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Tuscan dishes cooked alla diavola (devils style) are very spicy with lots of black pepper and/or chiles.
This recipe, which borrows the Tuscan technique of chicken cooked under a brick, uses jalapeño for its heat, adding a distinctly south-of-the-border flavor.
- Yield
- 4 people
- four 1-pound Cornish hens, butterflied, breasts boned
- 1 garlic clove, peeled, plus 4 cloves, peeled and thinly sliced
- 1 tablespoon plus 1 teaspoon hot red pepper flakes
- freshly ground black pepper
- juice of 6 lemons
- 1 cup extra virgin olive oil
- 2 fresh rosemary sprigs
- 4 fresh thyme sprigs
- 1 jalapeño pepper, split lengthwise, seeds removed, thinly sliced
- fine sea salt
- Rub the Cornish hens' skin, inside and out, with the peeled garlic clove. Discard the clove. Spread the red pepper between the skin and the meat. Season all over with black pepper. Place the hens in a nonreactive pan. Drizzle the lemon juice and olive oil over them then scatter the sliced garlic, rosemary, thyme, and jalapeño over them. Cover the pan with plastic wrap and let rest in the refrigerator overnight. The next day, remove the hens from the marinade, pat them dry, and season the skins with salt. Set a heavy cast-iron skillet large enough to fit all the hens (or use 2 or more smaller skillets) over medium heat. Place the hens, skin side down, in the skillet and press them with a spatula or weight (such as a cast-iron pan or foil-wrapped brick) to obtain a golden-brown crispy skin, then turn them over and repeat. Depending on the size of the hens, this will take 25-30 minutes altogether. Place each hen on a dinner plate and serve at once. VINO This spicy dish requires something that can stand up to its spice, like a full-bodied Brunello di Montalcino from Tuscany.
Related Video
Nutritional Info
- Calories1429
- Carbohydrates12 g(4%)
- Fat118 g(182%)
- Protein79 g(159%)
- Saturated Fat25 g(127%)
- Sodium1414 mg(59%)
- Polyunsaturated Fat19 g
- Fiber4 g(17%)
- Monounsaturated Fat67 g
- Cholesterol458 mg(153%)


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subset: commoncrawl


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According to the U.S. Marshals Service, the juvenile was taken into custody Friday at 5:40 p.m. at Valley Falls State Park. The juvenile is facing criminal charges, according to Fairmont Police. He was unarmed when authorities located him.
The U.S. Marshals Service, Mountain State Fugitive Task Force, FBI, Harrison County Sheriff's Department, Clarksburg City Police, Bridgeport City Police, and the Marion County Office of Emergency Services also assisted.
Original Story:
Area schools are on heightened alert after a Notre Dame High School student sent out a mass email early Friday morning.
Local law enforcement is actively looking for a 17-year-old white male, possibly wearing blue jeans, black athletic shoes, 5'10", 180 pounds, with sandy blonde hair. He may be armed and dangerous, according to an alert sent out by Fairmont State University.
Spokesman for the Catholic Diocese of Wheeling-Charleston Bryan Minor said that a young man sent a mass email in the early morning hours and expressed that he was depressed about something. There wasn't a specific threat made to students at Notre Dame, St. Mary's or the Fairmont Grade School, according to Minor.
"What really sticks out in the mind of administrators at the Diocese Office is that there has been outstanding cooperation and assistance with this young man's family, local law enforcement and teachers and faculty at the schools. There are plans in place for moments like these and they have worked out as planned," Minor said.
Notre Dame and St. Mary's students dismissed school at 1:15 p.m. Friday.
Police have asked the Marion County Board of Education to put its schools on lockdown as a precaution, according to a BOE official.
All Pierpont and Fairmont State employees and students have been asked to leave the main campus in Fairmont in a calm and orderly way as a precautionary measure, according to university officials. The campus is not on lockdown. Employees and staff are being asked to leave campus as a precautionary measure, university officials said.
Commencement rehearsal at Pierpont Community and Technical College was canceled Friday afternoon but officials said commencement will go on as scheduled, with line-up starting at 6 p.m. and commencement starting at 6:30 p.m.
If you have any information that might be helpful in this investigation, contact your local law enforcement.
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2 posts
Hi, I currently am teaching/designing a show that deals with identity and I need one thing said in a view different volumes. One in a whisper, one in a soft but audible voice, one in a medium/normal volume, and lastly one in a loud volume.
All I need recorded is the statement,
"Set Me Free".
If you could help me with that it would be great!
Thank you so much.
Any females able to help? It would be extremely appreciated! Please and thank you
2 posts


