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Conceived and designed the experiments: MK MM AV LB M. Gebbia IMW GG CN FP M. Grøtli. Performed the experiments: MK MM AV LB M. Gebbia IMW. Analyzed the data: MK MM AV LB M. Gebbia IMW GG CN FP M. Grøtli. Contributed reagents/materials/analysis tools: LB GG CN FP M. Grøtli. Wrote the paper: GG CN FP M. Grøtli.
Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1T100G). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.
Protein kinases have a crucial role in most, if not all, signaling pathways and regulate diverse cellular functions, such as cell-cycle progression, apoptosis, metabolism, differentiation, cell morphology and migration, and secretion of cellular proteins [1]. Our present understanding of the majority of cellular signal transduction takes the form of wiring diagrams in which many of the component parts have been identified, and to some extent the relative position of the components in a given pathway, but beyond this static snapshot view, little is known about the details of their dynamic operation. A critical piece of this puzzle is an understanding of how external and internal inputs are sensed in a time-dependent manner to effect a given signaling output. Highly selective, cell-permeable and fast-acting inhibitors of individual kinases would allow for the systematic investigation of the in vivo cellular function of a kinase in real time. Protein kinases share common sequences and structural homology in their ATP-binding site. The fact that many kinases share a highly conserved catalytic domain complicate the search for ATP competitive kinase inhibitors with sufficient specificity [2]. However, this conserved domain can be leveraged to deliver high selectivity by orthogonal targeting [3]. This approach involves modifying a kinase inhibitor to disrupt its binding affinity for its native target and subsequent mutation of a protein to allow it to recognize the orthogonal inhibitor. Shokat and colleagues have extensively used this "analog-sensitive" approach to study a range of protein kinases [4]. Recently, this chemical genetic approach has been used to identify four novel physiological substrates of Hog1 kinase [5], to show that the catalytic activity of Hog1 prevents cross talk between the high-osmolarity glycerol (HOG) pathway and both the pheromone response and invasive growth pathways [6], as well as to define the signaling properties underlying the HOG pathway [7]. We wanted to explore orthogonal targeting in order to develop selective and fast acting kinase inhibitors that would allow us to study the dynamic behavior of kinases in the HOG pathway. Herein we report the design, synthesis and evaluation of an orthogonal inhibitor that is able to inhibit as kinases efficiently and can be used to study signal transduction events that occur within minutes, e.g. gene expression and cell cycle studies.
The HOG pathway of the yeast Saccharomyces cerevisiae is a MAPK signaling pathway and is the functional homolog of the stress activated MAPK JNK and MAPK p38 pathways of mammals [8]. Because there is a high degree of conservation of these cascades, the yeast HOG pathway is a good model to study osmotic adaptation processes. The HOG pathway consists of two upstream osmosensing branches, the Sln1 and Sho1 branches, and a downstream MAP kinase cascade including the Ssk2/22, Ste11 MAP3K, the Pbs2 MAPKK and Hog1 MAPK [9]. Activation of the Hog1 MAPK elicits an extensive program required for cell adaptation which includes profound changes in gene expression. Specifically, Hog1 regulates gene expression by activation of specific transcription factors but also through chromatin binding, Hog1 recruits chromatin modifying/remodeling activities to stress-responsive genes altering their expression [8], [10]. In addition, environmental stressors (e.g. changes in osmolarity) critically affect progression through the cell cycle [9], [11], [12].
To develop an analog-sensitive inhibitor of an engineered Hog1 kinase, we selected the pyrazolopyrimidines as they represent an excellent scaffold for targeting the protein kinase family due to their structural similarity to the adenine moiety of ATP, furthermore, the scaffold has been shown to have activity against multiple kinase subfamilies. For example, different chemical substitutions around this scaffold result in increased selectivity in the inhibition of KDR [13], Src [14], and EGF [15] kinase families. Furthermore, this scaffold has previously been used to make orthogonal inhibitors [16]. We present here the design and synthesis of a novel orthogonal inhibitor based on the pyrazolopyrimidine that effectively inhibits a Hog1as kinase, and is able to dissect the transient cell cycle arrest and regulation of gene expression mediated by Hog1 in response to stress.
