are food items being investigated.
Dr Eric Hoskins, Ontario minister of health and long term care and Dr David Williams, acting chief medical officer of health, said they are working with Public Health Ontario and provincial partners to investigate the Listeria monocytogenes infections.
"Some of these cases are also part of a federally led national investigation," they said.
"To date, the source of these Listeriosis infections has not been confirmed. We will update the public as new information from the ongoing investigation becomes available.
"We will continue to work closely with our partners and will provide additional updates as more information becomes available."
Blue Bell update
Meanwhile, Blue Bell Ice Cream has entered phase five of its market re-entry plan.
The firm also gave an update on progress with its enhanced testing in production facilities.
It has identified suspected areas where bacteria may be present but has not found Listeria monocytogenes.
"We have identified locations where suspected Listeria species may be present in our facility, and we continue to extensively clean and sanitize those areas and make additional enhancements to the facility and our procedures based on the environmental test results," said the firm.
"To confirm that our robust environmental program is effective, and that our "seek and destroy" goals are being achieved, we expect to periodically find microbiological indications in our facilities.
"The entire purpose of our enhanced environmental testing is to identify locations where bacteria could be found in our facility in order to properly clean and sanitize the surface and prevent contamination.
"We have tested and will continue to test every batch of ice cream produced. No products produced have tested positive. No products are shipped to stores until tests confirm they are safe."
Related topics: Food safety and labeling
Salad contaminated with Listeria sickens 33
Dole halts production as part of Listeria outbreak investigation
Canadian listeria precautions exceed regulations - company
How food manufacturers are moving to clean labels
Oakland Innovation Ltd. | 01-Jul-2019 | Technical / White Paper
Today's consumers are more concerned with the integrity of the food and drink they consume so how should manufacturers of food and beverage products respond?
4 best practices to speed up product development
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New product development projects may appear to be unsolvable equations of ever-changing criteria: consumer demand, economic and political influence on...
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By GivaudanGivaudan Kitchen Ingredients – meeting consumer demand for clear and clean labelling


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Q: Mensagem quando excede tamanho de FileUpload Estou tentando colocar uma mensagem para quando exceder o tamanho do arquivo no FileUpload, já arrumei dentro do web config, porém sempre me retorna o seguinte erro:

Tamanho máximo de solicitação excedido.

Vi esse exemplo na internet, tentei adaptá-lo pro meu código porém continua aparecendo o erro acima:

<script type ="text/javascript">
        $(function () {
            $('<%= Anexo.ClientID %>').change(function () {

                //because this is single file upload I use only first index
                var f = this.files[0]

                //here I CHECK if the FILE SIZE is bigger than 8 MB (numbers below are in bytes)
                if (f.size > 8388608 || f.fileSize > 8388608) {
                    //show an alert to the user
                    alert("Allowed file size exceeded. (Max. 8 MB)")

                    //reset file upload control
                    this.value = null;
                }
            })
        });
    </script>

Já vi vários exemplos na internet, já tentei de várias formas, pesquisei aqui no fórum e em outros na internet, porém nenhum exemplo está ocorrendo a mensagem de que o tamanho foi excedido.

A: Na verdade este exemplo funcionou, estava faltando apenas colocar o # aqui: $('#<%= Anexo.ClientID %>') e deu certo.

redpajama_set_name: RedPajamaStackExchange


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Бирги́тта «Ги́ттан» Тёрн (; ) — шведская кёрлингистка.

Достижения
 : серебро ().
 : золото ().
 : золото (1979).
 : золото (, ), серебро (, ), бронза (, , ).
 : золото (1972).

Команды

(скипы выделены полужирным шрифтом)

Примечания

Кёрлингисты по алфавиту
Кёрлингисты Швеции
Чемпионы мира по кёрлингу
Чемпионы Швеции по кёрлингу


Featuring a longer wheelbase, plus a rear end that is short and sharply slanted, the Gran Coupe takes the convenience of four doors and adds the styling cues of a two-door model. It's a vehicle philosophy that seeks to combine luxury, performance, and functionality into one handsome package.The vehicle includes stylish exterior styling tweaks and additional options for the interior and body color paint scheme. Beyond that, all the luxury and performance you'd expect from a world-class Bavarian import is present and correct.


