text:
Foods we weren't meant to eat!
Tips on how to beat cancer, from 100-year-old farmer Percy Weston
We were not designed for this! These bodies we live in were Not designed for foods saturated with preservatives, antibiotics, artificial flavours, chemical fertilisers, pesticides and hormones, the doctor warned.
Nor were our bodies designed for foods with an altered molecular structure---- foods ruined by irradiation, microwave cooking, deep frying or scalding high temperatures (over cooked), according to David A Darbro MD. He wrote this in his foreword to a "commonsense guide for eliminating sickness through nutrition" a decade ago. Sixty years earlier a well-read farmer named Percy George Weston had a similar line of thought. A keen-eyed, diminutive dynamo who rarely became sick, found at the age of 31, in the prime of life, he was burnt out and drained of energy. His doctor diagnosed anemia.
Weston wondered if the typical Australian diet was to blame, perhaps lacking in certain mineral elements. Investigation revealed the pattern. His staple foods-such as porridge, bacon and eggs, meats, bread, beans, macaroni, pork, white rice, potatoes and tomatoes-contained a greater abundance of acid minerals such as phosphorus and chlorine than alkaline minerals such as calcium, potassium and magnesium. Was this constant diet making his body too acid to function properly? He suspected his growing the vegetables in a field that he regularly fertilized with superphosphate (to please Virginian leaf tobacco buyers), had made matters worse. He knew from his field experiments that "constant use of super-soluble phosphatic fertilizers could skew the take-up of minerals and trace elements in food crops, producing mineral-poor foods," as he was to write in his book Cancer: Cause & Cure. Nature's Secret Exposed (Bookbin) first published in 2001.
Percy Weston made a full and rapid recovery by switching to a set of foods with abundant alkaline minerals, including fruits and vegetables from his father's organic orchard. He became convinced of the need to formulate a cellular dietary supplement to neutralise acids in the body and to correct mineral imbalances in the food chain.
The Spur to its development came with a bout of arthritis in his forties, when he was able to put his studies of crop nutrition and observation of mineral preferences of sheep to good advantage. A largely vegetarian diet and the new mineral supplement worked wonders. It was the beginning. The fine-tuning of the formula was to take decades.
Concerns about health drove him on, especially important after World War 2 when agricultural chemicals flooded on to the market and he was advised by "experts" to use them.
Yet when he did so, he had problems with insect infestation and animal mortality. For the district as a whole, the cancer rate was rising. Finally he came down with cancer himself. Self-reliance was the name of the game as he had few financial resources and felt he could no longer trust the scientific community and doctors, whose advice so often turned out to be wHe cured himself with diet and the mineral powder.
As the fame of his Diet and Mineral supplement for humans spread, especially after dramatic recoveries from cancer of family and friends, documented in the book - Cancer Cause & Cure, he became very busy on his sheep and walnut farm at Eurobin, Victoria, Australia, opposite Mt Buffalo National Park. Hundreds of entries in his visitors' book are testimony to this. They came to buy Percy's mineral powder and they listened to his advice as one who speaks with authority on the subject.
Three years into the 21st Century, centenarian Percy, as few others could look back to a time when commercially grown fruits and vegetables possessed home grown qualities of distinct flavour and freshness. There were more varieties and flavours of apples available before the era of atomic weapon testing and the ages of mass production. Farmers had fewer problems with insects before chemists unleashed potent pesticides that killed both prey and predator and the chemical companies upset the biological balance of the soil web with new super-soluble fertilizers. Grazing animals thrived on natural pastures in good country that was not over-stocked and thereby not quickly depleted of trace minerals. Most of Percy's father's generation led active lives into their 70s and well beyond without succumbing to cancer, arthritis, diabetes, obesity and many other "modern" metabolic and degenerative diseases.
Percy's advice is, we eat "living food", i.e. organic food, preferably, freshly picked. You can tell the difference. High-quality produce gives you energy and you need less of it to feel replete. It is rich in minerals and will not readily rot. It will only dehydrate. Percy Weston saw from nature what man was doing wrong and provided a practical and affordable way to check disease and maintain wellness. He grew most of his food in good soil by organic methods and supplemented with sprinklings of his mineral formula that ionizes in water (Percy's Powder®), or better still, the powder stirred into a glass of cold water with juice of half a lemon. The mixture-key electrolytes present in exact ratios of bioavailable sulphates- is now made available to all.
Percy Weston's mission: To heal a sick world.
Percy Weston's mission is to inform you that: You do Not need to die, or suffer from the ill effects of cancer and any other degenerative disease. Percy shows you how you can claim back your health and lost vigor once and for all. Learn how to beat cancer and other diseases through dietary changes and specific mineral supplementation…..
Cancer, arthritis, obesity, heart disease, diabetes and depression - relatively unknown a century ago, have reached epidemic proportions globally. These modern day afflictions are ravaging the Western World at an alarming rate, and show no signs of slowing down.
Many health researchers would agree that the primary cause of modern day diseases and poor health has a lot to do with our: toxic & acidic diets; stressful lifestyles - both in the workplace and household; polluted environments; poor water & air quality; exposure to chemicals & pesticides; excess reliance on pharmaceuticals; the use of GE-grown crops & foods; and the consumption of empty, dead, or nutrient devoid foods.
It is a Fact that: One in Two men, and One in Three women will get some form of cancer in their life time - a frightening statistic indeed! However, this need not be the case for you or your loved one.
A 1.4g sachet of powder stirred into a glass of water with the juice of half a lemon makes a refreshing, effervescent alkaline drink.
Percy's Powder is a blood tonic, and helps maintain normal blood.
The mineral salts in Percy's Powder® readily dissolves in water (or fruit juice) to supply the body with a means of checking the build-up of acids and phosphate, and flushing them out in the urine. ( Acid build-up in the tissues causes infection and chronic disease.)
The minerals in Percy's Powder® are also involved in regulating water balance, in making new blood, in the operation of muscles and nerves, in the movement of oxygen and carbon dioxide, in transporting cell nutrients and regulating cell nutrition , in making enzymes involved in digestion, energy production and protein building, and in activating hormones that regulate many body processes.
Why take Percy's Powder® ?
Because modern diets, while containing these minerals, do not have enough of them to balance the abundant acid minerals coming from the soils conditioned by chemical fertilisers and processed additives.
Soft drinks, too ( i.e. Coke-Cola ) introduce acidity which the body has to work hard to neutralise in order for enzymes to function.
Percy's mineral salts are sulphates (unlike most others ). This means each one dissociates in water into ions, groups of atoms that the body can use immediately wherever they are needed, Sulphates disinfect the blood, resist bacteria, and detoxifies.
Each large display box of Percy's Powder contains 60 sachets ( Two months supply) of sulphates of magnesium, potassium, iron, zinc and manganese in the ratios perfected by Percy Weston over 60 years. The dosage is one a day, the ideal maintenance dose for people who weight up to 65kgs.
Dosage: (Adults and children over 12 years). Take one sachet of the powder daily in a glass of water. Stir well. Best taken with the juice of half a lemon, with grapefruit, or in grape or apple juice. May also be taken in tea.
Percy's Powder™ is best taken in conjunction with a vitamin supplement containing vitamin B12.