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Chemotherapy Research and Practice
Volume 2011 (2011), Article ID 965826, 9 pages
Development of an Oral Form of Azacytidine: Triacetyl-5-Azacytidine
1Nevada Cancer Institute, One Breakthough Way, Las Vegas, NV 89135, USA
2St. Jude Children's Research Hospital, Memphis, TN 38105, USA
3The Hong Kong Polytechnic University, Kowloon, Hong Kong
Received 12 July 2011; Revised 14 September 2011; Accepted 21 September 2011
Academic Editor: G. J. Peters
Copyright © 2011 Amy Ziemba et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Myelodysplastic syndromes (MDSs) represent a group of incurable stem-cell malignancies which are predominantly treated by supportive care. Epigenetic silencing through promoter methylation of a number of genes is present in poor-risk subtypes of MDS and often predicts transformation to acute myelogenous leukemia (AML). Azacitidine and decitabine, two FDA-approved DNA methyltransferase (DNMT) inhibitors, are able to improve overall response although their oral bioavailability complicates their clinical use. This study evaluated -triacetyl-5-azacitidine (TAC) as a potential prodrug for azacitidine. The prodrug demonstrated significant pharmacokinetic improvements in bioavailability, solubility, and stability over the parent compound. In vivo analyses indicated a lack of general toxicity coupled with significantly improved survival. Pharmacodynamic analyses confirmed its ability to suppress global methylation in vivo. These data indicate that esterified nucleoside derivatives may be effective prodrugs for azacitidine and encourages further investigation of TAC into its metabolism, activity, and possible clinical evaluation.
1. Introduction
Currently, it is estimated that between 12,000 and 20,000 new cases of MDS are diagnosed each year in the United States. Although MDS can affect all ages, the highest prevalence occurs in those over 60 years of age [1, 2]. Much of the population has indolent forms of MDS, making it one of the most prevalent hematologic malignancies of older adults.
MDS represents a heterogeneous group of hematopoietic disorders which are derived from an abnormal multipotent progenitor cell and are characterized by hyperproliferative bone marrow, cellular dysplasia, and ineffective hematopoiesis [3]. Morbidity and mortality result from anemia, bleeding, and infection, along with transformation to acute myelogenous leukemia (AML) in approximately one-third of patients [4, 5]. The basis of therapy is supportive care, including red blood cell or platelet transfusions and treatment of infections. Stem-cell transplantation remains the only chance for cure, but it is associated with significant treatment-related morbidity and mortality and is generally restricted to patients <60 years of age [6]. Similar limitations exist for the use of high dose chemotherapy. Given the limitations of existing therapies, there is a clear need for additional therapeutic options for patients with MDS.
Cancer cells are characterized by abnormal DNA methylation patterns, and DNA hypermethylation is suspected of being involved in MDS progression and leukemogenesis. Therefore, inhibitors of DNA methylation represent a useful approach to revert these epigenetic changes. 5-azacitidine (Vidaza) and its derivative 5-aza-2′-deoxycytidine/decitabine (Dacogen) are nucleoside analogs with DNA hypomethylating activity that have been FDA-approved during the past 5-6 years for MDS treatment [7, 8]. They appear to induce re-expression of key tumor suppressor genes in MDS [9]. Compared to supportive care, both agents show an improved overall response (60% versus 5%), a longer time to progression to AML or death, but still with limited overall survival advantage [10]. Azacitidine has been developed for the treatment of acute leukemia and is currently being evaluated in a variety of other disorders [11].
DNA methylation results in the addition of a methyl group at the carbon 5 position of the cytosine ring in CpG dinucleotides, which is critical to chromatin structure and genomic stability [12]. Since 5-methyl cytosine can be deaminated to thymidine, DNA hypermethylation also facilitates gene mutations in human cancers [13]. The transferring of methyl groups from S-adenosylmethionine to cytosine is catalyzed by DNA methyltransferases, the best known of which is DNMT1. The inactivation of DNMTs has been shown to be the most effective method of inhibiting DNA methylation although it is recognized that this approach lacks specificity. However, inhibiting the activity of DNMTs has resulted in the abrogation of tumorigenicity in murine cancer models [14].
DNMT-inhibiting nucleoside analogs require metabolism by kinases into nucleotides before their incorporation into DNA and/or RNA in order to inhibit DNA methylation. The modification at C5 prevents the release of DNMTs by forming a covalent complex, preventing further DNA methylation and consequently the DNA of the progeny cells is not methylated [15]. Azacitidine and Decitabine are extremely potent in inhibiting DNA methylation, but their short half-lives in aqueous solutions [16, 17] and low oral bioavailability complicate their delivery. For years, these drugs used as antineoplastic agents in leukemias were escalated to maximum tolerated doses (MTD) [18]; however, recent clinical trials have confirmed that low-dose exposures lead to greater responses and are associated with less toxicity [19, 20].
In an effort to overcome the stability and pharmacokinetic limitations, a number of groups are working to develop oral DNMT inhibitors, including oral forms of decitabine and azacitidine. As a proof of principle we have synthesized an acetylated derivative of azacitidine, 2′,3′,5′-triacetyl-5-azacitidine (TAC) to evaluate as an oral prodrug. While this is not a totally new approach, it was never utilized for this derivative and in this set of diseases. Our current study demonstrates that 2′,3′,5′-triacetyl-5-azacitidine (TAC) effectively inhibits methylation and improves life expectancies in murine models while demonstrating lower toxicity when compared to its parent compound, azacitidine.
2. Materials and Methods
2.1. Preparation of 2′,3′,5′-Triacetyl-5-Azacitidine (TAC) [21]
TAC was prepared through condensation of trimethylsilylated-5-azacytosine and 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose. In a 150 mL, 3-necked flask, a mixture of 5-azacytosine, hexamethyldisilazane, and ammonium sulfate was heated at reflux for 2 hours. A fresh amount of ammonium sulfate was added, and the reflux was continued for 6 hours. The excess hexamethyldisilazane was removed under reduced vacuum to afford trimethylsilylated 5-azacytosine as an off-white residue.
Tetra-O-acetyl-D-ribofuranose was prepared by adding acetic anhydride dropwise to D-ribose dissolved in dry pyridine. The solvent was removed by heating the solution at 50°C for 15 minutes, and tetra-O-acetyl-D-ribofuranose was crystallized from isopropanol [22].
Trimethylsilylated 5-azacytosine and 1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose were dissolved in acetonitrile, and the mixture was cooled at 0°C in an ice-water bath. Trimethylsilyl trifluoromethanesulfonate (TMSOTf) was added slowly at 0°C then stirred at room temperature for 3 hours. The reaction mixture was poured into a solution of Na2CO3 and NaHCO3 in ice water and then extracted with dichloromethane. The combined organic layer was washed with cold H2O, cold brine, and dried over anhydrous Na2SO4. The residue, after removal of the solvent, was recrystallized from a mixture of dichloromethane and hexane to provide the desired compound with an 85% yield.
2.2. LC/MS/MS Analysis of TAC and Its Metabolites Mono- and Di-Acetyl-5-Azacitidine and ACT
To monitor the in vivo absorption, distribution, and metabolism of TAC, a highly sensitive LC/MS/MS analytical method was developed that allows the quantitation of concentrations as low as 10 ng/mL of TAC and its metabolites. The samples were prepared by adding 200 μL of plasma to 400 μL of cold acetonitrile, vortexed, and centrifuged at 14,000 rpm for 10 minutes. The supernatant was placed into a mm glass test tube and evaporated to 50 μL under nitrogen at 37°C. The residue was then diluted with 1 mL of 2% phosphoric acid and vortexed for 10 seconds at 3,000 rpm.
Solid phase extraction was conducted using a Varian Bond Elute PLEXA PCX 1 mL columns placed on a vacuum manifold. Columns were activated by adding 1 mL of MeOH followed by 1 mL H2O and 1 mL of 2% phosphoric acid. All samples were allowed to drip by gravity flow. The extraction columns were then washed sequentially with 1 mL 2% phosphoric acid, 1 mL H2O, and 1 mL MeOH/AcCN (1/1) and eluted into a mm glass test tube containing 50 μL of 25% formic acid in MeOH with 2 × 600 μL of 2% NH4OH in MeOH/AcCN (1/1). The liquid was then evaporated to dryness with nitrogen at 37°C, and 100 μL of mobile phase A was added and vortexed for 10 seconds at 3,000 rpm and transferred to LC autosampler vials at 4°C.
HPLC separation was conducted on a reverse phase C18 column (Varian Pursuit C18, 3 micron particle size; 100 mm × 2.0 mm) using a Shimadzu SIL-HTc controller with dual LC-20AD pumps and DGU-20A3 degasser. The separation was achieved using a gradient of Mobile Phase A: 0.1% Formic Acid and Mobile Phase B: 0.1% Formic Acid, 90% MeOH with a flow rate of 400 μL/min at 40°C over 10 minutes. MS/MS fragmentation was performed on an Applied Biosystems API 3200 instrument with a Turbo V Ion Spray ion source and Positive ion polarity.
2.3. Drug Stability
ACT and TAC stabilities were determined after incubation at pH1 (2% Phosphoric acid), pH3 (0.1% Formic Acid), pH5 (K3PO4), and pH7.4 (Dulbecco's PBS). 100 ng of each compound was resuspended in 1.2 mL total volume, and 200 μL samples taken at 1, 3, 6, 29, and 48 hr. Each pH point was repeated 6 times in 2 dram vials with Teflon-lined screw caps at room temperature. Samples were extracted by solid-phase extraction using 100 ng decitabine as an internal standard and analyzed by tandem mass spectroscopy as described above.
2.4. Log P Determination
The OSIRIS Property Explorer software was used to generate initial cLogP values. The partition coefficient was determined by using an analyte concentration near the solubility limit for the compound in water and incubating in a water : octanol mix ranging from 0.5 : 1, 1 : 1, to 2 : 1. The pH of the water was 2 units above the pKa of the compound, preventing ionization. The experiment was conducted using the shake-flask method, after allowing octanol and water to equilibrate for 24 hours at 25°C. The initial drug concentration in water was 0.2 mM. Tubes were rotated at a 45 degree angle for 10 minutes with a 3 second rotation. The test was performed in a constant environment of 20°C–25°C. Tubes were centrifuged at 1000 g for 10 minutes, and the octanol phase was extracted. The UV/Visible absorption spectrum was evaluated from 550 nm to 210 nm to determine the absorption maximum. The analyte concentration was determined and the LogP calculated: Log = log(concentration in octanol/concentration in water).
2.5. Permeability across Caco-2 Monolayers
Caco-2 cells (ATCC) were maintained under standard conditions in Dulbecco's modified Eagle's medium (DMEM). Approximately 1 × 105 cells (passage number 25–35) were seeded onto polycarbonate cell culture inserts (Transwell, 0.45 μm, 12 mm diameter). The cells were allowed to grow and differentiate for three weeks changing the medium every 2-3 days. Lucifer yellow was used to ensure appropriate paracellular permeability levels [23].
The transport experiments were performed in Hanks' balanced salt solution (HBSS) containing 25 mM Hepes (pH 7.4) as thoroughly described [24]. The drugs were dissolved in transport buffer to a final concentration of 0.2 mM. The monolayers were washed in prewarmed transport buffer for 30 min. The drug solutions were added to the donor side of the monolayers, and fresh transport buffer was added to the receiver side. The plates were gently shaken (50–100 rpm) at 37°C. At time intervals ranging from 5 minutes up to 2 h, 0.2 mL samples were collected from the receiver chambers and replenished with fresh transport buffer. Samples containing the transported drug was collected for LC/MS analysis by immediately freezing at −80°C and subsequently extracted 2 : 1 with acetonitrile and analyzed as indicated in the LC/MS/MS methodology section.
2.6. Pharmacokinetics and Metabolism of TAC in Mice
To evaluate the in vivo pharmacokinetic properties of TAC, C57/BL6 female mice of 20 g body weight were dosed with 38 mg/kg TAC p.o. (equimolar dose to 25 mg/kg of ACT) or 25 mg/kg ACT i.v. via tail vein injection. TAC and ACT were solubilized in 1X PBS immediately before administration and dosed at 0.01 mL/g fasted body weight. Blood samples (two time points per animal, three animals per time point) were obtained via retro-orbital bleeding with Natelson pipettes at 0.5, 1, 2, 4, 8, and 24 hours after drug administration. The collected blood was then centrifuged for 4 minutes at 13,000 g. Plasma was immediately extracted with cold acetonitrile and stored at −80°C until LC/MS/MS analysis.
Noncompartmental plasma pharmacokinetic parameters for TAC prodrug and ACT after oral TAC or i.v ACT administrations were estimated using the sparse sampling option in WinNonlin Software version 4.1 (Pharsight Corp., Mountain View, Calif, USA). Briefly, the WinNonlin sparse sampling option applies the linear trapezoidal rule for area under the concentration-time curve (AUC) calculations and generates a standard error for the mean AUC estimate that accounts for correlations in the data resulting from repeated sampling of individual mice. Parameters of interest included the AUC from time 0 to the last measurable concentration (AUClast), observed peak concentration after oral administration (), estimated peak concentration after i.v. administration (C0), mean residence time to the last measurable concentration (MRTlast), and the half-life of the terminal phase (T1/2). To assess the multiphasic decline in plasma concentrations, an alpha (T1/2α) half-life was also estimated using a log-linear regression of the mean ACT concentrations through 2 hours after dosing.
2.7. Global Methylation Detection Assay
24 hours following drug administration, snap-frozen mouse tissues were homogenized and genomic DNA isolated using Promega's Wizard Genomic DNA Purification Kit. 100 ng of EcoRI linearized genomic DNA was spotted onto PVDF membrane. The membrane was air-dried and repermeabilized according to manufacturer's instructions. Nonspecific binding was blocked using 5% milk in PBS-T and washed twice in PBS-T for 5 minutes. The membrane was incubated with primary antibody (1 : 1000 anti-5-methylcytosine in PBS-T, Calbiochem) for 1 hour then washed four times for five minutes each with PBS-T. The membrane was incubated with the HRP-conjugated secondary antibody for 1 hour and washed four times for five minutes each with PBS-T. Signal intensity following ECL detection was quantitated using KODAK Image Station software. Assay results were confirmed using EpiGentek's Methylamp Global DNA Methylation Quantification Kit per manufacturer's instructions.
2.8. p15INK4B Methylation Status
Mouse leukemia cells (L1210) were purchased from ATCC. The published IC50 value for ACT (0.2 uM) was first confirmed using the Promega CellTiter MTS assay. Cells were treated with ACT (0.1 or 1 uM) or TAC (1 or 100 uM) for 48 hours, with fresh drug added daily. Genomic DNA was isolated after 48 hours using the Sigma GenElute Genomic DNA Kit, including the RNase treatment step to ensure no RNA carryover. The methylation status of p15INK4B was determined using a predesigned primer set and the EpiTect Methyl qPCR Assays MethylScreen technology (Qiagen, Frederick, Md, USA).
2.9. Antileukemic Activity of Triacetyl-5-Azacitidine
The in vivo L1210 lymphocytic leukemia model in C57BL/6 × DBA/2 F1(BDF1) female mice was used to evaluate the in vivo antiproliferative effect of TAC. L1210 cells were carried in BDF1 mice by weekly i.p. (intraperitoneal) passages. Leukemia cells from ascites fluid were diluted appropriately in RPMI 1610 medium and injected i.p. (1×105 cells/0.1 mL/mouse) into recipient mice. A daily oral administration of TAC (38 mg/kg via oral gavage) or ACT (25 mg/kg administered i.p.) diluted in PBS was given to leukemic BDF1 mice starting 24 hours from the inoculation of L1210 cells for a total of 5 days. Compounds were prepared fresh daily. A group of untreated leukemic mice received sterile water for injections and served as controls.
2.10. TAC Toxicology
The in vivo toxicity of TAC was evaluated in CD-1 mice using 3 groups of 3 male and 3 female CD-1 mice per group. CD-1 is the classical mouse strain chosen for toxicology studies including safety and efficacy. On Days 1–5 and Days 8–12, animals were administered vehicle (sterile water for injection, Group 1), 38 mg/kg/day TAC (Group 2), or 76 mg/kg/day TAC (Group 3) via oral gavage. Animals were not dosed on Days 6 and 7 and were euthanized on Day 13 for necropsy. Doses were prepared fresh daily from the drug stock. Criteria for evaluation included clinical observations, body weights, limited serum chemistry and hematology parameters, and histopathology evaluation.
3. Results
3.1. Synthesis and In Vitro Characterization of Triacetyl-5-Azacitidine
2′,3′,5′-triacetyl-5-azacitidine has been successfully synthesized through condensation of trimethylsilylated-5-azacytosine and 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (Figure 1). An HPLC detection assay was optimized for TAC and ACT as well as some potential analytes.