Because of its central role in cellular homeostasis and the implication of human homologs in diverse disease states, we selected Hog1 as the target of our mutant kinase-inhibitor pair design. Sequence alignment analyses identified the conserved T100 as a gatekeeper residue in Hog1 [5], [6]. Visual inspection of the binding pocket of an initial homology model of Hog1, using the structure of human p38 in the absence of a ligand (pdb code 1p38) for a template, indicated that a narrow path leads to a buried cavity within the ATP binding domain (Figure 1).
The cavity size and shape is comparable to that of a phenyl group, and mutation of T100 for a glycine would widen the pocket further (Figure 1). We therefore sought a compound that was based on the pyrazolopyrimidine structure, having a phenyl ring attached to it via a spacer of the appropriate length. Candidate compounds were manually docked into the binding site and the geometries were optimized in torsion space using an all-atom representation of both ligand and receptor, keeping the receptor fixed. 1-NM-PP1, a commercially available ATP competitive as-inhibitor was compatible with our model, but did not fit as well as other compounds into the ATP binding site of Hog1as. The resulting model complex that best matched our specifications included a two-carbon, triple-bonded linker (compounds with the general structure 6, Figure 1). The triple bound would place the benzene ring in such orientation that it fills up the lipophilic pocket that becomes accessible upon mutation. At the same time, the heterocyclic moiety can make similar interactions with the hinge area as would ATP. In the wild-type kinase the non-mutated gatekeeper residue should block access to the lipophilic pocket (indicated in red).
Previous published synthetic approaches for making 1,3-disubstituted pyrazolopyrimidines involves at least five sequential reaction steps, but more importantly, the R1 substituent is introduced in the first step [17], [18]. Therefore, the generation of analogues with varying C3 substituents is inefficient. We devised a convergent route for making 1,3-disubstituted pyrazolopyrimidines. This route involves the synthesis of a common intermediate, 4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine (3) that allows rapid derivatization of the heterocyclic core scaffold in two steps (Figure 2).
The common intermediate, 4-amino-pyrazolopyrimidine (2), was synthesized from (1) by a 4-step synthesis, on a multigram scale in 64% overall yield without the use of any chromatography. The corresponding 4-amino-3-iodopyrazolopyrimidine (3) was synthesized using N-iodosuccinimide (Figure 2) [19].
Starting from compound 3, a two-step derivatization process was developed. Initially, we found that alkylation of the N1 with 2-propanol using Mitsunobu conditions preceded only in moderate yield (58%). Treatment of 3 with 1.1 equiv of 2-chloropropan and 1.2 equiv of NaH in DMF for 3 h at 100°C gave a mixture of the N1 and N4 alkylated products. However, when the base was exchanged with K2CO3 and the reaction was carried out with microwave assisted heating at 200°C for 5 min, high region-selectivity was achieved and 4a was obtained in nearly quantitative yield. Using the same reaction conditions in combination with commercial available 2-(2-Chloroethoxy)tetrahydro-2H-pyran or 4-Chloromethyl-2,2-dimethyl-1,3-dioxolane compounds 4b and 4c were obtained in 70% and 79% yield respectively.
Compound 4 was then reacted with 4-halogenphenyl boronic acid (1.3 eq) Pd(PPh3)2Cl2, (3 mol %), 2M aq. K2CO3 (10 eq) in THF at reflux for 16 h to generate compounds with the general structure 5 [17] or with 1-ethenyl-4-halobenzene, Pd(PPh3)2Cl2, (3 mol %), Et3N (2 eq) and CuI (6 mol %) in THF at reflux for 16 h to generate compounds with the general structure 6 [20]. In all cases the reactions proceeded with high turnover of the starting material and the target compounds were obtained in high yields (75–90%). Removal of the acid labile protection groups in 6d and 6e were carried out with 2N HCl in THF, resulting in compound 7a and 7b in 92% and 90% isolated yield respectively.