text:
|Home | About | Journals | Submit | Contact Us | Français
The cyclin-dependent kinase (CDK) inhibitor p27Kip1 is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCFSKP2, KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.
We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21Cip1, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.
Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.
p27 is a cyclin-dependent kinase (CDK) inhibitor (CKI) that controls cell proliferation, cell motility and apoptosis [1]. It regulates the progression of cells from G1 to S phase by binding and inhibiting the cyclin E-CDK2 complex. A plethora of evidence has implicated downregulation of p27 in prevalent human carcinomas [1]. For example, downregulation of p27 is among the most frequent non-genetic molecular alterations in prostate cancer (PCa) [2]. In this disease, low p27 expression is correlated with a number of prognostic morphological features [3] and with decreased survival [4]. In contrast, ectopic expression of p27 can inhibit cell cycle progression in a human PCa cell line [5], suppress astrocytoma growth in nude mice [6] and induce the death of breast cancer cells [7]. Based on these findings, p27 has been denoted as a tumour suppressor.
The regulation of p27 during the cell cycle is very complex [1]. It involves regulation at the level of transcription, messenger (m)RNA translation [8] and protein stability. The distribution among different cyclin-CDK complexes [9], its sub-cellular localization [10] as well as phosphorylation of several residues in p27 are important mechanisms of control [11-13]. p27 levels are high in quiescent cells and decrease rapidly upon mitogenic stimulation. However, the cell cycle-dependent variations in p27 levels are not reflected by similar changes in p27 mRNA [8,14]. Unlike traditional tumour suppressor genes, the p27 gene rarely undergoes homozygous inactivation in cancer cells [15-17], a finding that points towards alternative mechanisms of p27 inactivation. Many aggressive cancers display decreased p27 protein levels in the presence of high p27 mRNA [18,19], suggesting that p27 depletion is primarily a result of ectopic proteolysis.
The p27 protein accumulates in cells when the ubiquitin proteasome system (UPS) is inhibited [20]. This system employs a cascade of enzymatic reactions that covalently attach a ubiquitin chain to a substrate protein [21], leading to the recognition by the proteasome for degradation. Biochemical studies identified SCFSKP2, an ubiquitin ligase complex that mediates phosphorylation-dependent p27 ubiquitylation in vitro [13,22]. Two other enzymes, KPC and PIRH2, have been also been implicated as E3s for p27 [23-25]. Whereas SCFSKP2 mediates the degradation of nuclear p27 throughout S phase and G2, KPC targets cytoplasmic p27 upon cell cycle entry from G0 [23,25]; PIRH2 instead targets nuclear and cytoplasmic p27 [24].
Considerable evidence suggests, however, that SKP2 is the prominent regulator of p27 levels in cancer cells [26]. SKP2 overexpression is frequent in human carcinomas devoid of p27 [27-32]. In addition, our own data have shown that SKP2 overexpression in LNCaP prostate cancer cells is sufficient to direct p27 ubiquitylation and degradation [33]. Furthermore, transgenic expression of SKP2 in the mouse prostate causes low-grade prostate carcinomas that coincide with p27 downregulation [32]. Conversely, RNA interference (RNAi)-mediated knockdown of SKP2 expression inhibits tumour growth in a mouse transplant model [34]. These findings validated p27 degradation pathways as promising cancer drug targets [35].
The complexity of p27 regulation presents considerable challenges to drug development because of the potential for redundancies. Thus, it is not readily apparent which enzyme involved in p27 regulation should be targeted in order to effect sustained p27 accumulation in cancer cells. In addition, pharmacological agents active in an enzyme assay in vitro are not necessarily bioactive in intact cells. Rather than targeting a preselected component, we have developed a method for the up-front identification of compounds that are bioactive in restoring physiological levels of p27 in prostate cancer cells. Using a validated cell-based assay, two compounds, small molecule inhibitors of p27 depletions (SMIPs) 001 and 004, were identified, which had the desired activity. SMIPs restored p27 to physiological levels, inhibited CDK2 activity and caused cell cycle delay or apoptosis selectively in prostate cancer cells but not normal cells.
Prostate cancers typically display an inverse correlation between the levels of p27 and SKP2 [29]. In contrast, the commonly used human prostate cancer cell line LNCaP, despite faithfully recapitulating many features of human prostate cancer [36], expresses relatively high levels of p27 but low levels of SKP2 when compared to HeLa cells (Figure (Figure1a).1a). In order to mimic the situation prevailing in primary prostate cancers, we created the LNCaP derivative cell line, LNCaP-S14, which stably overexpresses Myc-tagged SKP2 at six to eightfold excess over endogenous SKP2, a manoeuvre that led to maximal downregulation of p27 (Figure (Figure1b).1b). The same pattern was apparent by immunofluorescence staining; while nuclear levels of p27 are low in LNCaP-S14 cells, nuclear SKP2 levels are highly elevated (Figure (Figure1c1c).
We also determined whether known inhibitors of p27 degradation could upregulate p27 in LNCaP-S14 cells. Upon normalization to the loading control actin, the proteasome inhibitors MG132, epoxomycin and bortezomib caused between 1.2 and 1.7-fold upregulation of p27, while the CDK inhibitor roscovitine led to 1.5-fold accumulation (Figure (Figure1d).1d). The induction of nuclear p27 by MG132 and roscovitine was also apparent by immunofluorescence (Figure (Figure1e1e).
The immunofluorescence assay used in Figure Figure1c1c and and1e1e was adapted to 384 well plate format. All parameters, including the number of cells to be seeded, fixation, blocking conditions, antibody concentrations and incubation times with compounds were extensively optimized using positive (proteasome and CDK inhibitors) and negative (dimethyl sulfoxide; DMSO) controls, resulting in the reliable protocol described in the Methods section. Representative images of LNCaP-S14 cells treated with DMSO or roscovitine and stained with the above protocol in 384 well plates are shown in Figure Figure1f1f.
In order to evaluate the performance of our assay to reliably measure the percentage of p27 positive cells in a cell population, we determined the Z'-factor for the positive control reagent roscovitine. The Z'-factor measures the variation and separation bands of an assay thereby providing a statistical measure of its quality. It takes into account the dynamic range and variability of the positive and negative control measurements [37]. We determined a Z'-factor of 0.48 using the ImageXpress Micro and MetaXpress software from Molecular Devices (CA, USA) and 0.60 using the Cell Lab IC100 and Cytoshop software from Beckman Coulter (USA). Both values are deemed appropriate for a successful screen [37]. Figure Figure1g1g shows the signal-to-background ratios using both imaging platforms.
In order to assess the utility of the above protocol, we performed a pilot screen of 7368 compounds (derived from a variety of different compound libraries, see Methods) in duplicates for a total of 44 384-well plates. LNCaP-S14 cells were incubated with individual compounds (final concentration varied between ~5 and 35 uM depending on the molecular weights of the compounds) for 18 h, followed by fixation, staining, imaging (automatic microscopy) and analysis as described in Methods. We scored the percentage of cells positive for p27 in each well relative to the vehicle control (DMSO). Figure Figure2a2a shows the activity of positive control compounds (yellow triangles), the negative control compound DMSO (red squares), the non-specific staining (blue diamonds) and small molecules derived from compound libraries (blue circles) across all screening plates (44 plates) prior to normalization. Negative controls (DMSO) gave basal percentages of p27 positive cells ranging between 2% and 13% while the positive control roscovitine increased these populations up to 50%. As expected, the average of ~320 well measurements per plate - that is, the percentage of p27 positive cells in wells treated with individual compounds - was similar to the percentage of p27 positive cells in the negative control (DMSO), indicating that most compounds tested were inactive in causing p27 accumulation (Figure (Figure2a2a).
Normalization of raw data for 7368 compounds was performed by calculating Z scores, which represent the number of standard deviations by which a signal for a given compound differed from the mean signal of the entire plate [38]. It excludes control measurements altogether under the assumption that most compounds are inactive and can serve as intrinsic controls. The summed Z scores for one set of duplicates (22 plates) showed clear separation between the positive control compound roscovitine (column 2) and the non-specific staining (cells treated with DMSO or stained with secondary antibody only, columns 23 and 24; Figure Figure2b).2b). In addition, most summed Z scores had values similar to the negative controls (DMSO), indicating that we did not incur errors caused by plate position of compounds. This was further evaluated by visualizing every plate as a heat map of individual Z scores. As shown in the example of a single plate in Figure Figure2c,2c, potential hits were randomly distributed over the plate (green and yellow positions). The analysis also identified several false-positive compounds with very high Z scores, namely known DNA binding compounds of red colour (doxorubicin and derivatives, crystal violet, homidium bromide, propidium iodine, pararosalinine pamoate).
In the initial primary screen, we identified 249 compounds that increased the percentage of p27 positive LNCaP-S14 cells. Upon manual examination of microscopic images, 21 duplicate compounds (occurring in more than one library), seven obvious false positives (DNA stains) and 45 compounds with low quality staining (few cells left in well, background, indiscriminate whole cell staining) were sorted out. This resulted in a list of 176 candidates (Additional File 1, Supplementary Table Table1)1) with sixty compounds classified as strong (Z score > 5), 58 as medium (Z score 3-5) and the remainder as weak (Z score 2-3; Figure Figure3a).3a). At a Z score of 3, a compound has a theoretical probability of 0.0013 of being a false positive hit.
A number of experimental and clinically used chemotherapeutics such as camptothecin, thapsigargin, 17-N-allylamino-17-demethoxygeldanamycin, mitomycin C and etoposide were identified (Additional File 1, Supplementary Table Table1).1). The PI3 kinase inhibitor Wortmannin also scored positive, a finding that is consistent with the known role of the PI3K/AKT pathway in the downregulation of p27 through SKP2 [39]. We further validated some of the known bioactive compounds by immunofluorescence staining and immunoblotting (Figure (Figure3b3b and and3c;3c; data not shown).
The one hundred and nine most potent compounds (Z ≥ 3) were selected for confirmation by secondary screening using the primary screening assay. After statistical analysis, 60 compounds were still considered positive hits (Figure (Figure3d).3d). It is well established that compounds with high activity in the primary screen will appear less potent in the secondary screen because of a statistical effect known as 'regression towards the mean' [38]. Thus, weaker hits often fail to validate in the secondary screen due to random measurement error. Additional counter screening for autofluorescence identified another false-positive (pyrvinium pamoate).
Thirty-seven of the 60 validated compounds had known biological activity (for example, inhibitors of protein synthesis and topoisomerase activity, antibiotics and antihelmintics). Of the remaining 23 compounds, none of which had known bioactivity, 20 were prioritized for further characterization based on features of their chemical structures. These compounds were denoted small molecule inhibitors of p27 depletion, SMIPs.
Sixteen of the 20 SMIPs were obtainable as powders from commercial sources. The identity and integrity of all compounds was confirmed by high-performance liquid chromatography (HPLC) and mass spectrometry (data not shown). SMIPs were retested for activity in a dose-response experiment using the primary screening assay. Thirteen out of 16 purchased SMIPs induced a ≥ twofold increase in the percentage of p27 positive cells at the maximal dose with the remaining compounds having lower activity (data not shown). SMIP014 was identified as an additional false positive at this step (data not shown).