The solubility of TAC in comparison to ACT was evaluated at various pH. TAC and ACT were equally soluble at pH1 (40 mg/mL) and pH3 (25 mg/mL), while TAC was more soluble than ACT at pH5 (30 mg/mL versus 15 mg/mL) and pH7 (30 mg/mL versus 10 mg/mL). The stability of the prodrug across a range of pHs is indicated in Table 1. TAC shows remarkable stability at pH 3, 5, and 7.4 with no degradation to the parent compound and 30% hydrolysis to the mono- and diesters only at pH 7.4 after a 29 hr incubation at 25°C. At pH 1, we observed a moderate hydrolysis of TAC (26%) to the mono- and diesters in the first 3 hours of incubation increasing to 60% after 6 hrs. However, after 24, 99% of TAC was hydrolyzed, including 46% to the parent compound, ACT.
A similar experiment to establish the stability of azacitidine in solution resulted in significant degradation of the nucleoside analog throughout the pH range with maximum stability at pH 1. As previously determined, [25] the initial degradation to N-formylguanylribosylurea followed by the irreversible formation of guanylribosylurea resulted in two derivatives with no pharmacological activity (data not shown).
3.2. LogP Determination
The logP value of a compound is a well established measure of a compound's hydrophilicity. The theoretical physical-chemical properties of TAC were evaluated by measuring the partition coefficient of the un-ionized molecules in two immiscible phases of water and octanol. Azacitidine resulted in an experimental LogP value of −2.32 compared to the calculated cLogP values of −2.17 while the changes to the molecule by acetylation resulted in an improved LogP value for TAC of −0.61 versus a cLogP of −0.85.
3.3. Permeability across Caco-2 Monolayers
The human colon carcinoma Caco-2 cell model was used to evaluate the oral absorption of the prodrug across membranes and determine the permeability coefficient (absorption rate constant). A sigmoidal relationship exists between drug absorption rates as measured with the in vitro Caco-2 model and human absorption [26]. Permeability coefficients typically range from to cm/s. Drugs that are well absorbed in humans have permeability coefficients greater than cm/s, while drugs and peptides that are absorbed to less than 1% have permeability coefficients less than cm/s [27, 28]. Between these ranges, compounds are considered to have intermediate absorption rates. In these studies, ACT was determined to have a permeability coefficient between 1–, compared to 5– for TAC. This indicates a potential 2-3 fold improvement in intestinal absorption. The efflux ratio is defined as the quotient of the secretory permeability and the absorptive permeability. The efflux ratio was near 1.0, indicating these compounds are not being actively effluxed. No hydrolysis of the prodrug to azacitidine was observed during the course of this experiment.
3.4. Pharmacokinetics and Metabolism of TAC in Mice
TAC was administered at 38 mg/kg to C57/BL6 mice via oral gavage, an equimolar dose equivalent to 25 mg/kg of azacitidine. TAC and its active metabolite ACT were detectable in plasma at 30 minutes after the oral administration (Figure 2(a)). TAC appeared to be rapidly deacetylated leading to a minimal accumulation of the prodrug resulting in plasma concentrations below the limit of quantitation (10 ng/mL) at time points beyond 1 hour after administration. TAC-derived azacitidine reached a peak concentration of approximately 5,000 ng/mL (~20 μM) at 30 minutes with a pharmacologically relevant concentration of at least 0.5 μM being sustained for 24 hours after oral TAC dosing. The mean TAC and TAC-derived ACT plasma concentrations versus time plots after oral administration of TAC prodrug are depicted in Figure 2(a) with the pharmacokinetic parameters reported in Figure 2(b). ACT was also administered i.v. as a control to emulate the standard means of administration in the clinical setting. The T1/2 and MRTlast values for TAC-derived azacitidine were 9.2 hours and 7.7 hours, respectively, whereas i.v. administration of ACT resulted in T1/2 and MRTlast values of 6.8 hours and 1.1 hours, respectively. The AUClast values for plasma azacitidine were 73.5 hr-μM and 126 hr-μM after oral administration of TAC prodrug or i.v. administration of ACT, respectively. While the absolute bioavailability of derived azacitidine after oral TAC administration is 58%, the prolonged T1/2 and MRTlast values for azacitidine observed in plasma after oral TAC administration suggest a protracted absorption and conversion of the TAC prodrug at the gastrointestinal or presystemic level (Figure 2(b)). The log-linear regression analysis of ACT concentration data through 2 hours revealed an increased T1/2α value for ACT after p.o. TAC administration compared with i.v. ACT administration (0.73 hr versus 0.32 hr, resp.), also suggesting protracted absorption and both presystemic and systemic conversion of TAC.
3.5. Pharmacodynamic Effect of Triacetyl-5-Azacitidine
MTS assays (Promega, Madison, Wis, USA) were performed to determine the in vitro effects of ACT and TAC on L1210 cell proliferation. TAC had no effect on L1210 cell viability even at doses 100-fold higher than the known IC50 of ACT (0.2 uM) [29], and this is an indication that this compound does not get activated in vitro. This result was predicted, since cells in culture lack the necessary esterase activity to convert the prodrug to ACT that remains below the limit of detection using our LC-MS method.
Gene-specific methylation PCR was performed in order to confirm whether TAC could affect the methylation level of a specific target even in the absence of cellular toxicity. ACT, which is the active derivative of TAC, has been extensively studied by numerous research groups to determine specific cellular methylation targets and mechanism of action [30, 31]. P15INK4B is a classic target known to be hypermethylated in AML and MDS and attributes to a poor prognosis in those patients [32, 33]. 0.1 uM and 10 uM ACT decreased P15 promoter methylation by 39% (±3) and 49% (±17), respectively. In comparison, 1 uM and 100 uM TAC decreased P15 promoter methylation by only 19% (±8) and 23% (±16), respectively.
In an in vivo experiment designed to evaluate the effect of a prolonged administration of TAC on global methylation, TAC (38 mg/kg p.o.) or ACT (25 mg/kg i.p.) diluted in PBS was administered to C57BL6 mice every 24 hours for 5 consecutive days (four animals per group). Tissue methylation levels are reported in Figure 3. A significant reduction in global DNA methylation was detected in the gut and spleen after the administration of TAC, comparable to the methylation decrease detected after the administration of ACT. We did not observe a significant change in global methylation status in kidney or liver tissue (data not shown). The positive results in gut and spleen were confirmed using the EpiGentek's Methylamp Global DNA Methylation Quantification Kit. This quantitative analysis confirmed a comparable 50%–60% decrease in global methylation by both TAC and ACT (data not shown).
3.6. Antileukemic Activity of Triacetyl-5-Azacitidine
The L1210 lymphocytic leukemia model was used to determine the efficacy of TAC in an animal model. The L1210 model is a classical model used to predict for compounds that are effective against leukemias and lymphomas. A daily oral administration of TAC (38 mg/kg equimolar to 25 mg/kg of ACT and a higher dose of 50 mg/kg) or azacitidine i.p. (25 mg/kg) was given to leukemic mice for a total of 5 days. The median survival time for the untreated control group was 8 days, while the median survival time for the TAC treated groups was 12 days resulting in a 50% increased lifespan, demonstrating not only an epigenetic modulating effect, but also antileukemic activity. Azacitidine at the maximum tolerated dose of 25 mg/kg administered i.p. resulted in a 17-day survival with a 112% increased lifespan (Figure 4(a)). The toxicity curve (Figure 4(b)) indicates a nonsignificant weight loss for the TAC-treated groups at day 3 followed by a rapid recovery indicating the lack of toxicity of this prodrug coupled with a significant antileukemic effect, while the azacitidine-treated animal showed a 16% weight reduction with a significant recovery once the treatment was completed.
3.7. TAC Toxicology
The in vivo toxicity of TAC was evaluated by orally administering repeated doses to CD-1 mice for two weeks. On Days 1–5 and Days 8–12, animals were administered vehicle, 38 mg/kg/day TAC (Group 2, equivalent to 25 mg/kg of ACT), or 76 mg/kg/day TAC (Group 3) via oral gavage. Criteria for evaluation included clinical observations, body weights, limited serum chemistry and hematology parameters, and histopathology evaluation of a limited number of tissues from vehicle-treated and 76 mg/kg/day TAC-treated animals.
There were no deaths and no notable TAC-associated body weight changes during the study. Additionally, there were no TAC-associated changes in liver or renal function markers, including alanine and aspartate aminotransferases, alkaline phosphatase, bilirubin, blood urea nitrogen, and creatinine.
TAC-associated changes in various hematological parameters were observed (Figure 5). This included a reduction in white blood cell counts, absolute lymphocytes, and absolute leukocytes by 41%, 31%, and 24%, respectively, for the 38 mg/kg/day dosage group. The reduction values for the 76 mg/kg/day group only slightly increased by 44%, 31%, and 34%, respectively. There were TAC-related findings in the bone marrow (hypocellularity) and in the lymph nodes (inactive germinal centers) which reflect the decreased overall white blood cell count reductions. An additional microscopic TAC-related finding was duodenum apoptosis of the crypt epithelium in the 76 mg/kg/day-treated animals. Other observations included a decrease in myeloid and erythroid compartment cellularity correlating with microscopic findings in various tissues.
4. Discussion
Epigenetic modulators are appealing cancer therapeutics due to their potential reversibility and preference for highly proliferating cancer cells. Azacitidine and decitabine have demonstrated improved clinical success in hematologic malignancies with longer or continuous infusion [34]. However, the aza-pyrimidine ring is unstable in aqueous solutions and has poor bioavailability, limiting the use of oral formulations that would be more convenient for the patients, help in long-term dosing, and reduce local side effects when administered as subcutaneous depot.
We have successfully synthesized an azacitidine prodrug, 2′,3′,5′-triacetyl-5-azacitidine and shown its favorable physical-chemical characteristics. TAC has demonstrated higher solubility and stability in vitro across a wide pH range compared to its parent compound azacitidine, which is of great benefit for avoiding rapid degradation in the acidic environment of the stomach and the more neutral environment of the periphery. TAC has also shown a more favorable logP. While LogP values may not always be a completely accurate determination of the lipophilicity of ionizable compounds, the data confirm the efficient drug absorption at the gastrointestinal level and bioavailability observed.
In vivo analyses were performed comparing the desired oral version of the prodrug with the classical administration route of ACT (i.v. or subcutaneous). The stability and bioavailability of the prodrug was confirmed in vivo, where oral TAC-derived azacitidine had a terminal half-life of 9.2 hours versus 6.8 hours for i.v. administered azacitidine. Additionally, the alpha phase half-life (T1/2α) value after oral TAC administration was longer compared with i.v. ACT—0.73 hr versus 0.32 hr, respectively. The half-life values indicate a protracted absorption of the nucleoside prodrug at the gastrointestinal level and an effective conversion by serum esterases. This may allow maintenance of ACT concentrations over a 24-hour period as opposed to roughly 6 hours for i.v. ACT. The half-life of azacitidine in this murine model is longer in our study compared to some of the previous literature [35, 36]. This is likely due to an improved sensitivity of our analytical methodology that allows monitoring the concentration of azacitidine below 100 ng/mL.
TAC also demonstrated a pharmacodynamic effect in vivo. Oral TAC appears comparable to i.v. AC in its ability to suppress global methylation in normal tissues. Further analysis will be necessary to determine if this is a tissue-specific effect or effective in altering the methylation status of cancer cells and to identify specific gene targets. TAC was less effective than ACT at improving life span in the leukemic L1210 animal model, possibly due to a higher achieved by i.p. administration compared to oral, confirming the dichotomy between epigenetic therapy that is effective at low extended doses versus the cytotoxic effect achieved with higher doses and linked to a higher [37].
Animals administered TAC for 2 weeks resulted in no deaths and no weight loss, indicating minimal general toxicity. However, TAC did induce the expected changes in hematological parameters, bone marrow, and lymph nodes. Gastrointestinal toxicity was observed at the higher TAC dose with duodenum apoptosis of the crypt epithelium. This effect is possibly due to the localized hydrolysis of the prodrug with formation of azacitidine known to cause gastrointestinal toxicities in patients. Several have attempted to overcome the lack of stability and poor bioavailability of the current DNMT inhibitors. Oral formulations of azacitidine and decitabine have not been successful due to their hydrolysis in the aqueous environment of the intestinal tract and rapid deamination. An oral film-coated formulation of azacitidine has been recently evaluated in four human subjects resulting in 17.4% bioavailability for an 80 mg dose with a mean plasma half-life of 0.39 hr versus 0.68 hr for a subcutaneous formulation [38]. A very recent Phase I study of oral azacitidine showed a high interpatient variability in the bioavailability of azacitidine ranging from 6.3% to 20% (33) indicating the need for a more controlled delivery [39].
Other groups have proposed modification of the structure at the N4 position such as the addition of a 2-(p-nitrophenyl)ethoxycarbonyl group (NPEOC) to protect the azacytosine ring [40]. This derivative is similar to the 5-FU prodrug capecitabine. The N4-NPEOC-5-aza-2′-deoxycytidine has been shown to be activated to decitabine resulting in global and gene-specific DNA demethylation in cells expressing carboxylesterase 1 [41]. The NPEOC derivatives are characterized by very low solubility in water and their elevated hydrophobicity could lead to an improved oral bioavailability; however, in vivo data in animal models or humans are not available [40]. Protection of the N4 has also been achieved in sapacitabine by incorporating a N4-palmitoyl group of (1-(2-C-cyano-2-deoxy-β-D-arabino-pentafuranosyl) cytosine (CNDAC). This modification has resulted in better diffusion into gastrointestinal cells allowing the oral administration of the drug [42]. A Phase I study of oral sapacitabine confirmed its metabolism to CNDAC; however, it resulted in pharmacokinetic variability possibly due to differences in absorption or metabolism [43].
To improve the stability characteristics of decitabine and increase its intracellular delivery, the nucleoside analog has been incorporated into a guanine dinucleotide to generate S110 (5′-AzapG-3′). S110 has shown stability in aqueous solutions and toxicity comparable to that of decitabine with increased resistance to enzymatic degradation (deamination) before incorporation into DNA following endonuclease and/or phosphodiesterase cleavage [44].
We have synthesized and evaluated an acetylated derivative of azacitidine, 2′,3′,5′-triacetyl-5-azacitidine (TAC) as an oral prodrug. While this is not a totally new approach, it was never utilized for this derivative and in this set of diseases. A few examples exist in the literature of this type of derivatization for the oral delivery of pyrimidine analogs. Triacetyl-6-azauridine was developed to overcome the poor oral absorption of azauridine, an antipsoriasis drug [45]. 2′,3′,5′-tri-O-acetyluridine (PN401) was synthesized as a rescue agent for 5-fluorouracil-based therapies [46, 47].
Taken together, our data indicate the triacetylated derivative of azacitidine may be an effective prodrug that could allow continuous exposure to azacitidine at low doses resulting in a protracted DNA hypomethylation. The superior stability and solubility of TAC, together with its minimal toxicity, will encourage the further investigation of its mechanism of action, its epigenetic modulatory effect, and its ability to slow down the progression of MDS into AML.
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In less than 48 hours: Algerian army kills 16 alleged armed Islamists
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Algerian security forces have shot dead ten alleged armed Islamists in the western provinces of Algeria, KUNA reported Friday. Eight gunmen were killed in a first operation Thursday in the Saida province, 350 kms west of Algiers.
The two others were killed in a second operation the same day in a combing operation in the Gelizane province, more than 300 kms west of the capital.
The army seized arms and ammunitions in the two operations.
On Thursday it was reported that Algerian troops killed six alleged armed Islamists in the mountains of Assass in the province of Tlemcen, 650 kilometers west of Algiers. (Albawaba.com)