Using standard methods, we cloned, expressed and purified the glutathione-S-transferase (GST) fusion proteins of Hog1wt and Pbs2wt as well as the mutated kinases (Hog1as and Pbs2as). The analogue sensitive mutant allele of each kinase (as mutant) was created by the replacement of a conserved bulky residue with a glycine (T100G) in Hog1 or an alanine (M435A) in the active site of Pbs2.
Based on the analyses of their in vitro kinase activity, both Hog1as and Pbs2as were more active when the phosphodonor is the analogue Phenyl-Ethyl-ATP (PE-ATP) versus ATP, consistent with previous reports [5]. It is worth noting that, Hog1 expressed from E. coli was not inhibited by 1-NM-PP1 (9, Figure 3) [16], [21], [22], [23], while Hog1 produced from yeast was sensitive to this inhibitor (data not shown). This observation suggests the E. coli protein is inactive/only partially active and that full function of Hog1 depends on association with another yeast protein, such as a chaperone or that the conformation or postranslational modification of the Hog1 protein in E. coli is different from that produced in yeast.
We tested the 1,3-disubstituted pyrazolopyrimidines (6a–6c, 5a, 5b), as well as SB203580 (8, a known inhibitor of p38α, p38β and AKT/PKB. p38 is the mammalian homologue of Hog1) and 1-NM-PP1 (9, Figure 3). Hog1as was totally inhibited in vitro by 8 and 9, and almost totally inhibited (>89%) by 6a–6c, with the two last (5a and 5b) the least efficient. Pbs2as was inhibited by all the compounds in vitro, but not as efficient as for Hog1 as. Protein levels were similar in all assays based on inspection of coomassie stained gels. As expected (as it is its homologue p38 in mammals), Hog1wt was inhibited by 8, and poorly (range 8–25%) inhibited by the rest of compounds except for 5b (94%). Finally, none of the inhibitors had an effect on Pbs2wt.
The in vitro IC50 values for compounds 9 and 6a that target Hog1as and Pbs2as were determined (Figure 3F). Compound 9 was 4-times more efficient than 6a when targeting Hog1as, while 6a is less than two times more efficient when 9 when targeting Pbs2as. Thus, both inhibitors inhibited the kinases in vitro at a similar range of concentrations.
As the mutated proteins were functional in vitro, yeast strains expressing the modified kinases were generated. Once we had checked that hog1as or pbs2as strains were osmoresistant (data not shown), we followed the transcriptional response to osmostress using the STL1::LacZ reporter (Figure 4). Cells were incubated with the different inhibitors for 8 hours at a fixed concentration of 5 µM, before an osmotic shock with 0.4 M NaCl. The results agreed with those observed in the in vitro assay, with the exception of 8, that was less effective in vivo.
To further characterize 6a we performed a time course at a concentration of 2 µM (end-point analysis), a concentration that does not manifest a fitness defect in a HOG1 strain, but is sufficient to almost totally inhibit Hog1as (Figure 5 and data not shown). At 5 minutes of incubation with 6a the β-galactosidase activity was inhibit by 86%, with an IC50 of 238 nM (Figure 5A and 5C). This rapid inhibition of gene expression by 6a suggests that this compound could be a key, fast-acting tool to study some of the aspects of the mRNA biogenesis regulated specifically by the Hog1 MAPK as by signaling kinases in general.
To complement the end-point analyses of the inhibitors effect on transcription we also carried out a time course experiment for both hog1as and pbs2as (Figure 6). The addition of 6a five minutes before stress completely blocked the expression of STL1, which in the absence of the inhibitor, increased up to 30 minutes in response to osmostress. Similar results were obtained by inhibition of Hog1as and Pbs2as.