Several SMIPs also induced p27 accumulation as determined by immunoblotting (Figure (Figure4a4a and and4b),4b), although the effects were generally weaker (~twofold at 40 μM). While seemingly small, such an increase is biologically significant because p27 levels do not vary more than two- to threefold during a normal cell cycle ([40] and our own unpublished observation). This apparent discrepancy between the immunofluorescence and immunoblotting data, which we had encountered previously during assay development (with positive control compounds roscovitine and MG132; Figure Figure1c1c and data not shown), is explained by the different metrics of the assays: While the immunofluorescence assay determines the percentage of cells with nuclear p27 staining above a threshold determined by staining with secondary antibody alone, the immunoblotting assay measures the total amount of p27 that can be extracted from a cell population (no threshold).
Further prioritization of the remaining 15 SMIPs was achieved by counter screening against another unstable prostate tumour suppressor (NKX3.1) [41,42]. In order to address target specificity, LNCaP cells stably overexpressing NKX3.1 fused to yellow fluorescent protein (NKX3.1-YFP) were used. Cells were treated with a single concentration (40 μM) of SMIPs in 384-well plates and analysed by automated microscopy (see the Methods section). The Z' factors for this assay, as determined by positive and negative controls, were between 0.62 (epoxomycin) and 0.67 (MG132), while the signal-to-background ratio was five in both cases. Although some SMIPs (005, 006, 016) caused a ~twofold increase in the percentage of NKX3.1 positive cells, the effect was subtle in comparison to proteasome inhibitors (~fivefold; Figure Figure4c)4c) and could not be confirmed by immunoblotting (data not shown). Likewise, when the expression of endogenous NKX3.1 was evaluated in DU145, another prostate cancer cell line with very low levels of NKX3.1, none of the SMIPs scored positive (data not shown). The minor effects of some SMIPs in the NKX3.1-YFP fluorescence assay were most likely an artifact of compound autofluorescence at the wavelength used to excite YFP.
We also determined the effect of SMIPs on a HeLa cell line stably expressing p27 fused to firefly luciferase (p27-luc) [40,43]. While proteasome inhibitors led to an increase in p27-luc expression, which was readily apparent by luciferase assay (Figure (Figure4d)4d) and immunoblotting (data not shown), none of the SMIPs were active. This finding suggests a certain degree of cell type specificity of SMIP action.
Since p27 can drive cell cycle arrest, senescence, and apoptosis [7,44,45], we assessed the effect of SMIPs on cell proliferation and viability. LNCaP-S14 cells and IMR90 normal human fibroblasts were exposed to increasing concentrations of SMIPs for 72 h and scored using the MTT assay followed by calculation of IC50 values (concentration required for 50% inhibition) (Table (Table1).1). Whereas SMIP012 and 016 were moderately toxic in normal fibroblasts, SMIPs 001 and 004 showed substantial cancer cell specificity being at least five times more potent in LNCaP-S14 than in IMR90 cells. Although SMIP005 also showed good cell type selectivity, it was excluded at this point due to structural features that suggested potential unspecific reactivity with cellular macromolecules. Based on their apparent cancer cell specificity and their chemical structures, SMIPs 001 and 004 were selected for additional studies.
Figure Figure5a5a shows the chemical structures of the two compounds. We next confirmed the accumulation of p27 upon treatment with 40 μM SMIP001 and 004 in LNCaP-S14 cells (Figure (Figure5b).5b). The CKI p21, which was described as another ubiquitylation target of SKP2 [46], was also upregulated by SMIPs (Figure (Figure5b).5b). The effects on p27 and p21 were also observed with the CDK inhibitor roscovitine. Interestingly, SMIP004 caused a decrease in the levels of both the endogenous and the exogenous, stably expressed SKP2. In contrast, treatment of normal human fibroblasts with SMIPs did not affect p27 or p21 levels (see below).
In order to evaluate the dose-response of the effect on p27, LNCaP-S14 cells were treated with increasing concentrations of the respective SMIPs (5 - 40 μM), and the percentage of cells with nuclear p27 was measured by immunofluorescence and automated microscopy. Both SMIPs led to a dose-dependent increase in p27 positive cells (Figure (Figure5c).5c). Dose-dependent increases in p27 and p21 were also observed by immunoblotting (Figure (Figure5d).5d). Whereas SMIPs 004 led to ~threefold induction at 40 μM, SMIP001 was less active in this experiment causing only a modest accumulation of p27 (30%-40%).
In order to begin to determine the mode of action of SMIPs, we measured their effects on p27 protein stability in a cycloheximide chase experiment. Whereas the half-life of p27 was ~ 1.29 h in cells treated with DMSO, a value that is in good agreement with published reports (for example, [47]), treatment with either MG132 or SMIP004 increased p27 half-life to > 6 h (Figure (Figure5f).5f). No effect on p27 stability was seen for SMIP001. We also evaluated the stability of p21 and SKP2 but did not see any effects of SMIPs on these proteins. In summary, these data revealed a striking correlation between the ability of SMIP004 to downregulate SKP2 and to induce p27 stabilization. In contrast, SMIP001 appears to regulate p27 and p21 primarily at the level of mRNA expression (Figure (Figure5f5f).
Since SMIP004 replicated the effect of MG132 on p27, we tested whether it acts as a proteasome inhibitor. Total cell lysate from LNCaP-S14 cells treated with 20 and 40 μM of the SMIPs were analysed by immunoblotting with anti-ubiquitin antibody to assess the abundance of polyubiquitylated proteins. None of the compounds caused the accumulation of polyubiquitylated proteins that was readily seen with the proteasome inhibitors MG132 and bortezomib (Figure (Figure5g).5g). Likewise, none of the SMIPs inhibited the proteolytic activity of purified 26S proteasomes toward fluorogenic peptide substrates even when assayed at 40 μM concentration (data not shown).
Since SMIPs robustly upregulated p27 and p21, we asked whether this resulted in G1 delay. Indeed, SMIPs 001 and 004 induced a G1 delay with a concomitant decrease in the S phase population (Table (Table2).2). We next asked whether the cell cycle delay coincided with inhibition of CDK2, one of the major cellular target kinases of p27 and p21. CDK2-cyclin complexes were immunoprecipitated from lysate of LNCaP-S14 cells treated with DMSO, the CDK inhibitor roscovitine or SMIPs and assayed for activity toward histone H1 in vitro. As with roscovitine, application of SMIPs led to strong inhibition of the CDK2 activity retrieved from cells (Figure (Figure6a).6a). In order to test the possibility that SMIPs act like roscovitine as direct inhibitors of CDK2 kinase activity, SMIPs were added to CDK2 complexes purified from untreated cells. Unlike roscovitine, none of the SMIPs inhibited CDK2 activity when added to the kinase reaction in vitro (Figure (Figure6b),6b), indicating that the two SMIPs are not kinase inhibitors.
We also evaluated the effect of SMIPs on the levels of p27, p21 and cyclins E and A complexed with CDK2 by coimmunoprecipitation. Both SMIP001 and 004 led to a strong increase in the recruitment of p27 to CDK2, while SMIP001 also slightly increased coprecipitation of p21 (Figure (Figure6c).6c). SMIP004 also reduced the amounts of cyclins E and A retrieved with CDK2 (Figure (Figure6c).6c). This was paralleled by a marked downregulation of cyclins E and A upon SMIP004 treatment (Figure (Figure6c).6c). A more variable downregulation of cyclin A and CDK4 was observed with SMIP001 (Figure (Figure6e).6e). Taken together, these findings suggested that SMIP-induced inhibition of CDK2 activity might be a combined consequence of p27/p21 upregulation and cyclin E/A downregulation.
In order to determine the specific contribution of p27 and p21 to SMIP-induced cell cycle delay, we performed siRNA-mediated knockdown studies. The depletion of p21 and p27 led to a decrease in the G1 population of untreated cells by 6 - 13 percentage points (Additional File 2), a finding that indicates biologically significant effects at the knockdown efficiencies achieved (~80%, Figure Figure6e).6e). Surprisingly, however, neither individual nor combined knockdown of the two CKIs was able to abrogate SMIP-induced cell cycle delay (Figure (Figure6d6d and Additional File 2). Likewise, CDK2 activity in SMIP-treated cells was not rescued by knockdown of p27 and p21 (data not shown). Finally, neither the downregulation of cyclins E and A nor that of CDK4 was consistently affected by knocking down the CKIs, suggesting that these effects of SMIPs probably account primarily for their G1 delay activity.
In addition to G1 delay, treatment of LNCaP-S14 cells with SMIPs for 24 h caused apoptosis as determined by the cleavage of poly-ADP ribose polymerase (PARP, Figure Figure6f6f and and6g).6g). The apoptotic effect of SMIPs was independently evaluated by measuring the amount of cytoplasmic histone-associated DNA fragments. Cells treated with 40 μM SMIP001 and 004 from 24 to 72 h showed a three- to fivefold increase in the amount of mono and oligonucleosomes, thus confirming the apoptosis-inducing activity of SMIPs.
As shown in Table Table2,2, SMIPs also induced robust G1 delay in parental LNCaP cells as well as in two other prostate cancer cell lines, DU145 and PC3. No such effect was seen with HeLa cells or IMR90 fibroblasts. In parental LNCaP cells, G1 delay correlated with an increase in the levels of p27 and p21 as well as a downregulation of cyclins A and E and CDK4, which was particularly prominent with SMIP004 (Table (Table33 and Figure Figure7a).7a). Unlike in SKP2 overexpressing LNCaP-S14 cells, SKP2 was downregulated by both SMIP001 and 004 in parental LNCaP cells, although the suppression was substantially greater with SMIP004 than 001 (Figure (Figure7a).7a). Less pronounced effects on SKP2 were also observed in PC3 and DU145 cells and in IMR90 fibroblasts. In contrast, when averaged over four independent experiments, SMIPs had no consistent effect on p27 levels in PC3 and DU145 cells, although a minor increase in p21 levels was observed (Table (Table3)3) that coincided with a modest reduction in the levels of cyclin A and CDK4 (Figure (Figure7a).7a). None of the latter effects were apparent either in HeLa or IMR90 cells. Despite a lack of induction of apoptosis (PARP cleavage, Figure Figure7a)7a) or overt cytotoxicity (Table (Table4),4), the SMIP-induced G1 delay of PC3 or DU145 cells resulted in the same robust inhibition of colony formation in soft agar as observed in LNCaP-S14 and parental LNCaP cells (Figure (Figure7b7b).
The intimate link between p27 depletion and cancers deriving from the prostate and many other tissues renders pathways controlling p27 abundance attractive targets for the development of novel cancer therapeutics [48]. At the same time, the complexity and apparent redundancy of p27 regulatory pathways raises doubts as to whether targeting a single enzyme or proximal regulator can lead to sustained p27 accumulation in tumour cells. Rather than screening for inhibitors of SCFSKP2 or other enzymes controlling p27, we have developed a cell-based phenotypic screening system to identify compounds that can modulate the p27 regulatory network in cancer cells such that normal nuclear p27 levels are restored. The platform was validated in a pilot screen that identified two compounds of previously unknown biological activity that efficiently reversed the depletion of p27 in prostate cancer cells at low micromolar levels. One of these compounds, SMIP004, increased p27 levels by prolonging the half-life of the p27 protein. Therefore, p27 depletion can be reversed by small molecules in cancer cells overexpressing SKP2.
Whereas the cell-based format readily revealed compounds that robustly modulated the screening endpoint - nuclear p27 levels - in intact cells, our pilot screen also exposed the disadvantages of this approach, namely that the molecular targets of SMIPs remain unknown and that the modulated screening endpoint is not necessarily causal to the ultimate cellular effects of the compounds identified. For example, our RNA interference studies showed that neither p27 nor p21 were required for SMIP-induced G1 delay in LNCaP-S14 cells. It is unlikely that inefficiency of the small interfering RNA (siRNA)-mediated knockdown obscured such a requirement for SMIP action, since depletion of p21 and p27 decreased the fraction of cells in G1 thus indicating that the knockdown efficiencies achieved here were functionally consequential. In addition, p21 and p27 levels in SMIP-treated knockdown cells were still lower than or equal to the levels in untreated control cells, suggesting that the minor accumulation of the CKIs observed upon SMIP administration to knockdown cells will be insufficient to cause a cell cycle arrest.