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Suzann Pettersen leads LPGA Championship; Pressel, Sorenstam in contention
Norwegian Suzann Pettersen shot a Friday round of 67 to lead the LPGA Championship. Yet all anyone wants to talk about is whether Pettersen is still haunted by her Kraft Nabsico collapse earlier this year.
Go to article or click photos to enlarge.
Suzann Pettersen - LPGA Championship
Suzann Pettersen hasn't let the Kraft memories stop her smiling.
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Struggling to live up to annually high expectations since the lockout, the odds are finally in favour of the Calgary Flames meeting or exceeding them.
Thats because people arent anticipating much.
The buzz on the first day of training camp Friday will have a much different feel than it did a year ago when the Flames were considered a Stanley Cup contender.
And maybe thats just what this veteran team needs.
Motivation in the form of proving people wrong, rather than right.
Considered a division if not conference favourite the last five seasons, theyve been less than stellar in four first-round knockouts before missing the playoffs altogether last spring.
So here we are heading into camp with a roster not entirely unlike the one that entered last fall as a favourite to edge the Vancouver Canucks for the Northwest Division title, but armed with expectations much lower than that.
Just make the playoffs again.
Some dont dont even give them that much credit.
Gambling on players like Alex Tanguay, Olli Jokinen and Ales Kotalik rebounding from substandard seasons, and others like Matt Stajan, Niklas Hagman, and Rene Bourque having career years, Flames GM Darryl Sutter has done what he believes he can to help the club rebound from a brutal offensive season.
It could work, but good luck finding someone who will bank on it.
Coming into camp a year ago with Las Vegas odds pegging them at 18/1
to win the Stanley Cup, the Flames have seen their numbers drop to 35/1.
They were considered a top-10 bet last fall.
Theyve fallen to the middle of the pack, sharing odds with teams like the Tampa Bay Lightning, Ottawa Senators and Anaheim Ducks and well behind teams like the Boston Bruins and Los Angeles Kings.
While theres little doubt people inside the locker-room would love to drop some dough on themselves if that kind of thing werent frowned upon in the world of pro sports and take advantage of their longshot status, theres even less question theyd be offended at the idea few are taking them seriously this season.
They still have stud goaltender Miikka Kiprusoff and winger Jarome Iginla, who should get at least a little more help in the goal-scoring department this year.
Jay Bouwmeester and Robyn Regehr are anchoring the defence, and Mark Giordano has sufficiently replaced Dion Phaneuf as the young up-and-comer. Theres plenty of experience behind them.
Or is that age?
It all depends on how much last years fall has affected your feelings toward the Flames, whether your glass is half full or half empty.
Another month from now when the first week of regular-season action is in the books, that perception could change.
So could the odds on them winning the Cup.
For now, though, they have to prove to their coaches in camp they can get the job done, and in the process, convince their fans that last year was an anomaly and not the new standard.
Or substandard.
Their own expectations havent changed.
The players will take the same game-by-game approach to the playoffs.
Their motivation to make it there again, however, has deepened.
From individuals like Tanguay, Kotalik and Jokinen who feel the need to prove they can still produce at a high level, to the core that may have begun to take the playoffs for granted, competition is a word that should extend well beyond camp.