To be honest, I was too lazy to find out if this has been discussed already somewhere.
Like others I try to avoid infills as they are time consuming and don't add much strength to the part. Main reason for using them is to provide some substructure for large top surfaces.
I don't like the small "enforcement" zig-zag infills close to slanted walls very much either. When not printed very slowly the corners tend to curl up, giving a quite rough surface which may cause additional trouble.
And I don't like the additional retractions caused by infills. Less retractions are always better.
So why not add an additional innermost perimeter line that oscillates inwards (in direction of the path normal) instead of infills?
Amplitude and frequency should be adjustable. With high amplitude it would provide support for top surfaces. It also adds structural strength because it is partially fused with the perimeter. With high frequency (and low amplitude) it would replace the enforcement infills for slanted walls. And there are no additional retractions.
As the path follows the topology of the perimeter it should be as aligned to the layer beneath as the perimeter (... at least hoping that, I haven't studied maths ...). If calculations for smooth corners are too complex, sharp corners should work as well. Of course the oscillating would have to be clipped at certain places like tight bends.
- Is one layer enough to make it stable?
- Is it visible on the outside of the print (there is such an effect with injection moulding)?
- What is the most suitable type of oscillating curve?
I assume a thickness of 0.4 mm should work quite well. If not, two oscillating lines will do it.
It might be visible, depending on wall thickness and material color, like the normal infill.
The curve is an interesting question. I like the idea of having longer segments in touch with the perimeter and peaks facing inwards, like bracings. However the peaks should be printable without any risk due to the sharp corners.
Some narrow Gaussian might do the job. Practically it would be rather a convolution of a Rectangle with a Gaussian. The smooth tip should lead to shorter print times compared to a sharp peak.
One of the things I would really like to see, is an infill that automatically adjust so that it is less dense at the bottom and increases in density over height to support the top layer. Something where you would tell the computer the maximum unsupported span and maximum angle of an overhang and it would calculate a suitable branching infill pattern.
That would be a very nice feature, indeed!
What about randomly rotating the infill pattern with every layer? Sort of making a birds nest infill.


text:
Deposit Information:
Pay/View Your Bill
Click Here for Tombigbee Fiber >>>
Tombigbee Electric Power Association (TEPA) is a non-profit, customer owned utility dedicated to providing electric service to residential, commercial, industrial, and public facilities.
On February 26, 1934, a charter was granted to the Lee County Rural Electric Power Association. In June 1934, the Tennessee Valley Authority (TVA) took over from the Mississippi Power Company lines serving electric customers at Guntown, Saltillo, Sherman, Shannon, Nettleton, Plantersville, Verona, East Tupelo, Auburn, Chesterville, and Belden in Lee County and Fulton in Itawamba County. The Lee County EPA opened for business with 6 employees.
On June 25, 1935, Mr. Louis Cook was appointed manager of the Lee County EPA, and during that year the name was changed to Tombigbee Electric Power Association. On October 10, 1935, TEPA bought from TVA all of their distribution lines in the area and began operation as a co-operative with 168 miles of line and 151 members.
On November 1, 1967, Mr. James M. Allen succeeded Mr. Cook as manager of TEPA. As of December 1, 1969, TEPA had 1933 miles of line and 16,505 members and 95 employees.
On June 30, 1995, Mr. William W. Long became the third manager of TEPA. As of April 2007, TEPA has 3,814 miles of line and 40,094 customers and 133 employees.
TEPA has sustained 4 severe ice storms and numerous tornadoes. TEPA has always been a distributor of TVA power.
SERVICE PRACTICE POLICIES
Stay up to date with our company policies.
Find out if there are any job opportunities available at TEPA.
Click here to see our By-Laws.
Check out our company's photo album.
Click here to see our current board members.
Click here to see our contact information.
Tupelo Office: (662) 842-7635
Fulton Office: (662) 862-3146
© 2020 Tombigbee Electric Power Association


wan·der·lust 🌍✈👸🏼 /ˈwändərˌləst/ noun a strong desire to travel. "a woman consumed by wanderlust"
The light at the end of the (ice cave) tunnel 🚇❄ • • • Getting to see an ice cave was well worth the day of traveling and less than comfortable drive on a glacier. It's as ... cool 🤣 ... as it looks!
23:23 do dia 23 de abril de 2019. Nossa viagem de lua de mel. Nosso presente. 💚 Eu acho que nunca irei conseguir definir o que foi esse dia. Só sei que foi perfeito, como Deus quis que fosse.
A unique sight where you feel connected to the power of the planet.


Content marketing is a strategic marketing approach focused on creating and distributing valuable, relevant and consistent content to attract customers and ultimately drive profitable customer action. Every company has different type of content depending on the product it offers in the market. Content Marketing furnishes a platform to communicate with the customers instead of selling to them directly. For, a content marketing to work efficiently, it is important that it must work simultaneously with a good social media strategy. This is also true in the case of web content. However, content marketing faces several challenges like measuring the Right of Information (ROI) is very difficult and it is essential to have a call-to-action so as to attract customer for purchasing the products.


Renee interviewed two dog trainers today: Nannette Morgan and Jo Jacques. Both trainers are extremely knowledgeable about Siberian Husky sled dogs. They discussed an incident that occurred after the 2010 Winter Olympics where 100 sled dogs were inhumanely destroyed at a tourist center in Vancouver, British Columbia. Nannette and Jo also talked about how sled dogs can easily be re-homed despite the fact that they've lived outdoors on a tether all of their lives. If you love Huskies, you'll enjoy this program!
This entry was posted in Specific Breeds on March 24, 2011 by renee.