Two potential explanations can be offered to rationalize the dispensability of p21 and p27 for SMIP-induced G1 delay induced by SMIPs: First, it has been well established that mouse embryonic fibroblasts lacking p27 and p21 remain proficient in responding to negative proliferation signals with cell cycle arrest because the pocket proteins p107 and p130, which have CKI activity themselves, compensate for the loss of the p27/p21 [49,50]. Secondly, SMIPs induced downregulation of various positive cell cycle regulators, including cyclins E and A and CDK4, which was fully maintained upon p27 and p21 knockdown. Thus, it is conceivable that the combined effect of cyclin/CDK downregulation and CDK inhibition by pocket proteins and other CKIs accounts for SMIP-mediated cell cycle delay in the absence of p27 and p21.
Although it appears that the compounds identified in the present pilot screen caused p27 upregulation as a secondary consequence of cell cycle delay, using nuclear p27 accumulation as a screening endpoint readily enabled the identification of cell permeable compounds with antiproliferative activity. Notably, both SMIPs show cancer cell selectivity as they induce cell cycle delay and/or apoptosis in four different prostate cancer cells but not in normal human fibroblasts. Moreover, they inhibit colony formation in soft agar, which is a hallmark of the transformed phenotype of cancer cells and considered a stringent surrogate for in vivo tumour formation [51,52]. Finally, when applied to a more diverse set of compounds with refined drug-like structures, our screening platform may lead to the identification of more potent and direct modulators of nuclear p27 accumulation, similar in characteristics to the recently identified proteasome inhibitor argyrin A, which induces cell cycle arrest and/or apoptosis that is strictly dependent on p27 [45].
The high content screening platform described here also enables chemical genetics strategies for the identification of novel cellular pathways and targets impinging on p27 - for example, the observation that SMIP004 strongly downregulates SKP2 appears significant with regards to its mechanism of action. In a previous work we demonstrated that SKP2 is a rate-limiting component for p27 degradation in LNCaP cells [33]. A recent study in mice revealed that inactivation of SKP2 induces tumour cell senescence that is dependent upon p27, p21 and the transcription factor ATF4 [53], which is an integral component of the unfolded protein response (UPR) that is activated in response to endoplasmic reticulum stress. Although the exact mechanism by which SMIP004 downregulates SKP2 is presently unknown, we have observed a robust activation of UPR signalling by the compound (ERB and DAW, manuscript in preperation). A similar downregulation of SKP2 has been noticed with the proteasome inhibitor bortezomib, which is also an UPR inducer [54]. Mechanism of action studies on SMIP004 are therefore beginning to suggest a link between UPR-mediated downregulation of SKP2 and the accumulation of p27 (and perhaps p21). However, a substantial future effort will be required to decipher the exact molecular targets of the compounds identified here. Once those are revealed, target directed high throughput screening campaigns could be initiated to identify a more diverse set of compounds with improved potency toward p27 and clinical potential.
The results shown here provide proof-of-principle that the cell-based screen we developed provides an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. The advantage of a cell-based screening format is, however, offset by the limitation that the modulated endpoint is not necessarily causal to the ultimate cellular effects of identified compounds, thus requiring additional pathway deconvolution studies. Nevertheless, this approach can be applied to larger and more diverse sets of compounds with refined drug-like properties, revealing both unknown cellular pathways globally impinging on p27 and novel chemotherapeutic lead agents.
NINDS, Prestwick Peakdale 1, Mixed Commercials, Antimitotic, and Enamine and Known Bioactive Compounds libraries were obtained from The Institute of Chemistry and Cell Biology (ICCB) at Harvard Medical School (MA, USA). Information about the libraries can be found at http://iccb.med.harvard.edu.
SMIP001 to SMIP018 were purchased from Ryan Scientific (SC, USA). SMIP0019 was purchased from ChemDiv, Inc (CA, USA). Roscovitine was purchased from Enxo Life Sciences (formerly Biomol International; NY, USA), bortezomib from LC Laboratories (MA, USA), cycloheximide (CHX) and camptothecin from Sigma (MO, USA), MG132 and epoxomicin from BostonBiochem (MA, USA). All drugs were dissolved in DMSO and kept at -80°C.
Anti-p27 antibody (610241, 1:5000 for immunoblotting and 1:1000 for immunofluorescence) was obtained from BD Biosciences (NJ, USA). Anti-SKP2 (32-3300, 1:2000 for immunoblotting.) and anti-ubiquitin (13-1600, 1:2000) was from Zymed Laboratories (CA, USA). Anti-SKP2 (sc-7164, 1:500 for immunofluorescence), anti-CDK2 (sc-163, 1:1000 for immunoblotting, 2 μg/500 mg protein for immunoprecipitation) and anti-CDK4 (sc-260, dil 1:500) from Santa Cruz Biotechnology, Inc (CA, USA). Antibodies for Cyclin E Ab-2 (clone HE12, dil 1:200) and Cyclin A Ab-6 (clone 6E6, dil 1:100) from Thermo Scientific (CA, USA). Anti-p21 (2947, 1:2000) and anti-PARP (9542, 1:1000) were from Cell Signaling Technology (MA, USA). Anti-Tubulin (T5168, 1:5000) was from Sigma Life Science. Anti-Actin (69100, 1:20000) was from MP Biomedicals, LLC (CA, USA). HRP Donkey anti-mouse IgG (715-035-150, 1:5000) and HRP Donkey anti-rabbit IgG (711-035-152, 1:5000) was obtained from Jackson ImmunoResearch Laboratories (PA, USA).
Parental LNCaP cells were stably transfected with a plasmid driving the expression of Myc epitope-tagged SKP2 or empty vector (pcDNA3). Briefly, 5 x106 cells were transfected with 20 μg of each plasmid using Lipofectamine 2000 reagent (Invitrogen, CA, USA) following the manufacturer's protocol. Cells were selected in 600 μg/mL geneticin (G418) until individual colonies were visible. Colonies were picked, passed to 24 well plates and expanded. The levels of p27 and SKP2 were analysed by immunoblotting as described below. LNCaP-S14 (positive clone for SKP2 overexpression), LNCaP-P10 (vector control cell line), LNCaP (parental cell line), PC3 and DU145 cells were maintained in RPMI supplemented with 10% fetal bovine serum and 5% penicillin/streptomycin solution. IMR90 and HeLa cells were maintained in DMEM containing 10% fetal bovine serum, 5% penicillin/streptomycin and 4 mM glutamine. Cells were grown as a monolayer in a humidified incubator at 37°C and 5% CO2.
The screen relies on the detection of differences in the levels of endogenous p27 by immunofluorescence. LNCaP-S14 cells were seeded at a density of 100,000 cells onto 15 mm glass cover slips and allowed to attach for 1 day. Cells were treated with vehicle (DMSO) or positive controls (roscovitine, MG132) for 18 h. After the incubation, cells were fixed by the addition of 10% paraformaldehyde in phosphate buffered saline (PBS; final concentration 4%) and incubated for 20 min at room temperature (RT). Cells were permeabilized by washing 3 × 5 min with PBS, 0.1% Triton X100, followed by blocking in 5% nonfat dry milk in PBS, 0.1% Triton X100 for 1 h at RT. Cells were incubated with 50 μl primary antibody (anti-p27, 1:1000 in blocking buffer) for 1 h, followed by one wash with blocking buffer. 50 μL of secondary antibody (Alexa Fluor 594 goat anti-mouse, Invitrogen A11005, 1:200 in blocking buffer) was added for 1 h. Cells were washed 3 × 5 min in PBS, 0.1% Triton X100 and stained with Hoechst dye (1 μg/mL in PBS) for 2 min. Cells were washed twice in PBS and mounted for imaging with a Nikon Type 120 inverted fluorescent microscope using 60× magnification. Compounds used as positive controls included MG132, epoxomycin and roscovitine, while DMSO was used as negative control. Cells stained with secondary antibody only were used to assess the non-specific (NS) staining background.
The above protocol was adapted to 384 well plates as follows: 4000 LNCaP-S14 cells per well were seeded into 384-well plates (black with clear bottom) in 30 μL RPMI, 10% fetal bovine serum (FBS). Nuclear p27 staining was done under the same conditions as above but with reducing the volume of solutions to 20 μL/well for 10% para-formaldehyde in PBS, 30 μL/well for blocking and wash solutions and 15 μL/well for primary and secondary antibody solutions and Hoechst dye. All liquid handling was done with an eight-channel multidrop liquid dispenser (Wellmate, OH, USA) and a wand aspirator (VP Scientific, CA, USA; VP-186L). After staining, plates were sealed and stored in the dark at 4°C until scanning.
Images from 384 well plates were acquired by using the ImageXpress Micro inverted epifluorescent automated microscope (Molecular Devices, CA, USA) at dual wave length to detect Hoechst and p27. The microscope was equipped with the Photometrics CoolSNAP ES digital CCD camera and an automated objective and filter cube changers. Two images per well at a 20× magnification were obtained at each wave length.
Images were analysed with the MetaXpress cellular imaging analysis software using the cell scoring module. Cells were scored positive for Hoechst, if the integrated pixel intensity was 210-fold above local background and positive for p27 when staining was 30-fold above background (signal obtained with secondary antibody). MetaXpress processing of the raw images provided quantitative measures of the total cell number and the number of p27 positive cells in a given field. The data from both images of each well were combined to get a single number of positive cells. The percentage of positive cells was calculated relative to the total number of cells. Background correction was done by subtracting the number of p27 positive cells in wells stained with secondary antibody only.
The staining protocol was also evaluated using another imaging and software package, the Cell Lab IC100 and Cytoshop software (Beckman Coulter, CA, USA). Minor changes were introduced to the protocol: The cell number was reduced to 3000/well and the secondary Alexa Fluor 568 goat anti mouse antibody was diluted 1:500 in blocking buffer. Four images per well were taken using 10× magnification with a numerical aperture of 0.25 and a camera binning of 2 × 2. A manual threshold was established by comparison of the total nuclear intensity in the positive control and the total intensity obtained in cells stained with the secondary antibody only (unspecific staining) and/or the vehicle control (DMSO). Processing of the raw images provided quantitative measures of the total cell number and the number of p27 positive cells in a given field.
Z'-factor calculation: This parameter was used to assess the quality of the assay in the HTP optimization (HTP Company, CA, USA) [37]. In three independent experiments, LNCaP-S14 cells were seeded in 384 well plates followed by treatment with 0.3% DMSO (192 wells, negative control) or 20 μM roscovitine (192 wells, positive control) for 18 h. The percentage of p27 positive cells was determined as described above and the Z' factor calculated from 576 replicates (each condition) as follows: Z'= 1- (3SD+ + 3SD-)/(Ave+ - Ave-), where SD+ is the standard deviation for the positive control, SD- the standard deviation for the negative control, Ave+ the average for the positive control and Ave- is the average for the negative control [37].
The cell-based screen was performed at the Institute of Chemistry and Cell Biology, Harvard Medical School, using libraries of uncharacterized compounds (Peakdale 1, Mixed Commercials, Antimitotic and Enamine, total of 4888 compounds) plated in 5 mg/mL stock solutions in DMSO, the NINDS library (1040 compounds, 10 mM in DMSO), the Prestwick Library (960 compounds, 2 mg/mL) and a library of 489 known bioactive compounds (plated at 5 mg/mL, 1.1 mg/mL, and 0.25 mg/mL in DMSO) for a total of 7638 compounds. LNCaP-S14 cells were seeded onto 384 well plates as described above. Compound stocks (100 nL) were pin-transferred using a robot-controlled stainless-steel pin array, resulting in a 300-fold-dilution (5 to 35 μM final screening concentration, depending on individual compound molecular weight) followed by incubation for 18 h. All compound plates were screened in duplicates. Each individual plate contained at least eight positive controls (roscovitine, 20 μM), eight negative controls (DMSO, 0.3%) and eight wells of cells treated with 0.3% DMSO and stained only with secondary antibody (non-specific staining). Staining of p27, imaging and analysis was performed as described above. Potential hits were classified according to their Z score. The Z score is a simple and widely known normalization method calculated as follows: Z = Xi - X/Sx, where Xi is the raw measurement on the ith compound, X and Sx are the mean and the standard deviation, respectively, of all measurements within each plate [38]. Potential hits were classified as weak if their Z score was between 2 and 3, medium if the Z score was between 3 and 5 and strong if the Z score was greater than 5.