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Colin P. Doherty
Colin P. Doherty, Ph.D., works as a Consultant Neurologist and as an Honorary Senior Lecturer in neurology at St. James's Hospital in Dublin, Ireland. He was trained in the Partners Neurology Residency Program at Harvard Medical School, Boston, USA, and subsequently completed fellowships in epilepsy and cognition. He has published widely the areas of brain imaging in epilepsy, the genetics of epilepsy, functional imaging of language, and clinical epileptology and neurology. In the last few years, he has become interested in the challenges of population health, health services research, and care delivery in epilepsy. He was a Member of the Irish government's expert group on resource allocation in the health sector which produced its report in 2010. He is a Member of the national clinical effectiveness committee in Ireland and a Board Member of the main government funding body for clinical research in Ireland, the Health Research Board. He is the Irish representative to the International League Against Epilepsy (ILAE) committee for the Global Campaign Against Epilepsy. Recently, he has been appointed as the National Clinical Lead for the Epilepsy Care Program in Ireland, with a charge to develop epilepsy services nationally to improve quality of care, access for patients, and value.
Biography Updated on 18 December 2011


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Republicans in Northern Ireland look to Taliban for weapons
Dissidents have links to advanced technology never seen before in Ulster
TALIBAN-INSPIRED technology is boosting the capacity of dissident republicans to wage war against the security services, with the discovery of advanced weaponry never seen before in Northern Ireland, The Independent on Sunday can reveal.
The degree of technical sophistication is "unprecedented", and experts are warning that it is part of a worsening picture that could include a sustained bombing campaign.
Police managed to foil an attack which had been planned in South Armagh using what the Police Service for Northern Ireland (PSNI) described as two "mortar type" devices. Dissident republicans had planned to bring down a helicopter using the rocket launchers, which took army bomb disposal experts three days to examine.
In the wake of the discovery, security sources approached Democratic Unionist MP Jim Shannon with their concerns. The weaponry, found in August, was unlike anything seen in Northern Ireland before. It is understood that it could be detonated remotely using an infrared laser – a tactic used by the Taliban in Afghanistan.
"This is a different level of terrorism. When it comes to the sophistication, when it comes to the technological detail, these are things that have never been seen in Northern Ireland," said Mr Shannon.
He said the "deeply worrying" discovery confirmed that there are links between people in Afghanistan and Pakistan and those that made the bomb and mortar attack weapon in Cullyhanna.
The Northern Ireland Office, PSNI and Ministry of Defence declined to comment. But one security expert, who would not be named, said: "There is information to suggest it had an infrared detonator. There is a genuine anxiety about this."
Independent MP Patrick Mercer, a former army officer who has served in Northern Ireland, speculated last night that another possibility was that military personnel who have served in Afghanistan and Iraq may be responsible for passing on details about the technology. "I have heard about this. This is all to do with light-sensitive devices," he said. "But of course it's no more or less than the fact that they've got people coming back from Afghanistan who have served over there who are able to pass on this expertise. There are many Irishmen serving in all branches of the services. It's not unknown for loyalties to be split."
Speaking under condition of anonymity, a senior military figure who commanded troops in Northern Ireland, admitted: "It is almost inevitable that 'leakage' of military skills from 'us' to 'them' happens over time and is disturbing and definitely of concern to the hierarchy."
But it is possible that "information exchange" between dissidents and the Taliban is taking place, according to Colonel Richard Kemp, former commander of British forces in Afghanistan. "We did see in the past co-operation between Islamist extremists in the Middle East and the Provisional IRA."
Earlier this year The IoS revealed how dissidents are using armour-piercing horizontal mortars similar to those used by the Taliban.
There is a "growing sophistication in terms of capability" and the ranks of the dissidents are being swelled by former Provisional IRA members who have lost patience with the peace process, according to Professor Richard English, a terrorism expert at the University of St Andrews. He said: "Given the capacity which seems to be emerging from the dissident republicans, it would be surprising if there were not more fatalities in the near future in Northern Ireland. The conditions are there for a sustained, occasionally very lethal, republican bombing campaign."
There have been big advances in the skills of dissidents in the past few years, and they are winning support due to a strategy of low-level vigilante attacks, according to Professor Andrew Silke, director for terrorism studies at the University of East London. The threat is being "downplayed" in Britain, and "as a result people are unaware how bad it's getting and the direction it's going", he added.
Recent activity by dissidents includes an attempt to blow up a 150lb bomb in Belfast city centre last Sunday. Earlier this month, a former police officer and his 12-year-old daughter were targeted when a booby-trap bomb was placed below his car in County Down. It was one of more than a dozen attempted bombings across the province in recent weeks.
Jonathan Owen, Independent.co.uk
Online Editors


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On the way to Windy Gap
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Scenery along the short but steep approach canyon to Windy Gap from the north. The view is at the western slope of the canyon. On top of this slope the Pacific Crest Trail winds its way to Little Jimmy Campground. San Gabriel Mountains, July 2011
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Wayne Rooney pays price for missing penalties
Wayne Rooney has been stripped of penalty-taking duties for Manchester United.
Sir Alex Ferguson's decision produced almost immediate dividends, with Ryan Giggs scoring from the penalty spot to give United a third minute lead in their FA Cup fourth-round tie against Fulham at Old Trafford.
Rooney missed the tenth penalty of his United career during the FA Cup third-round replay at home to West Ham United ten days ago.
Ferguson had stayed tight-lipped about his plans when asked in the wake of that miss whether Rooney would keep the job but it has since emerged that the United manager told his striker that he would be relieved of the responsibility.
It is understood that Robin van Persie has been installed as United's new primary penalty-taker,…


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10 Smart Social Media Tactics to Try
March 9th, 2013
Did you know that there is a whole world of social media out there beyond Facebook and Twitter? That really, social media is any sort of website or digital space where people can talk about content. Your blog, YouTube, Pinterest and Instagram are also networking sites that deserve consistent updates so get ready to use them all in the best way possible with these 10 smart social media tactics.
10 Smart Social Media Tactics to Try
- Talk less and share more. Start sharing other peoples content.
- Just be yourself. I know you've probably heard that a million times – but it's true! Your authentic self is your best self.
- Try to help solve problems. If you're followers ask a question and you can help find a solution – do it.
- Become a part of the conversation. Don't just talk about engagement, do it.
- Get in the habit of retweeting. People love to be retweeted – it lets them know that that they're being heard.
- Understand who and where your content is being read and shared.
- Try a social media monitoring tool to know when and where you're being discussed.
- Offer your readers weekly roundups – gather all your favorite content from the web each week and share the links.
- Step up your game with lots and lots of photos.
- Share quotes to inspire your readers and followers.
What do you think – can you try these 10 smart social media tactics?