To analyze whether the solubility of the molecule could affect the inhibition by 6a, we tested two derivatives with one or two hydroxyl functions (7a and 7b). We reasoned that increased solubility could result in better uptake and therefore more potent compounds. On the other hand, too polar compounds could be less bioactive if they are unable to cross the lipophilic cell membrane. However, we did not observe either a faster uptake or a faster inhibition with these two new derivates (Figure 7). Thus, although in vitro these compounds were able to inhibit Hog1as as efficiently as 6a (Figure 7A), they did not improve the inhibition of the MAPK in vivo (Figure 7B).
The Stress-Activated Protein Kinase (SAPK) Hog1 elicits a program for cell adaptation that includes the control of gene expression and the modulation of cell-cycle progression. As recent studies have shown that monitoring SAPKs activity in vivo by reversable inhibition, we wanted to know if 6a, is a suitable tool to study the transient cell cycle arrest mediated by Hog1 activation in response to stress.
Both high osmolarity and inactivation of Sln1 activity will result in activation of Hog1. It is known that cells manifest a transient cell cycle arrest in response to Sln1 inactivation, a phenotype that can be followed by flow cytometry [12], [24]. A temperature sensitive allele of SLN1, (sln1ts4) arrests at G1 phase following synchronization at G1/S with mating pheromone and release into the restrictive temperature (Figure 8, lane A). This arrest can be circumvented by mutations on the HOG1 gene (in hog1 cells) or if cells are pre-incubated with 6a for as little as 10 min.
Our results demonstrate that 6a is a powerful tool to study transient cell cycle arrest or gene expression mediated by Hog1 in response to stress. In addition, 6a was recently used to demonstrate that dynamic signaling in the Hog1 pathway relies on high basal signal transduction [7]. However, the general applicability of this approach depends, in part, on the selectivity with 6a inhibit the mutant protein kinases compared with the other wild-type protein kinases that are expressed endogenously in the same cells [25]. We therefore examined the specificity of 6a by chemical genetic profiling of the yeast deletion mutant collection and scored for mutants with reduced growth in the presence of 500 µM 6a and without osmotic stress (Figure 9). It should be noted that the concentration of 6a used in this experiment was 100 times higher than what was required to get efficient inhibition of Hog1as (5 µM) in osmotic stressed cells and only off-target effects as well as secondary effects of these was expected to be identified. This analysis revealed that 60 strains that showed a significant depletion from the pool of 1200 essential heterozygotes and 4800 non-essential homozygous diploids (i.e. with a log2 ratio greater than 1) when cultured competitively in the presence of 500 µM 6a. Notably, of the 60 gene deletion strains (see table S1, Supporting Information), 50 could be classified into five functional groups; kinases (6), other enzymes 14), cytoskeleton (17), transcription regulation (10), and cell wall (3), clearly demonstrating that several off-target effects takes place at this concentration of 6a. A similar experiment has been reported using compound 9 (500 nM) targeting cdc28-as, showing excellent selectivity for the targeted kinase [26]. However, when the inhibitor concentration was increased to 5 µM several strains came up as sensitive, with several of these having a catalytic/nucleoside triphosphate binding role. Our results are consistent with these data. Together, these results demonstrate the importance of using the lowest possible dose and understanding the specific activity of each particular inhibitor.
To summarize, a potent, cell-permeable as-inhibitor of the yeast Hog1 MAP kinase has been developed and its utility has been demonstrated by studying various roles of the Hog1 kinase. The inhibitor can be regarded as a "sister-compound" of the commercial 1-NM-PP1. The inhibitor will be a very useful tool to study important model signal pathways in yeast.
1H and 13C NMR spectra were obtained from a JEOL JNM-EX 400 spectrometer. Column chromatography was performed on wet packed silica (0.040–0.063 mm) using flash chromatography. Microwave reactions were performed in a Biotage Initiator reactor with fixed hold time. Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine was prepared following a literature procedure [19].