Target specificity was analysed using LNCaP cells stably expressing the homeobox transcription factor NKX3.1 fused to YFP. In brief, 4000 LNCaP-NKX3.1-YFP (clone 4A) cells were seeded in a 384 well plate and treated with SMIPs for 18 h. Proteasome inhibitors were used as positive controls. Images from 384 well plates were taken using the Cell Lab IC100 automated microscope (Beckman Coulter), and the percentage of cells positive for NKX3.1-YFP was quantified using Cytoshop software (Beckman Coulter). The percentage of NKX3.1-YFP positive cells was determined relative to DMSO treated cells.
Cell specificity was evaluated using HeLa cells stably transfected with a p27-luciferase (p27Luc) construct [43]. Briefly, HeLa-p27Luc cells were seeded in 96 well plates and treated with 40 μM of the respective SMIPs or positive controls (proteasome inhibitors). After incubation for 18 h, cells were lysed using Cell Culture Lysis Reagent from Promega (WI, USA) and luciferase activity was determined by the Luciferase Assay System (Promega) using a Veritas microplate luminometer (Turner Biosystems, CA, USA). Protein concentration was measured in a parallel plate using Bio-Rad Protein Assay (Bio-Rad, CA, USA). Luciferase activity was normalized against protein concentration and compared to the activity recovered from DMSO-treated cells.
Total cell lysate from cells treated with vehicle (DMSO) or compounds were obtained by incubating cells in lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, protease inhibitors) for 15 min at 4°C. The supernatant was collected by centrifugation and the amount of protein was determined with a Bio-Rad protein assay (Bio-Rad) using BSA as standard. Equal amounts of protein were subjected to SDS-PAGE (4%-20% gels, Invitrogen) and transferred to PVDF membranes. The membranes were blocked with TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) containing 5% non-fat dry milk or bovine serum albumin (BSA) for 1 h at room temperature, followed by incubation in primary antibodies 4°C overnight. Membranes were washed and incubated with secondary antibodies conjugated to HRP (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Signals were visualized using the ECL Western Blotting Substrate (Pierce Biotechnology, IL, USA) according to the manufacturer's instructions. Membranes were stripped using Restore™ Western Blot Stripping Buffer (Pierce Biotechnology). Films were scanned and quantification of the bands was performed using Image J software http://rsbweb.nih.gov/ij/.
Total lysates of cells treated with vehicle (DMSO), SMIPs or positive controls were prepared by incubation in immunoprecipitation (IP) buffer (25 mM Tris-HCl, pH 7.4; 150 mM NaCl, 0.5% Triton X-100, protease inhibitors) for 15 min at 4°C. Upon centrifugation, the supernatant was collected and the amount of protein was determined with a Bio-Rad protein assay (Bio-Rad Laboratories) using BSA as standard. Five hundred micrograms of protein was adjusted to 1 mL with IP buffer and incubated with 10 μL anti-CDK2 antibody (sc-163, Santa Cruz Biotech) at 4°C for 3 h with constant rotation, followed by addition of 100 μL protein G-sepharose beads (10% slurry) for 2 h. After four washes for 5 min each with 1 mL IP buffer, beads were resuspended in 2X-SDS Laemmli buffer, followed by SDS-PAGE and immunoblotting.
Histone H1 kinase assays were performed as described in reference [55]. Briefly, the total cell lysates from LNCaP-S14 cells treated with the respective SMIP (40 μM) for 24 h were prepared in IP lysis buffer supplemented with protease inhibitors followed by IP. Kinase reactions were performed by adding histone H1 (1 μg) and 7.5 μCi [γ-32P]ATP (800 Ci/mmol Perkin Elmer Life Sciences, MA, USA) in kinase buffer (20 mM MgCl2, 10 mM EGTA, 40 mM Hepes, pH 7). After incubation at 30°C for 20 min, the reaction was stopped by adding 20 μL 2× SDS gel loading buffer. Samples were separated by electrophoresis, gels were stained and dried, followed by exposure to X-ray film. DMSO was used as negative control and roscovitine (CDK2 inhibitor) as positive control.
LNCaP-S14 (10,000 cells), PC3, DU145 or IMR90 cells (5000 cells) were seeded in 96-well plates and treated with increasing concentrations of the respective SMIPs for 72 h. Cell viability was assessed using the MTT cell proliferation assay (ATCC®, Middlesex, UK) according to the manufacturer's protocol. IC50 was calculated using the BioDataFit 1.02 software http://www.changbioscience.com/stat/ec50.html.
LNCaP-S14 cells were treated with SMIPs (40 μM) for 18 h followed by the addition of 100 μg/mL cycloheximide (CHX). Cell extracts were obtained as described above at different times after CHX addition. Protein lysate was subjected to SDS-PAGE and immunoblotting for p27, p21 and SKP2. Tubulin was used as loading control. Protein half-lives were calculated using a web based calculator http://www.1728.com/halflife.htm.
Total RNA was isolated from LNCaP-S14 cells treated with 40 μM SMIPs or vehicle (18 h) using the RNeasy Mini Kit (Quiagen, CA, USA), followed by first-strand cDNA synthesis using Omniscript® Reverse Transcription (Quiagen). Real-time PCR analysis was performed on the Stratagene Mx3000p detection system (Stratagene, Agilent, CA, USA) using the SYBR Green PCR Master Mix (ABI, CA, USA) at the Microarray and q-PCR Facility of the Sanford-Burnham Medical Research Institute. The primers used were:
5'-ACTGCAGAGACATGGAAGAG-3' and 5'-ATGCGTGTCCTCAGAGTTAG-3' for p27 (to amplify a region from nucleotides 608 to 852); 5'-CGATGGAACTTCGACTTTG-3' and 5'-GGGTACAAGACAGTGACAGG-3' for p21 (to amplify a region from nucleotides 142 to 355); and 5'-TGAGCTGCTAAAGGTCTCTG-3' and 5'-ATGTGCTGTACACGAAAAGG-3' for SKP2 (to amplify a region spanning from nucleotide 527 in exon No. 2 to 739 in exon No.4). Briefly, reactions were done in a 25 μL reaction volume of q-PCR mixture containing 2 μL of cDNA (40 ng) and 250 nM of each primer. Activation of the enzyme was done at 95°C for 10 min followed by 40 cycles of amplification at 95°C for 30 s, 56°C for 1 min and 72°C for 30 s. All reactions were done in duplicates and normalized using GAPDH as control. Primers were design using Primer 3 http://frodo.wi.mit.edu/primer3/ and synthesized by Valuegene, Inc, CA, USA.
Cells were exposed to SMIPs (40 μM) for 24 h and 48 h. Cells were trypsinized, washed with PBS and suspended in 600 μL PBS. Cells were fixed by adding 1.4 mL cold absolute ethanol and kept at -20°C for at least 12 h. After washing once with PBS, the cells were resuspended in 250 μL PBS containing 2.5 μg RNAse A and incubated for 45 min at room temperature. Staining was done by adding 40 μg/mL of propidium iodine followed by incubation for 15 min at room temperature. DNA-bound fluorescence was read at 564 to 606 nm using FACSort and FACSCanto flow cytometers (BD Biosciences) at the Flow Cytometry Facility of the Sanford-Burnham Medical Research Institute. Distribution of cells in the different cell cycle phase was determined with ModFit LT 3.2.1 or FlowJo 8.6 software. Aggregates were excluded from the analysis manually using pulse shape or identified by automated modelling in ModFit LT.
Apoptosis was measured using the Cell Death Detection ELISA (Roche, Basel, Switzerland) following the manufacturer's instructions. Briefly, LNCaP-S14 cells (0.1 × 106) were seeded in 12-well plates and treated with SMIPs (40 μM) for 24 h. Cells were collected in medium, spun at 1000 rpm, and resuspended in 1 mL PBS at the specified time points (day 1 to day 3). The cell suspension was divided into two parts. The first half was used for determination of cytoplasmic histone-associated DNA fragments (ELISA), the second for protein determination to normalize the ELISA data to the amount of input protein.
LNCaP-S14 cells were seeded in 6 cm dishes or six-well-plates coated with poly-lysine the day before transfection. 10-20 nM of siRNA was transfected using DharmaFECT 3 (Thermo Scientific) according to the manufacturer's instructions. Briefly, siRNAs were dissolved in siRNA suspension buffer (Quiagen) at 20 μM, heated to 90°C for 1 min and incubated at 37°C for 1 h. siRNAs were added to the DharmaFECT 3 reagent diluted in Optimem media (1:100) and incubated for 20 min at room temperature. The mix was added to the cells for 24 h. Treatment with SMIPs (40 μM) or vehicle was carried on for another 24 h. Cells were obtained for FACS or lysed for immunoblotting analysis as described above. Untransfected cells as well as cells transfected with non-specific siRNA were used as controls. Silencer® Negative Control siRNA No.1 from Ambion (Applied Biosciences, CA, USA) was used as non-specific control siRNA. siRNA target sequences for p27 and p21 synthesized by IDT (Integrated DNA Technologies Inc, Iowa, USA) were as follows; p27 siRNA: AAGGUUGCAUACUGAGCCAAG, and p21 siRNA: AACAUACUGGCCUGGACUGUU [56].
Agar Noble (Difco, MD, USA) was suspended at 6% in water and autoclaved. A dilution 1:10 was made with RPMI culture medium and added to six-well plates (2 mL). We suspended 20,000 cells were in 0.5 mL RPMI culture medium (containing drugs), added to 0.5 mL of agar (0.6%) and poured immediately into a six-well plate containing hardened bottom agar. Cells were fed with fresh medium containing DMSO, SMIP001, SMIP004 or bortezomib every third days. Images were taken after 14 days using a Nikon Eclipse E600 Microscope.
ADT: androgen deprivation therapy; CDK: cyclin-dependent kinase; CHX: cycloheximide; DMSO: dimethyl sulfoxide; IB: immunoblotting; IP: immunoprecipitation; PCa: prostate cancer; q-PCR: quantitative polymerase chain reaction; siRNA: small interfering RNA; SMILES: simplified molecular input line entry specification; SMIP: small molecule inhibitors of p27 depletion; UPR: unfolded protein response; UPS: ubiquitin proteasome system; YFP: yellow fluorescent protein.
The vast majority of the experiments in this study were carried out by the principal author, ERB. She developed the screening assay, performed all screening and counter screening, carried out the bulk of the validation assays and drafted the manuscript. CCY generated the LNCaP-NKX3.1-YFP cell line used for counter screening and assisted with advice. LL generated and characterized the LNCaP-S14 cell line used for screening. GPR assisted with advice in prioritizing SMIPs for follow-up based in their chemical features and directed the HPLC analysis of SMIPs. DAW developed the concept for this work, participated in study design, secured funding, assisted with kinase assays and edited the manuscript.
Supplementary Table 1. The table shows 176 small molecule inhibitors of p27 depletion (SMIPs) identified in the primary screening. Compounds were classified according to their Z factors as strong (S), medium (M) or weak (W). Their position in the screening (stock plate and well), simplified molecular input line entry specification (SMILES), name and vendor are also listed. A column with known biological actions is also shown for some compounds.
Supplementary Table 2: The table shows the cell cycle distribution of LNCaP-S14 transfected with small interfering RNA for p27 and p21 and treated with SMIP004 (40 μM) as described in the Methods section. Cell were analysed by flow cytometry and the percentage of cells in the different phases of the cell cycle were calculated.
The authors would like to acknowledge the support of staff members of the ICCB at Harvard Medical School (Sean M Johnston and David Wrobel) and the Conrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute (Susanne Heynen-Genel). We are grateful to Alfred L Goldberg for his assistance with the proteasome activity assays and Dr Bill Kaelin for the HeLa-p27-luc cell line. This work was funded by NIH grants R01CA116571, R21NS061675 and RO1GM59780 to DAW and by the Department of the Army, Grant 00475063, Prostate Cancer Training Award to ERB. The generous support of Jeanne and Gary Herberger for this work is gratefully acknowledged.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.