text:
Son of Popular Israeli-born Evangelist Among Victims of Downed Malaysian Jet
27-year-old Ithamar Avnon was the son of Dov Arnon, a popular evangelist born in Israel who converted to Christianity.
One of the victims of the Malaysian airline disaster on Thursday was Ithamar Avnon, 27, son of Israeli-born Dov Arnon, a convert to Christianity and...
Want to enjoy 'Zen' reading - with no ads and just the article? Subscribe todaySubscribe now


text:
finic helps users manage their finances. It can be used on a local network (as a client-server system for many users) or on local computer (client only for one user), with separation of accounts, clauses, and customers. It can encrypt information and manage different users with different access rights. Reports can be generated as HTML, PDF, or XLS files or through an internal print preview system. An API is available for creating extensions.
DBReplicator is a powerful application for network-based multi-master heterogeneous database replication or filtered synchronization. It supports heterogeneous replication, bi-directional data synchronization between any of the supported database backends, application independence, automatic conflict detection and resolution, a scheduling facility, verbose debugging using Apache log4j, special characters, and automatic table creation.
The TREE Data Server captures real-time financial data from one or several data feed services (e.g. IB TWS API), archives data in a historical database, and makes both live and archived data available to client applications. The system can be used in real-time charting, an ATS, a tick feed simulator, or any situation in which multiple clients need real-time access to the tick stream or archived tick data. In addition, the archived tick stream and tick data are available for offline data analysis and backtesting.
python-money provides carefully designed basic Python primitives for working with money and currencies. The primary objectives of this module is to aid in the development of financial applications by increasing testability and reusability, reducing code duplication and reducing the risk of defects occurring in the code. The module defines two basic Python classes: a Currency class and a Money class. It also pre-defines all the world's currencies, according to the ISO 4217 standard.


text:
Here is a step by step guide on how to finish your Farmville 2: Riled in the Wild Mission.
"Some creature has been chewing on my crops at night. Have you seen this mysterious midnight nibbler?"
Walter said: "I think a wild puff-ball rabbit has been munching on my produce? Please help me to catch it and stop it!"
You will need 3 things to complete the first part of the Riled in the Wild Mission:
- Collect 5 Barbed Wire
You can ask your friends for Barbed Wires or take advantage of the Zynga Community Post to acquire these.
- Perform 15 neighbor actions
Visit 3 friends and use up your visit energy to help them out.
- Buy a Cotton Tail Rabbit
Cotton Tail Rabbit is available on the General Store for 9 Farm bucks or you can buy a Baby Cotton Tail Rabbit for 4000 coins. Baby Cotton Tail Rabbit needs 2 Baby bottles to grow as an adult Cotton Tail Rabbit and produces (1) Wool when fed with 4 feeds. Wool sells on the Market Stand for 90 coins
When you have completed the requirements for the first part you will be rewarded with
- +70 Experience
- +1200 coins
Walter: "Hmmmm. It appears our mystery creature may be much larger than an ordinary rabbit. We'll need to be careful!"
Riled in the Wild 2/4: The Carrot Trap
The second part of the mission starts with Walter saying:
"Most night feeders love root vegetables". Perhaps our mystery creature is the fabled giant rabbit! Lets lure it out!"
You will need 3 things to complete the second part of the Riled in the Wild Mission:
- Plant 20 Carrots
We all know that rabbits love carrots. Go to general store and buy seeds of carrots and place them on your farms plots. Carrot seeds cost 60 coins, it needs 24 hours to harvest and sells for 188 coins.
TIP: Use your farm hands to speed up the time between harvests.
- Use Fertilizer on 10 Carrots
You need 10 Fertilizers to use on your planted carrots. You can obtain Fertilizers 3 different ways, by asking your friends and neighbors, when you feed your animals and you can take advantage of the Zynga community neighbors. Fertilizers are very helpful, when you Fertilize a crop you'll get double the quantity of your harvest, therefore saving time to harvest another one, specially when you need bigger quantities.
- Sell 15 Recipes to the Villagers
Go to your kitchen and craft some easy to craft Recipes. Examples are Flour, Batter, Lemon Water sell them sell them on your Market Stall or if you already have crafted some earlier get 15 of your choice and hit the market.
When you have completed the requirements for the second part you will be rewarded with
- +80 Experience
- +1200 coins
Walter: "Ha! No creature can resist beautiful carrots like those! Let's see if it comes out tonight!"
Riled in the Wild 3/4: The Carrot Trap
Walter: "Still no luck catching that rabbit! You said the folks suggested crafting Carrot Bread? I had no idea rabbits had such refined tastes!"
You will need 3 things to complete the third part of the Riled in the Wild Mission:
- Plant 15 Wheat.
Wheat is an ingredient to make Flour. You will need Flour later on to make Carrot breads. Wheat seeds cost 10 coins and sells for 14 coins. It takes 4 hrs to harvest, i suggest that you also Fertilize your crops.
- Help Walter out on his Farm.
Go and visit your neighbor Walter and spend 5 visit energy co complete this requirement.
- Craft 2 Carrot Breads.
Proceed to your kitchen and craft or bake your Carrot Breads. You will need 2 Carrot's and a Bread to make one Carrot Bread. Bread can be crafted and it will need 2 Salts and a Dough. Obtain Salt by asking your friends and craft the though. To craft a Dough you will need a Flour and 2 water. Make use of the wheat you just harvested and craft a Flour.
When you have completed the requirements for the third part you will be rewarded with
- +90 Experience
- +1200 coins
Walter: "I knew it! It was Marie's lost black mare eating my crops all this time! Why was it so hungry?
Riled in the Wild 4/4: Unbridled Attraction
Walter: "It turns out it wasn't a giant rabbit eating everything, it was Marie's lost black mare. She was pregnant and had a baby!"
You will need 3 things to complete the fourth part of the Riled in the Wild Mission:
- "Adapt" the baby Black Arabian Horse.
Buy a Baby Black Arabian Horse on the General Store It will cost you 22000 coins so save up.
- Raise a Black Arabian Horse.
Feed the Baby Black Arabian Horse you bought earlier with 6 Baby bottles to grow it to adulthood.
- Feed an adult Black Arabian Horse
An adult Black Arabian Horse produces 3 Saddles and 2 Horseshoes and some Fertilizers randomly when fed, which are very profitable.
When you have completed the requirements for the third part you will be rewarded with
- +100 Experience
- +1200 coins
Marie: "Thank you so much for finding my lost horse and raising the baby! Drop by and see mama horse anytime!"
You have finished the Farmville 2: Riled in the Wild Mission
Total Gain:
Experience from completing the mission: 340
Gold gained from completing the mission: 4800
+ experience and gold from planting and harvesting crops.


text:
Old 280 Boogie
April 16, 2016
April 16, 2016
Standard Deluxe
The Boogies assemble great Southern musical acts and put them on a stage that Peek built under a shed out in the yard. When you're in Waverly and someone tells you it's time to "go out in the yard and boogie," they mean it.
Folks bring their kids, listen to music, eat barbecue and pickles from the local pickle maker, Wickles Pickles, and shop for T-shirts and posters. They hang out and just generally have a high old time.
Get Directions using Google Maps
Quick Links
Lake Martin News
Lake Martin Photos
Lake Martin Videos


text:
Nifmon is a curses-based tool for FreeBSD which can be used to monitor network
interfaces (also tun devices).
It displays continously updated statistics for the following quantities:
- total amount of bytes/packets transferred in/out
- bytes/packets transferred in/out during the last second/minute/hour
- a stripped down tcpdump output
You will need super-user permissions in order to use nifmon.
WWW: http://www.freebsd-support.de/misc/
make generate-plist
To install the port: cd /usr/ports/net/nifmon/ && make install cleanTo add the package: pkg install nifmon
cd /usr/ports/net/nifmon/ && make install clean
pkg install nifmon
PKGNAME: nifmon
SHA256 (nifmon-1.4.tar.gz) = c7f3faec467eb8c4f913d481975317d8dafd08e42d1cc92aeb0e0445f5648c44
SIZE (nifmon-1.4.tar.gz) = 10038
There are no ports dependent upon this port
No options to configure
Number of commits found: 9
net/nifmon: USES+= ncurses, link to libnurses, not libcurses
Remove indefinite articles and trailing periods from COMMENT, plus minor
COMMENT typos and surrounding whitespace fixes. A few Makefiles where not
included as they contain Latin-1 characters that break the Phabricator
workflow. Category N.
Approved by: portmgr (bapt)
Support staging
Add NO_STAGE all over the place in preparation for the staging support (cat:
- Get Rid MD5 support
Reset email@example.com due to several months of inactivity.
Hat: portmgr
Add SHA256 for my ports (that don't already have it).
Change email address in ports maintained by me to email@example.com
Approved by: arved (mentor)
Add nifmon 1.4,
nifmon is a curses application which displays counters and
statistics of one particular network interface in one part of
the screen and a stripped down tcpdump(8) output for this
interface in the other part of the screen.
PR: ports/63358
Submitted by: Simon Barner <email@example.com>
Servers and bandwidth provided byNew York Internet, SuperNews, and RootBSD
No vulnerabilities have been reported in the past 14 days
* - modified, not new
All vulnerabilities


text: Sports Business Journal reported that new Jets quarterback Geno Smith, who fell to the second round of the NFL draft, will hire a new agent after parting ways with the folks from Select Sports Group. Smith confirmed the story to SiriusXM NFL Radio, but said it is "not because of the whole draft experience." Smith threatened to leave New York after the draft's first round, then guaranteed that the Jets would make the playoffs. He sure is making a lot of noise and bringing a lot of drama to New York, so he'll fit right in with the Rex Ryan circus. … "I think Tim can learn a lot from me." Those are the words of Omaha Beef quarterback , according to The Associated Press. The Beef, a professional indoor team in Nebraska, has reportedly offered a standard $75 a game contract to Tim Tebow, who was cut by the Jets on Monday and went unclaimed on waivers Tuesday. Credit the Beef for trying to draw attention to the franchise. Who knew there was an arena team with such a cool nickname?