A solution of malononitrile (22.6 g, 0.344 mol), triethyl orthoformate (83 mL, 0.499 mol), and acetic anhydride (77 mL, 0.791 mol) was heated to 100°C for 5 h. After cooling to rt the solution was concentrated on the rotary evaporator. The solution was left to crystallize at rt overnight and the yellow solid was recrystallized from EtOH to give 36.0 g (98.5%) of 1,1-dicyano-2-ethoxylethene as yellow crystals. This material (36.0 g, 0.339 mol) was added carefully and in small portions to cold (0°C) hydrazine hydrate (99.5%, 26 g, 0.807 mol). The solution was heated to reflux for 1 h and then left to cool at rt whereupon the contents of the flask solidified. Water (25 mL) was added to the solid material and the mixture was left in the refrigerator overnight. The mixture was filtered and the solid was washed with 10 mL of cold water and suction dried for about 5 minutes. The product was dried in a vacuum desiccator over calcium chloride and was obtained in 75.5% (27.7 g). This material (27.6 g, 0.255 mol) was added to formamide (42 mL). The solution was vigorously boiled for 30 minutes (216°C oil bath). The creamy suspension was left to cool at rt, diluted with water (70 mL), and filtered. The light tan colored solid was washed with water, suction dried, and dried in a vacuum desiccator over calcium chloride to give the target compound in 86% yield (29.6 g) that gives an overall yield of 64% (calculated over three steps). NMR was in agreement with published data [27].
Compound 3 (1 eq) and K2CO3 (2 eq) were suspended in 12 ml of dry DMF in a 20 ml microwave vessel. To this mixture, R1-Cl (1.1 eq) was added and the sealed tube was heated to 200°C for 5 min. (20 s of pre-stirring and fixed hold time: on). After cooling of the reaction mixture to room temperature, additional R1-Cl (0.5 eq) was added and the microwave vessel was heated again to 200°C for 5 min. After cooling to ambient temperature, the reaction mixture was diluted with DMF and filtered. The solvent was removed in vacuo at 80°C and the residue was co-distilled with toluene three times. The crude product was purified by flash column chromatography on silica gel.
Compound 3 (2.00 g, 7.66 mmol) was converted to the target compound using general procedure A. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=120) to give 97% (2.25 g) of 4a as fine yellow needles. 1H NMR (400 MHz, CDCl3) δ 1.55 ppm (d, 6H), 5.09 (m, 1H), 6.17 (bs, 2H), 8.32 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 22.27 ppm, 49.89, 85.62, 104.25, 153.17, 155.88, 157.63. Anal. Calcd for C8H10IN5 (303.00): C, 31.70; H, 3.33; N, 23.11. Found: C, 31.84; H, 3.40; N, 23.19.
Compound 3 (2.00 g, 7.66 mmol) was converted to the target compound using general procedure A. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=120) to give 70% (2.09 g) of 4b as a yellow powder. 1H NMR (400 MHz, CDCl3) δ 1.27–1.64 ppm (m, 6H), 3.30–3.39 (m, 1H), 3.48–3.58 (m, 1H), 3.77–3.86 (m, 1H), 3.99–4.09 (m, 1H), 4.42–4.59 (m, 3H), 8.22 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 18.93 ppm, 25.30, 30.21, 47.26, 61.71, 64.85, 86.59, 98.12, 103.77, 154.14, 155.79, 157.89. Anal. Calcd for C12H16IN5O2 (389.03): C, 37.03; H, 4.14; N, 17.99. Found: C, 37.04; H, 4.15; N, 18.04.
Compound 3 (2.00 g, 7.66 mmol) was converted to the target compound using general procedure A. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=120) to give 73% (2.09 g) of 4c as fine yellow needles. 1H NMR (400 MHz, CDCl3) δ 1.33 ppm (d, 6H), 1.40 (s, 3H), 3.92–3.96 (m, 1H), 4.04–4.08 (m, 1H), 4.56–4.65 (m, 2H), 6.24 (bs, 2H), 8.34 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 25.52 ppm, 27.00, 50.12, 67.34, 74.28, 86.91, 104.17, 110.17, 154.59, 156.45, 157.69. Anal. Calcd for C11H14IN5O2 (375.02): C, 35.22; H, 3.76; N, 18.67. Found: C, 35.24; H, 3.79; N, 18.65.