subset: commoncrawl


text:
Website development (Backend) Intern
Classroom OutfitDelhi
Internship DescriptionThe selected intern(s) will work on following during the internship: 1.Upload products to the website 2.Edit photos of the products 3.Write few details about the product # of Internships available: 1 Who can apply: Anyone with basic know-how of a computer and some photoshop skills plus a flair for writing can apply No specific degree or diploma is necessary but the person should have basic knowledge of SEO. Additional Information: Anyone who wants to work with a cool team and gain experience from a bootstrapped company may apply!
About Company
An online firm committed to making clean-cut uniforms.
We are running a uniform design and e-commerce portal.


text:
Dixon of Dock Green: Collection Three DVD
Starring Jack Warner, Peter Byrne, Geoffrey Adams, Nicholas Donnelly, Jocelyn Rhodes, Peter Thornton, Richard Heffer, Gregory De Polnay, Stephen Marsh
Directed by Michael E. Briant
Third collection of episodes from the long-running BBC police drama.
The adventures of the mild-mannered policeman, Constable George Dixon (Jack Warner), filled British TV screens from 1955 to 1976, when he came to the aid of many members of the public.
In these later episodes, Dixon is now retired and working as a collator for Dock Green, advising the younger officers on how to catch the bad guys.
The episodes included in this collection are: 'Domino', 'The Job', 'Vagrant', 'Everybody's Business', 'Alice', 'Jackpot', 'Legacy' and 'Reunion'.
- Format: DVD
- Region: 2
- Certification: (PG)
- Catalogue number: AV3199
- Studio: Acorn Media UK
- Release date: 11/05/2015
- Genre: Drama
- Colour: Colour


text:
MOVIE SYNOPSIS [ General Audiences ] Starred by Eddie Becton, this film directed by Steve Rudolf was premiered in TV or cinema in 2009. The film "Good Ear" had an original production of USA. You can watch Good Ear online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2009.
Rating: 5 by 10 users./
Runtime: 67 minutes
Eddie Becton, Motoko Honda, Wayne Horvitz, Sergio Mielniczenko, Rocco Somazzi and Leni Stern.
67-minute film (full movie) directed by Steve Rudolf.
The original audio of Good Ear has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2009.
Steve Rudolf.
Steve Rudolf and Rocco Somazzi.
This film was produced and / or financed by Good Ear Productions.
Without commercial breaks, the full movie Good Ear has a duration of 67 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Good Ear in FULLTV Movies.


text:
Forgot your password? Enter the email address you signed up with and we will send you a link to create a new password.
Your password reset has been submitted to the address provided.
Colors could not be determined.
Design plus words, letters, and/or numbers
leather pouches; pouches made of suede
Business in LOS ANGELES, CA
Business in Los Angeles, CA


text:
We recommend upgrading to the latest Internet Explorer, Google Chrome, or Firefox.
If you are using IE 9 or later, make sure you turn off "Compatibility View".
Handpicked brands for you
Just one day left until our @FRENDS festival favorites giveaway ends! Find out what you and a friend could win below! 2 sets of each item will be included: @frends Taylor Silver/White headphones with Etched Oil Slick interchangeable caps...


text:
SoundPlayer in the .NET Compact Framework
[This documentation is for preview only, and is subject to change in later releases. Blank topics are included as placeholders.]
The .NET Compact Framework version 3.5 supports the SoundPlayer class, which provides an intuitive API for playing sounds in applications such as casual games. To enable casual game development, the .NET Compact Framework pushes each SoundPlayer object through WaveOut, which is the audio rendering filter in Windows Embedded CE and Windows Mobile powered devices. This filter enables the device to mix sounds.
The .NET Compact Framework 3.5 supports the following types:
For a code example that demonstrates how to use this feature, see SoundPlayer.
The differences between the .NET Compact Framework implementation and the desktop implementation of SoundPlayer include the following:
The .NET Compact Framework supports only PCM-encoded files (uncompressed sound).
On a device, more than one sound can play at the same time. On the desktop, only the last sound plays.
On a device, the application will not terminate until playback is completed. If a sound is playing in the application loop, the application will never exit. To avoid this situation, you can explicitly terminate playback by calling the Stop or Dispose method on the SoundPlayer object.


text:
Girl's SHOCK ACT on balcony caught on Snapchat as teens run riot
AS the boozing continues on Australia's Gold Coast shocking footage has surfaced of a teenage girl.
The alarming Snapchat clip surfaced over the weekend and shows the girl on a balcony of the Beachcomber Resort at Surfers Paradise.
The teen climbs around the edge of the balcony – several floors up – and appears to sway from side to side.
Her friends behind the camera laugh and say "this isn't going to be funny when she falls".
"Police take this behaviour extremely seriously as a fall from a balcony can result in death"Inspector Bruce Khan
Inspector Bruce Khan told The Courier Mail the incident was a "life-endangering act".
"Police take this behaviour extremely seriously as a fall from a balcony can result in death," he said.
Four years ago a teen plunged to her death in the same part of town.
Thousands of teenagers descended on Surfers Paradise for a booze-soaked week to celebrate leaving school.
As the years have gone by the behaviour has become more hedonistic and cops are out in force on the lookout for underage boozing and worse – illegal drugs.
One 17-year-old boy was arrested after cops allegedly found a small number of pills – thought to be MDMA, an ecstasy derivative – and a sum of cash on Friday (November 17).
Club promoters have been heavily targeting students with offers of cheap booze and even here McDonald's in a bid to get them through the doors.
Yesterday a video surfaced of some pranksters cutting the ties of a girl's bikini and placing a live lobster on her stomach.
And a mass brawl broke out during a beach party in which a series of arrests were made.


text: hello, considering using such a device on a vehicle weight 3300lbs Vehicle max tow capacity is 6600lbs [way to much in my opinion ] trailer weight 4000lbs trailer single axle with brakes 12 x 6 As i understand it a single axle is a lot less stable than a dual axle . From previous experience also . Given the understanding that a single axle influences the vehicle alot more . Does anybody who tows have a single axle trailler with such a device fitted .Your experiences . Dual axle owners r welcome to reply Another point is to remember is that increasing the load capacity to the rear springs is not the absolute answer as in this application .Remember the trailer pushes down on the hitch therefore using the rear axle as a pivot point . This results in the front suspension unloading [poor braking and steering] LOad leveling devices apparently redistribute weight evenly over tow vehiclef and r axles. Iam assuming stability would then return to also. tomo


text:
Former NChas Housing Authority Dir. charged with embezzlement
NORTH CHARLESTON, S.C. (WCIV) -- South Carolina Law Enforcement Division agents have arrested the former executive director of the North Charleston Housing Authority.
George Luis Saldana, 58, is charged with embezzlement and faces up to five years in prison if found guilty.
According to the affidavit, from April 2007 to May 2011, public funds were used via a NCHA Visa credit card on numerous occasions and used to buy gas on personal trips taken by Saldana in{} four different states.
The affidavit also says Saldana used the card to buy gas and other items while on vacation, on sick leave, on weekends and other times he wasn't performing official duties and that he admitted to using a NCHA vehicle and the Visa for personal use.
The affidavit lists the total taken over four years as adding up to $6558.04.
His bond was set at $10,000.


text: > Let me express my appreciation and gratitude for Debian. Reading debian-devel during the last weeks, I had the same feeling that some positive counterpart to the recent discussions is needed to somehow keep the "balance". I intended to post the top 5 reasons, why I love Debian, but you were faster than me, so let me just add this to your thread: 1. From a users perspective, Debian just works! I recently installed and upgraded a lot of systems to Lenny without the slightest problem. My oldest system has been dist-upgraded from Woody to Etch to Sarge to Lenny - and it still works! 2. apt-get install <whatever-you-need> 3. Debian is around for quite some time now, so we must be doing something right, mustn't we? 4. Debian has a pretty large community. And for a non-profit, volunteer-driven project, this works really well and it's fun to be part of it! 5. Debian values software freedom. It's nice to get tons of software for free and it's even nicer to have the freedom to take the ideas and knowhow expressed in this software to create something new. That's how it has always been in art and science. The world needs free software and it needs Debian :-) And as a bonus: 6. We have a world wide financial crisis and it's not Debian's fault :-) Tobias


text:
EDGE Group Announces Fast, Low -Cost Delivery for NEC Residential Plasma Products
Innovative Regional Program for Big-Screen Home Theater-style Displays Creates New Service Opportunities for Dealers and Custom Installers
J. B. Stanton Communications
860/542 -1234 Voice • 860/542 -0005 Fax
Bryan Stanton / email@example.com
Stan Pinkwas / [718] 788 -7777 Voice
EDGE Group Announces Fast, Low -Cost Delivery for NEC Residential Plasma Products In Partnership with Watkins Motor Lines
Innovative Regional Program for Big -Screen Home Theater -style Displays Creates New Service Opportunities for Dealers and Custom Installers
DENVER, CO, Feb. 9, 2005 - The EDGE Distributors Group, a nationwide coalition of consumer electronics distributors serving the custom installation and specialty retail markets, has announced a new program to provide a fast, flexible, low -cost shipping solution for NEC's high -performance Showcase Series plasma TVs and related products. Created in partnership with Watkins Motor Lines, Inc., the nation`s largest privately held less -than -truckload (LTL) carrier, the innovative program offers rapid delivery of plasma displays at a highly affordable flat rate.
The new program opens up fresh customer service opportunities for dealers and custom installers alike. It also shows how The Edge Group's pro -active and dynamic approach works to supports its dealers. In partnering with Watkins, The EDGE Group has not only demonstrated its awareness that high freight costs can impede profitability, but taken the initiative to lower costs and speed deliveries. In many cases, the new program makes next -day delivery possible to all but the most far -flung reaches of the domestic U.S., and at a rate significantly lower than those from almost all other carriers. At the same time, EDGE customers retain full access to Watkins` renowned premium level services, such as Watkins Online Tools.
"We're impressed with Watkins' planning abilities and vision in creating this program with us," said EDGE Group President Michael Levy. "It's possible explosives might be more dangerous to move, and crystal glassware more fragile, but our customers are just as nervous when we ship plasma displays. After all, NEC's Showcase Series plasmas are precision components, faced in glass, that need to be delivered rapidly to fit our customers' demanding timelines, and in perfect condition. Fast, careful delivery is a powerful tool for us and our customers."
Because the shipping rate is per pallet, almost limitless product combinations are possible. For example, a dealer can assemble two 120 -pound 50 -inch plasma TVs, two 70 -pound swing -arm plasma brackets, and two pairs of 20 -pound speakers on a single pallet, and have them delivered in many cases the day after they're ordered, all at a single low rate.
Watkins' innovative service is made possible by the company's unique ability to pick up both regional and long -haul business on the same truck, then separate the regional freight at the terminal of origin and run it via dedicated equipment to a Regional Express hub. This innovative capability is a first for long -haul LTL carriers.
Since Watkins is a trucking company, not a moving company, its drivers do not carry the plasma displays up stairs or inside homes. However, Watkins offers a variety of additional services, from lift gates to inside white -glove delivery.
About The EDGE Group
Founded in 1999, the EDGE Group (www.edgegroup.org) consists of 12 regional distributors that primarily serve the premium U.S. custom installation market and select retailers. EDGE Group partners sell and support products aimed at residential value -added installers and specialty retailers. They encourage growth by their members and customers alike by carefully evaluating, then bringing to market the finest installation products available - emphasizing those that are reliable, innovative and exciting. Product lines the EDGE Group distributes and supports includes Channel Vision™, D -Tools, Focalpoint™, Isotec, NEC, Peerless Industries®, Optoma, and F -Conn Industries.
# # #
Featured Product
Aperion Audio - Award Winning Home Audio for less
Aperion is a group of dedicated audio enthusiasts committed to helping you create the sound experience of your life, at a price that won't break the bank. We make award-winning speakers that look as good as they sound and sell them online so you get unbeatable quality for the price. You always get a 60-day in-home audition with FREE SHIPPING. If you don't like your speakers for any reason, send them back and we'll pay the shipping. Really. You get unlimited free support for your entire system and we back up our products with generous 5-and-10-year warranties. Since we've been around for over fifteen years, you're covered.