text:
345 Main Ave., Bay Head, New Jersey
The Grenville Hotel came to life in 1890.
The Grenville is known as one of the most luxurious summer hotels that populated the shore during the late nineteenth century.
For many families it would become an annual voyage to stay at the Grenville and enjoy its cool ocean shore.
Today, the Grenville still operates, and is creating more memories for visiting guests. While most guests enjoy a quiet uneventful stay, some come away with a story of how they encountered one of Grenville's ghosts.
The employees of Grenville have many stories about the ghosts they have encountered in Grenville's hallways and lobby. Employees have stated that they have heard the sound of footsteps in rooms that are unoccupied, and also stomping down hallways in which they are alone. Many have stated the sound of children, playing and laughing, can be heard throughout the lobby, typically at night. Like the disembodied footsteps, the sound of children has also been heard traveling down the hallways.
Guests have experienced the same phenomena as the employees, however, some have encountered strange ghostly occurences in their rooms. Some guests have claimed to see apparitions of people dressed in nineteenth century clothing walking through their rooms. Others have said that they have witnessed an impression in their beds as if someone were laying there.
4 comment(s) on this page. Add your own comment below.
stayed in room 306 last night...have never even thought about ghosts ever....about 10 pm i heard children giggling and laughing and it sounded like it came from my bathroom....i turned down the volume on tv and still heard it....went to bathroom and assummed it was coming from the room next to mine....then at midnight i see this and it totally freaked me out.....a man in a 3 piece mid century suit with a beard and a top hat standing near the bathroom door with his hand outstretched to me....like he was asking me to come with him or help him.......i blinked and it was gone...i was freaking out.....then i call downstairs around 1am and ask if there are children staying on the floor and am told that no children are staying at the hotel....i then tell him about the man i just saw and he assures me that many have seen things and they are harmless....around 2 am i hear my door knob rattling...i go out into hallway and nobody is there...i go to end of hall and hear a baby crying....and kids giggling.....i never believed in ghosts...never thought about them before and now i am googling hauntings at grenville b and b......weird day!
In1967 Labor day weekend I was a young man sitting alone on Karge bbeach Bay Head betweeen 2:00-3:oo A.M. It was a dark night. Two siiloette ghost were gliding accross the water approx.10-15 feet out close. They were having a conversation. I heard a male and female speaking---not english. As I stood up and yelled Hey they turned and headed out to Sea.
My phone # 845-635-8584 if you want to discuss etc.
Tom petrronella
A few years stopped for dinner at the Grenville because we were in the area. Having been in CA never heard of the hotel. The dining room was SO quiet with only one other couple in the room. I just had the strongest sense of a small group of young people timidly standing at the lobby/entrance to the restaurant. I did NOT hear or see anything, so I'm aware my account is less than remarkable. But that very strong feeling has stayed with me, so I just googled the restaurant to see if there were any reports on the place. I'd like to go back to visit, but now my husband won't go back. Anyone else have the experience of strongly sensing rather than seeing or hearing what I felt to be a sweet rather than sinister presence?
PS I did NOT have these feelings because of the age/look of the building. I live in a 1790's historic home, all the restaurants in the area are in old inns, historic buildings etc.and never feel that way.


text:
Unexpected Challenges
By Alicia Bessette
Summer internships turn out potential leaders.
The First Job
By Tasneem Paghdiwala '04
Young alumnae choosing their career paths carefully.
Carrying Social Justice Forward
GSSWSR: leadership, community, radical roots and tradition/innovation.
Value added or subtracted?
By Alicia Bessette
Sociological model used to study popular music.
By Sophia Olender '12 and Bryce Pope '10
Speaking out
Kathryn Roth-Douquet '86
Military families need support and appreciation.
Bryn Mawr College
Alumnae Association
101 N. Merion Ave.
Bryn Mawr, PA 19010
Phone: 610-526-5227
Fax: 610-526-5228
Toll Free: 800-BMC-ALUM


text:
Disney Trivia Question #9: What did Walt Disney propose as the main mode...?
Posted by + Show Post
Let's play Disney Trivia!
I'll make some posts with various Disney World related questions. See if you can answer the questions without searching for the info.
The 9th question is....
What did Walt Disney propose as the main mode of transportation for his original vision of EPCOT, the city?
a. Walking
b. Bicycles
c. Electric cars
d. PeopleMovers
If you're not sure, take a guess!:)
One member who replies to all of our Disney trivia posts will be chosen at random to win a $50 gift card to the Disney Store!
Click here for links to all of the Disney Trivia Posts:
Let's Play DISNEY Trivia! Behind the MAGIC! Win a $50 Gift Card PRIZE!
Click here to see the answers to the Trivia questions:
Disney Trivia ANSWERS - Behind the Magic!
Replies (31-40):
Add your quick reply below:
You must be a member to reply to this post.


text:
Gay, Felix continue gold rush
OSAKA, Japan - Tyson Gay won his third gold medal at the World Championships. For Allyson Felix, it is two - and counting.
With his ninth race in eight days, double sprint champion Gay ran a devastating final curve on the US 400-meter relay team yesterday to set up his triple. Only Carl Lewis and Maurice Greene had previously won three golds at a single World Championship.
"The 4-by-1 put the icing on the cake," Gay said. "This was the 'funnest' medal I got."
Felix can join the select group of triple titlists if she can help the US 1,600 relay team to victory today. The 200 winner won gold on successive days when the American women won their sprint relay late yesterday.
"It was my first relay tonight, it was a great feeling, and I can't wait to do it again tomorrow," she said.
The women held back their celebration until the men streaked across the line in first place soon after. Then they set up the biggest group hug of the week.
After winning the 100 and 200, Gay showed no fatigue as he set up anchor Leroy Dixon for victory over the favored Jamaicans.
"I'm extremely tired, but my teammates told me to go and do it one more time and get it done," Gay said.
As he handed the red baton over to Dixon, Gay screamed "go," urging the anchor runner on amid the din of the capacity crowd of 50,000 at Nagai Stadium.
And go Dixon did, doing enough to hold off surging world record-holder Asafa Powell, who ran a blistering leg to earn Jamaica the silver.
Gay was already pumping the air before Dixon crossed in 37.78 seconds, 0.11 ahead of Powell. Britain was third.
The three golds set up Gay as the US star heading into next year's Beijing Olympics. Lewis won three world golds in 1983 and 1987, combining the 400, 1,600 relays, and long jump. Greene's three golds were in the 100, 200, and 400 relay in '99. Lewis also won four golds at the Los Angeles Olympics in 1984.
"I didn't come into these championships thinking about three gold medals," Gay said, arguing that the rivalry with Powell in the 100 had created enough pressure as it was. "I think that's why I got it accomplished."
To make the US night even better, Brad Walker won the pole vault by clearing 19 feet, 2 3/4 inches on his first attempt. France's Romain Mesnil took silver, jumping the same height on his second try. Germany's Danny Ecker took bronze at 19- 3/4.
A night after winning the 200, Felix helped the 400 relay team build up a lead that even 100 world champion Veronica Campbell could not close on the anchor. Torri Edwards threw her arms up in celebration as she held off the Jamaican by 0.03 seconds to win in 41.98.
Only Marita Koch of the former East Germany achieved triple golds among women at the Worlds, at the inaugural meet in 1983.
Lauryn Williams left the blocks like a bullet, before handing over to Felix, who was already well into her elegant stride. With a good handoff to Mikele Barber, the US runners had built enough of a cushion for Edwards to hold off Campbell.
After the relays, the US led the medal table with 11 golds and 22 overall. Russia had four golds and 13 overall.
Kenya's Catherine Ndereba won the women's marathon this morning, quashing Japan's best chance of gold at the meet.
Ndereba, the 2003 world champion and silver medalist in Helsinki, became the first woman to win two marathon titles at the worlds.
She won in 2:30:37 - 8 seconds clear of No. 1-ranked Zhou Chunxiu of China. Reiko Tosa finished third for Japan's first medal in Osaka.