text:
Gold Star family of slain California soldier booed on flight
The family of a slain soldier was booed on a plane as they flew to pick up their son's remains. Perry said that he and his wife and daughter were delayed on an American Airlines flight going from Sacramento to Phoenix, but were let off the plane first so that they'd make their connecting flight to Dover Air Force Base in DE where they were to meet their son's body. [Full Article...]
Two men charged with kickback scheme at Valeant
They are accused of being involved in a multi-million dollar fraud and kickback scheme to defraud Valeant. U.S. Attorney Preet Bharara will hold a noon news conference in Lower Manhattan. They said the investigation of Laval, Quebec-based Valeant and Philidor is continuing. "Citing emails, prosecutors said Davenport evoked images from the old Western "Butch Cassidy and the Sundance Kid", thi... [Full Article...]
Time for Guardiola to settle on a regular Man City starting XI
But a laughing Guardiola said: 'It's impossible to play good football if you don't have sex with your partner. Do you think Nasri has the talent to play for a team such as Barca? Crystal Palace have lost seven of their last eight Premier League games against Manchester City, winning the other in April 2015. [Full Article...]
Zika Virus No Longer International Public Health Emergency
The decision to "call off the global emergency" could prompt governments and donors to "pull back even more" from what Gostin described as a "lethargic" worldwide response. Several countries, such as Venezuela and Haiti, have not been vigilant about reporting cases. Maintaining vigilance against the virus is critical, said Dr. [Full Article...]
Scientists develop new mouse model to aid Zika virus research
Thailand has reported more than 680 cases of Zika since January but its Department of Disease Control has said the virus is manageable. A small lab study that exposed mosquitoes to blood infected with varying combinations of dengue, Zika and chikungunya proved that the mosquitoes were able to pick up two viruses - Zika and chikungunya - at the same time. [Full Article...]
Trump lauds Electoral College as Clinton wins popular vote
The rest of the contacts came in phone calls and handwritten letters. But what if California's vote was in line with all the other Democratic states , where Clinton beat Trump 53.5% to 40.2%? Anuzis says he thinks it's deceptive to say Hillary Clinton would have won under a system similar to the one he's proposing, because he says the campaigns didn't adopt strategies based on the nation... [Full Article...]
FB Stock: This Could Be a Big Problem for Facebook Inc
One source said high-ranking officials were briefed on a planned News Feed update that would have identified fake or hoax news stories, but disproportionately impacted right-wing news sites by downgrading or removing that content from people's feeds. [Full Article...]
World Diabetes Day observed
Another challenge confronting the authorities is that 16.51 percent of residents over 35 are prediabetes, which means their glucose level is higher than healthy people but they are not yet suffering from diabetes. According to World Health Organisation, one in three adults aged over 18 years is overweight and one in 10 is obese. In Type 2 diabetes, weight loss and diet can help. [Full Article...]
Trump pledges restraint with Twitter from White House
US President-elect Donald Trump said he will keep parts of President Obama's health care law after campaigning on a promise to immediately repeal it in full. That figure refers to a broader group of people that includes not just unauthorized immigrants but also people who are lawful permanent residents or those with temporary visas, Washington Post fact-checkers reported in September. [Full Article...]
Ten To Air Trump's First Post-Election TV Interview
President-elect Donald Trump says he will deport or incarcerate 2 or 3 million undocumented immigrants who have criminal records. The billionaire businessman said that once the border is secured, immigration officials will "make a determination" about remaining undocumented immigrants in the country. [Full Article...]
Mother of suspect in 7 killings says he is 'not a monster'
The news comes almost a week after authorities discovered a living woman who had allegedly been bound and chained for two months inside a metal storage container on Kohlhepp's 95-acre property in Woodruff, authorities have said. She and Johnny were reported as missing on December 22 after they stopped returning messages to family. What would take everyone by surprise was when Kohlepp also admitt... [Full Article...]
It's a baby girl for Rob K, Blac Chyna!
A source told the HollywoodLife.com: "No doubt Tyga and Chyna have had their ups and downs, but he's insane excited for her and Rob and can't wait to meet the baby". Little Dream gently holds on to a hand, which appears to belong to her Papa, already Daddy's little girl! I'm relaxed. [I've had] no cravings, no morning sickness - thank God! The couple was surrounded by close family and frie... [Full Article...]
Portman calls for unity after fractious election
Portman has run a campaign based around creating jobs for the Buckeye State and trying to stop the state's opioid addiction problem. He beat Democratic challenger Ted Strickland by more than 20 percentage points statewide amid strong voter turnout. [Full Article...]
Colorado Voters Approve Right-To-Die Initiative
According to this new directive, terminally ill adult patients who have less than six months to leave may opt to obtain prescription drugs from their attending physician and administer them themselves. Department of Veterans Affairs. Supporters of the measure had raised $5.7 million as of October 31, buoyed by $4.5 million from the Compassion and Choices Action Network, Ballotpedia reported. [Full Article...]
Ohio's presidential pick will win or make history in loss
Guard members are also at the ready to be activated by Republican Gov. John Kasich of OH should there be any violence on election day. But over the weekend a Court of Appeals said OH law already covers such behavior and rescinded his order. [Full Article...]
Nebraska Restores the Death Penalty After Eliminating It
Former state Attorney General John Van de Kamp says in the petition that it would create confusion and upheaval in the courts. Those projects could be sent back to voters for approval if the proposition passes. With almost 6.5 million ballots counted, Proposition 60 was losing by a margin of 54 percent to 46 percent , and Steven Hirsch, co-chairman of Vivid Entertainment said he was confident... [Full Article...]
Massachusetts goes to pot, Bay State approves recreational marijuana
On Question 3, voters decisively approved a ban on the sale of eggs and other food products that come from farms where animals are confined to overly restrictive cages. Rahsaan Hall, of the American Civil Liberties Union, said there have also been isolated complaints of translation services not being provided for non-native English speakers and poor access to polling locations for disabled residen... [Full Article...]
LSU's ragin' Cajun: Orgeron adds extra seasoning to interim role
Pettway became the first Auburn player since Bo Jackson in 1985 to have at least 150 rushing yards in four consecutive games. The biggest matchup in college football this week, and the site of College Gameday, also resides in the SEC: the slugfest between No. [Full Article...]
Dizzying fall for Hertz Global after earnings
Hertz slashed its adjusted earnings per share outlook to between 51 cents and 88 cents per share from the previous outlook of $2.75 to $3.50 per share. Teachers Advisors holds 0.06% of its portfolio in Hertz Global Holdings, Inc (NYSE:HTZ) for 3.35M shares. Swiss Bank accumulated 0.01% or 591,698 shares. [Full Article...]
Janet Reno, former U.S. attorney general, dies at age 78
In 1995 Reno was diagnosed with Parkinson's disease, a progressive disorder of the central nervous system that caused trembling in her arms. Never married, she retired to her modest family home near the Everglades and declared herself a private citizen. [Full Article...]


text:
Outdoor Research Uberlayer Hooded Jacket
- Extremely breathable
- Excellent mobility
- Well featured
- Limited wind resistance
- Heaviest jacket of the test
- Low water resistance
This Outdoor Research Uberlayer Hooded Jacket is ideal for intermittent, high output activities in cold conditions. The Polartec Alpha insulation and shell materials do a standout job of allowing heat and vapor to escape while moving, and then trapping the heat when stopped. Theis Jacket can cut the hassle and time loss of adding then removing a layer during breaks, as long as it's dry and winds aren't strong.
Breathability is where the Outdoor Research Uberlayer Hooded Jacket shines. During high intensity activity in cold conditions, the combination of a nylon 30d stretch woven outer shell, polyester stretch mesh inner lining and Polartec Alpha insulation provides plenty of air movement to abate heat and moisture accumulation. This construction is effective over a wide range of aerobic intensities, from the low aerobic demands of rock climbing to the high demands of trail running.
The Outdoor Research Ublerlayer hooded jacket is one of the more durable pieces in this category. Both outer shell and inner lining fabrics survived haphazard treatment in mountain and rock climbing environments without a nick. Zipper sizes, cordage diameter and other hardware also reflected a nod towards increased durability and usability. Classic baffled construction ensures insulation stays in place over the long haul.
This jacket suffers in weatherproofing, like others in this test, due to the focus on breathability. Although slightly more windproof than some in this test, the Outdoor Research Uberlayer Hooded Jacket lets anything other than the slightest breeze cut through. The main zipper has a wind resistant flap behind it to protect that area. The outer shell, although DWR coated, can only withstand drizzle due to the porous and breathable nature of the fabric. This was particularly noticeable where the jacket made contact with shoulder or hip straps.
The fit is another strong point of the Outdoor Research Uberlayer Hooded Jacket. The cut is definitely on the trim side; all girths are geared toward the slimmer set. Torso and arm length worked well for overhead activities, only causing slight ride up and wrist creep. Internal volume accommodates heavy base layers. Mobility is excellent as the stretch materials and slippery lining enhance the jacket's ability to keep up with aggressive movements. A single cord lock adjusts the elastic in the lower hem and elasticized cuffs seal at the wrists. The hood adjusts via a single cord lock in the rear and the stretchy fabrics allow it to move well with the head. It does accommodate a climbing helmet but is tight; some binding occurs when fully zipped.
The Outdoor Research Uberlayer Hooded jacket is well appointed and these extra features limits compressibility compared to more pared down pieces. This jacket will cinch down to the size of a cantaloupe; not superior but still manageable for such a versatile garment.
The list of features on the Outdoor Research Uberlayer Hooded Jacket will be appreciated by anyone venturing out in cold conditions. The main zipper can open from either end, has a storm flap and every zipper pull is robust and easily managed by gloved hands. There are two zipped handwarmer pockets (too low to clear pack hip belts), and a vertical zipped chest pocket that all work well as vents. There is a stretch mesh "Shove-It" pocket on the interior of each torso that work well for keeping gloves, hats and snacks warm and accessible. There is a key clip in the left handwarmer pocket and the hood's brim has a moldable wire insert.
How We Tested It
The jackets in this test were used over a three-month period in Central Texas winter/spring conditions while guiding, hiking, rock climbing and mountaineering in Central Mexico. This tester is 6 feet tall, has a 32" waist and weighs 167 lbs.
For more reviews beyond this 2017 test, check out our Best Softshells of 2017, Best Synthetic Sweaters of 2017, along with our insulated jacket tests, softshell jacket tests, and other related hiking and camping gear tests.
The products featured in this test have been loaned to the Gear Institute. For more on our policies regarding editorial objectivity and sample returns, see here.
About the Author
Shop for the Outdoor Research Uberlayer Hooded Jacket
Commentscomments powered by Disqus
Jump To
> Climbing Gear Reviews
> Camping & Hiking Gear Reviews
> Fishing Gear Reviews
> Running Gear Reviews
> Skiing Gear Reviews
> Watersports Equipment Reviews


text:
United Visiting Nurse Association
About United Visiting Nurse Association40 Lindeman Drive
Trumbull, CT 6611
Phone: (203) 330-5522
Fax: (203) 396-0506
Service Area for United Visiting Nurse Association
We don't have the exact service area for United Visiting Nurse Association. Based on the average area most agencies cover, the agency is very likely to provide services to the area in green and less likely to provide services further outside this area. Be sure to contact the agency to make sure they provide services in your area.
Other Nearby Home Health Care Agencies


text:
Back in the mid- to late-90s, there were a couple of guys in the Charleston Daily Mail newsroom who gave us great work, great personality and a great community reputation.
Actually, there was more than a couple. But the guys I'm thinking about in this case were Chris Stadelman and Jody Jividen.
Chris, who was business editor, then city editor, and then managing editor, was whipsmart — as his rapid ascension to the top might attest. He was confident. I don't think "brash" is too strong a word. "Stubborn" was used this week, and no one seemed to disagree. But also personable — really widely liked.
His last name took on a one-word life of its own — like Prince, Cher or Sting: Stadelman.
Jody walked slow, and he talked slow. He was lanky. He'd lean over your desk and sigh. He'd cackle, not unlike a donkey's bray, and then wipe his forehead at the absurdity of what had been said.
In print — and in person — he was a master storyteller.
He had a special name, too: Uncle Jody.
Both returned to newsprint this week thanks to a story by another special, veteran reporter, Charlotte Ferrell Smith.
Chris, who is battling cancer, is the honorary chairman of the annual Run for Your Life event to raise awareness of colorectal cancer as well as provide education and funding for screenings. The event is organized by the Charleston Area Medical Center Foundation.
Run for Your Life was established in honor of Jody, who died August 9, 2002, of complications of colorectal cancer. Jody was 44.
I treasure my time working with Chris and Jody in a newsroom on a regular basis. If you have smart, fun people to work with every day that's a true gift. These guys continue to be a presence in our newsroom through a fun atmosphere, a tradition of hard work and great journalistic products.
Plus, they actually still are a presence in our newsroom. There's a black and white staff photo of Chris from when he was a very young reporter, just lying around on a desk. And Jody continues to have a place on our "unofficial business" bulletin board.
As Jody would say, "Peace unto ya."


text:
Gluten Free Ireland is your gateway to the Coeliac and Gluten Free community, living, visiting and travelling in Ireland and Northern Ireland.
From just £8 a month you can upgrade your listing to highlight your business, bring it to the top of search results and add valuable information such as a website, e-mail, photograph and social media links.
Your listing will also appear in our guide, Eating Out in Ireland, available as a Digital eBook or PDF Download.
Enhanced listing on website, in print and mobile from just £8 per month.
Pay Annually: £96 (That's just £8 per month)
Alternatively, email: email@example.com with your venue details and an invoice will be issued to you.
Producers and Manufacturers:
To get a Premium listing as a Producer contact Derek Thompson directly on +44 (0)28 9263 9992.


text:
These Former Boise State Players Could be Going to the Big Game
Sunday, February 5th, two of the NFL's top teams will square off in the biggest sporting event of the year.
Three of the top eight teams vying for a spot in the big game have former Broncos on their squads.
The Seattle Seahawks, New England Patriots, and Dallas Cowboys all have Boise State graduates on their current rosters. So who can we look out for during the big game?
Seattle Seahawks
Jeron Johnson – #23 – Safety
Reese Odhiambo – #70 – Guard
New England Patriots:
Shea McClellin – #58 – Linebacker
Dallas Cowboys
Kellen Moore – #17 – Quarterback (injured, out for season)
Orlando Scandrick – #32 – Cornerback
Demarcus Lawrence – #90 – Defensive End
Tyrone Crawford – #98 – Defensive End


text:
Stock Graphic >> Product Mockup - Dessert Box Mock Ups 299024 by JSquarePresents @ CreativeMarket
File Size: 0.45 KB; Requirements: Adobe CS4+; Layered: Yes; Vector: Yes; Dimensions: 4800 x 3197 px.
Dessert Box Mock Ups - Made from Real Photos by JSquarePresents
In this package.
- 2 fully editable Photoshop PSD files
- 1 bonus PSD .pat preset (4 metallic foils + 1 craft paper + 1 hot air balloon)
- 1 bonus dessert box die cut EPS file
- You can stamp, foil emboss your logo via smart object
- You can add illustrations to the front panel via smart object
- Easy changeable box texture & color via Effect- Pattern Overlay & Color Overlay
- Easy changeable box pattern/print via Smart Object
- Organized layers and folders
- Works with Photoshop CS4+
- Image size: 4800 x 3197 pixels
- IMPORTANT!!! You will find a link in the .txt file you download from Creative Market leading you to the balance files.
- Help file included.
- In order to provide you the maximum edibility to these mock ups, there are quite a bit of organized layers. Please kindly refer to your Help file for step-by-step walk-through, or send me a message for questions. I'll try my best to help!
For more of my mock ups, please visit: http://crtv.mk/ahfx
Thank you for stopping by!
Copyright ? J Square Presents | All Rights Reserved.
Buy Now ~ $8
Dessert Box Mock Ups
by JSquarePresents
Keywords: hero images, dessert box mock ups, packaging mockups, editable mockups, presentation, promotion, image display, realistic mock ups, showcase template, rustic mockups, organic mockup, logo stamp, foil emboss logo, color block, pattern preset, gold silver foil pattern, craft paper texture, box die cut eps, smart object, branding mock ups.


text:
Shalani Soledad Wedding
January 23, 2012 by staff
Shalani Soledad Wedding, Councilor Shalani Soledad finds "home and happiness" in Congressman Roman Romulo, "You are my home. You are my happiness…" This was part of Shalani Soledad's wedding vow, which she recited late this afternoon, January 22, at the St. Benedict's Church in Ayala Westgrove, Sta. Rosa, Laguna.
As for the groom, he told his bride, "I waited 44 years before God gave me this favor…"
And with some 500 guests as witnesses, Councilor Shalani Soledad was officially pronounced wife of Congressman Roman Romulo at around 6:45 p.m.
One of the highlights of the ceremony, which began a little past 5:30 p.m., was the homily of His Eminence Cardinal Gaudencio Rosales.
Cardinal Rosales stressed that it was love that brought the couple together, and not "economic welfare" or "political status."
He also advised the couple to be "kind," "patient," "understanding," "not to be jealous," and "do what God calls you to do."
SIDELIGHTS. The wedding was the first time that Shalani saw her father, Adolfo Aguirre, and her mother, Evelyn Soledad-Yumol, together.
But the bride initially walked down the aisle alone.
Halfway through her march, Mr. Ramon Soledad—her mother's brother, who was instrumental in bringing the bride into the world of politics—accompanied her.
Next, her biolgical father joined the bride.
And the last man who walked with her before reaching the altar was Dan Yumol, her stepfather.
The background music, "If It's Love," "Fields of Gold," and "Just The Way You Are," was sung by Gail Blanco.
Celebrities in attendance were Senator Bong Revilla and wife Lani Mercado, Richard Gomez and wife Lucy Torres, Mariel Rodriguez, Migz Zubiri and wife Audrey, Valerie Concepcion, Marco Alcaraz and girlfriend Lara Quigaman.
Reception was held at the Evoliving Center Dockside in Nuvali, Sta. Rosa, Laguna.
PARENTS' MESSAGE. The program was simple and short, as the family said they wanted the guests to enjoy the food prepared by Florabel Catering.
"Nagustuhan niyo ang pagkain?" asked the groom's sister, Mons Romulo, twice, to which we readily answered, "Oo naman!"
The menu had smoked salmon and romaine lettuce salad with herb croutons, Parmesan flakes and longganisa bits; chicken binakol with Baguio spinach; roast U.S. Angus rib eye with mashed potato; queso de bola cheesecake; suman latik with Batangas tsokolate; plus malunggay iced tea.
About 800 people joined the newlyweds at the reception, which showcased a 10-minute fireworks display.
Two of the highlights were the parents' messages.
Please feel free to send if you have any questions regarding this post , you can contact on
Disclaimer: The views expressed on this site are that of the authors and not necessarily that of U.S.S.POST.


text: I have three cards (for my kids) of the R4i chip before I knew that they were knockoffs of AKAIO. The latest software version that I can find is 1.86a - does anyone know of an update? I see that AKAIO is now on 1.89 and I would like to update. Thanks for your help. As soon as money allows I will be buying us AceKards to alleviate this problem. TF


text:
sort by best latest
There are 4 programs available with HubPages to earn money.
1) Google adsense
2) Kontera
3) Ebay
4) Amazon
If you have an ID (membership) of above affliate programs simply you can enter those IDs in "Affliate Settings" (My account --> Affliate Settings). Then 60% of time you will get revenue from those programs when someone read your hubs.
If you do not have any of this you should sign in with those programs. Kontera & Amazon is easy to sign in.
You can help the HubPages community highlight top quality content by ranking this answer up or down.