text:
UVU's Woodbury Art Museum Presents Russian Soviet-Era Pastoral Exhibit
October 9, 2012
For Immediate Release
University Marketing & Communications: Mike Rigert (801) 863-6807
Written by: Cheryl Kamenski (801) 863-6351
Utah Valley University's Woodbury Art Museum is partnering with the Center for Global & Intercultural Engagement to present "Global Spotlight Russia: Pastoralism" starting with an Oct. 16 reception at 7 p.m. The exhibition will be on display at the museum through Dec. 15.
The exhibition brings together more than 60 paintings and drawings from several private collections in Utah, many of which have never been displayed publicly. The artworks span over three decades of Soviet-era landscapes and pastoral scenes from the 1950s to the 1980s.
"We are once again glad to be part of the Global Spotlight program, offering a view into Eurasian cultures through art," said Melissa Hempel, UVU Woodbury Art Museum interim director/creator. "We hope many students and residents have a chance to experience the exhibition, which wouldn't have been possible without the generous collectors who loaned their priceless works of art."
"Global Spotlight Russia: Pastoralism" is part of UVU's greater Global Spotlight: Eurasia program. A second rotation of Soviet era works will follow the pastoralism show. "Global Spotlight Russia: Military" is scheduled to open in January 2013.
The museum's Linda and Malan Jackson Collection of Mongolian Art will also be on display in the Permanent Collection galleries. Many opportunities to learn more about the artwork and cultural components are available. Art Talks will take place at the museum on Oct. 30 and Dec. 4 at 7p.m.
These collections are free and open to the public. UVU's Woodbury Art Museum is open Tuesdays, 11 a.m. to 8 p.m., and Wed.-Sat., 11 a.m. to 5 p.m. The museum is located in the University Mall, 575 W. University Parkway in Orem, on the second floor between the Gap and the former Nordstrom store that is currently vacant.
About UVU
Utah Valley University is located in Orem, Utah, and is home to more than 33,000 students. UVU began as a vocational school during World War II, and in the seven decades since has evolved into a technical school, community college, state college and, finally, a comprehensive regional teaching university. UVU is one of Utahís largest institutions of higher learning and offers programs ranging from career training to high-demand master degrees, with emphasis on undergraduate education.


text:
Wal-Mart stock plunged Friday after Bloomberg quoted an internal memo from a company vice president that called their early February sales "a total disaster."
"In case you haven't seen a sales report these days, February MTD sales are a total disaster," Jerry Murray, Wal-Mart's vice president of finance and logistics, said in a e-mail, according to Bloomberg. "The worst start to a month I have seen in my ~7 years with the company."
Shares in the world's biggest retailer fell 2.1 percent, the stock's largest decline since December.
USA Today reported Wal-Mart responded, "As with any organization, we often see internal communications that are not entirely accurate, that lack the proper context and represent individual opinions."
Later this month, the company is due to report its fourth-quarter earnings, USA Today said.
Bloomberg reported Wal-Mart executives blamed the poor sales on the country's still high unemployment and the fact customers are paying higher payroll taxes, which is eating into the paychecks.


text:
'Just lucky to be alive' tenant says after Fargo apartment fire
Emily Staiger doesn't know why she woke up, but she's glad she did.
The 20-year-old had removed the batteries from the smoke alarm in her south Fargo apartment unit Sunday evening because it kept beeping after hamburger grease briefly flared up in a frying pan.
She awoke about 1 a.m., smelled smoke, opened her bedroom door and found the kitchen ablaze.
"I'm just lucky to be alive," she said.
No one was hurt as Staiger and about 30 others escaped the brick-façade building at 2601 14th St. S., said Fargo Fire Capt. Leroy Skarloken, who saw tenants fleeing out first- and second-floor windows when he arrived.
Adults stood on each others' shoulders to pass down children from a second-floor window. Firefighters used ladders to help other tenants escape.
"It could have been a real serious issue there," Skarloken said. "There were quite a few young children and infants in that building. It was pretty intense for a little while."
Staiger said she initially ran outside but then tried to return to the second floor when she saw a neighbor pounding on a window. When she opened the hallway door, she said she "couldn't see, like, 2 inches in front of me," so she and others helped the woman down through the window.
"That's all I cared about was making sure that everybody got out safely," she said.
Investigators found the fire started on or near the stove in Staiger's apartment next to the east stairwell, Skarloken said. Crews had the fire under control by 1:15 a.m.
The cause of the fire is under investigation and will be difficult to determine because of the severe damage, he said. The flare-up was a possible cause but hadn't been linked to the blaze, he said.
Staiger said a friend of hers was cooking chicken in the apartment Sunday night and may have forgotten to turn off the stove, but he wasn't certain.
The apartment building sustained moderate smoke and fire damage and light water damage, mostly on the east end.
Staiger said she was able to retrieve some pictures, books and her purse and wallet. She was already slated to move into a different apartment on June 1, and she doesn't have renter's insurance.
"So, it's just kind of hard because now I don't have anything for my apartment," said Staiger, who took refuge with her parents in south Fargo.
Her roommate, 20-year-old Sabrina Boe, wasn't home during the fire and had closed her door. Her room sustained water damage and minor smoke damage, its white walls in stark contrast to Staiger's charred room. On Monday, Boe stuffed her clothes into plastic garbage bags and tossed them out the window toward a pickup headed to her aunt's house.
"I was going to be staying here through the summer. So, yeah," she said.
The Minn-Kota Chapter of the American Red Cross assisted four families with food, shelter and clothing. The Salvation Army also assisted tenants and firefighters at the scene.
A damage estimate wasn't available.


text:
Michael Allain, a clinical nurse specialist at Crouse Hospital, has received the Excellence in Research Award from the Omicron Alpha Sigma Theta Tau International Honor Society of Nursing. The award recognizes the accomplishments of an active member of Omicron Alpha in the area of nursing research. The recipient must meet one or more of the following criteria: conducts distinguished research in nursing; communicates nursing research findings through publications, presentations at conferences/ workshops/seminars, consultation, or teaching; gives evidence of a history of nursing research activities. Allain currently serves as a CNS in the hospital's ICU and Emergency Department. Allain recently replicated a study related to the use of a specialized dressing to prevent pressure ulcers for high-risk patients who meet specific criteria in the intensive care unit. Through his leadership and collegiality, he enlisted staff members to perform routine 'skin rounds' in the ICU, identifying and implementing interventions that have significantly decreased nosocomial pressure ulcers in the ICU. Omicron Alpha is committed to advancing nursing leadership, scholarship and research in clinical practice and in public policy.
Latest News
American College of Radiology (ACR) Designates Crouse Hospital as a Lung Cancer Screening Center
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© 2017 Crouse Hospital / 736 Irving Avenue / Syracuse, NY 13210 / 315-470-7111


text:
About Us
Clearwater Lingerie & More Store
Contact Us
Women's Lingerie
Baby Doll Lingerie
Chemise & Gown Lingerie
Corsets & & Bustier Lingerie
Crotchless Lingerie
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Teddy Lingerie & Bodysuits
GarterBelts & Garter Sets
Panties, Thongs & G-Strings
BDSM & Fetish Lingerie
Bridal Lingerie
Costume Lingerie -Role Play
His & Hers Lingerie
Sale & Clearance Lingerie
Christmas - Holiday Lingerie
Hanukkah Lingerie
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Baby Dolls, Gowns & Chemises
Corsets & Bustiers
Crotchless Lingerie
Crotchless Panties
Garterbelts & Garter Sets
Teddy Lingerie
Panties, Thongs, G-Strings
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Men's Bikini Underwear
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His & Hers Lingerie
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Fishnet Pantyhose, Fishnet Stockings & Net Hosiery
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Stockings for Garters
Thigh High Stockings
Hosiery Sales & Clearance
Gloves & Arm Accessories
Hose & Heels Accessories
Plus Hosiery
Crotchless Pantyhose-Plus/Queen
Crotchless Pantyhose XXL Miss O Luxury Euro Imports
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Women's Lingerie
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> Fishnet Teddy & Gloves Set
Sultry Patterned Fishnet
Teddy Lingerie
Fishnet Teddy & Gloves Set
Fishnet Teddy has a floral pattern and sultry one shoulder styling. Matching arm sleeves complete the look (the manufacturer refers to them as gloves, but they end at the wrist). Elegant Moments #1147. One Size Fits Most recommended for 90-160 lbs.
Item #:
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Your Savings $5.00 (25%)
One Size Fits Most
Usually ships the next business day
Product Description & Sizing Recommendations
Fishnet Teddy has a floral pattern and sultry one shoulder styling. Matching arm sleeves complete the look (the manufacturer refers to them as gloves, but they end at the wrist). Elegant Moments #1147.
One Size Fits Most recommended for 90-160 lbs.
Teddy Lingerie
Rose Dust Teddy Lingerie
Teddy & Stockings Set Sheer Red & Black
Teddy Lingerie in Red
Romper Teddy Playful Pink
Fishnet Teddy & Gloves Set
Inner Goddess Teddy Lingerie
Teddy Lingerie Brilliant Blue & Gold
Teddy Lingerie Tantalizing Teal
Teddy Lingerie Spider Web Net
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SINGAPORE, Jan 11 (Reuters) - U.S. 10-year Treasuries slipped in Asia on Friday but their moves were limited, with traders looking to upcoming economic data for clues on direction.
* Ten-year notes fell 3/32 in price to yield 1.910 percent , rising 1 basis point from late U.S. trade on Thursday and inching back in the direction of an eight-month high of 1.975 percent set last Friday.
* The 10-year yield has climbed more than 15 basis points so far in January, having gotten a lift after U.S. policymakers reached a deal to avoid the "fiscal cliff" of tax hikes and spending cuts that could have pushed the economy back into recession.
The 10-year yield's rise gained added momentum after minutes of the Federal Reserve's December meeting, released last week, raised concern that the Fed could stop its asset purchases, known as quantitative easing, before the end of the year.
* The 10-year yield has now probably moved into a range of 1.65 percent to 2.15 percent, said a trader for a European brokerage house in Tokyo. "New data has to come in for the range to expand," he added.
Investors will be closely watching a speech that Fed Chairman Ben Bernanke is due to give on Monday at the University of Michigan for any further indications of how long the Fed's latest bond purchase program will last.
Much, however, is likely to depend on the economy, as Bernanke has said he wants to continue stimulus until the economy is on surer footing and unemployment drops significantly.
Next week will also bring a slew of economic indicators, including data on retail sales and consumer prices.