Compound 4 (1 eq), Ar-B(OH)2 (1.3 eq) and Pd(PPh3)2Cl2 (3 mol %) were suspended in DMF (2 ml) and 2 M aq K2CO3 added. The reaction vial was sealed and heated in a microwave reactor to 140°C for 10 min. After cooling to ambient temperature, the reaction mixture was diluted with DMF and filtered. The solvent was removed in vacuo and the residue was co-distilled with toluene three times. The crude product was purified by flash column chromatography on silica gel.
Compound 4a (2.00 g, 6.60 mmol) was converted to the target compound using general procedure B. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=135) to give 70% (1.33 g) of 5a as fine yellow needles.1H NMR (400 MHz, CDCl3) δ 1.59 ppm (d, 6H), 5.18 (m, 1H,), 6.17 (bs, 2H), 7.48–7.54 (m, 2H), 7.63–7.68 (m, 2H), 8.35 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 22.19 ppm, 46.16, 98.71, 129.70, 130.03, 135.28, 142.78, 173.77, 155.62, 158.10. Anal. Calcd for C14H14ClN5 (287.75): C, 58.44; H, 4.90; N, 24.34. Found: C, 58.46; H, 4.91; N, 24.36.
Compound 4a (2.00 g, 6.60 mmol) was converted to the target compound using general procedure A. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=135) to give 79% (1.41 g) of 5b as fine yellow needles. 1H NMR (400 MHz, CDCl3) δ 1.58 ppm (d, 6H), 5.16 (m, 1H,), 6.19 (bs, 2H), 7.18–7.24 (m, 2H), 7.66–7.71 (m, 2H), 8.32 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 22.06 ppm, 48.96, 98.58, 116.42, 129.77, 130.46, 142.89, 153.52, 155.41, 158.16, 163.24. Anal. Calcd for C14H14FN5 (271.30): C, 61.98; H, 5.20; N, 25.81. Found: C, 61.99; H, 5.22; N, 25.84.
Compound 4a (1 eq), Ar-CCH (1.3 eq), Pd(PPh3)2Cl2 (3 mol %) Et3N (2 eq) and CuI (6 mol %) in THF was refluxed for 16 h. After cooling to ambient temperature, the reaction mixture was filtered. The solvent was removed in vacuo and the residue was co-distilled with toluene three times. The crude product was purified by flash column chromatography on silica gel.
Compound 4a (0.20 g, 0.66 mmol) was converted to the target compound using general procedure C. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=130) to give 81% yield (148 mg) of 6a as fine white powder. 1H NMR (400 MHz, CDCl3) δ 1.55 ppm (d, 6H), 5.09 (m, 1H), 6.17 (bs, 2H), 8.32 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 22.27 ppm, 49.89, 85.62, 104.25, 153.17, 155.88, 157.63. Anal. Calcd for C16H15N5 (277.13): C, 69.29; H, 5.45; N, 25.25. Found: C, 69.35; H, 5.46; N, 25.21.
Compound 4a (0.20 g, 0.66mmol) was converted to the target compound using general procedure C. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=130) to give 79% yield (154 mg) of 6b as fine white powder. 1H NMR (400 MHz, CDCl3) δ 1.58 ppm (d, 6H), 5.16 (m, 1H), 6.22 (bs, 2H), 7.07–7.11 (m, 2H), 7.56–7.60 (m, 2H), 8.36 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 22.20 ppm, 49.67, 80.97, 92.91, 102.07, 116.23, 117.92, 125.92, 134.04, 152.71, 156.37, 158.02, 163.32. Anal. Calcd for C16H14FN5 (295.30): C, 65.07; H, 4.78; N, 23.71. Found: C, 65.10; H, 4.73; N, 23.77.
Compound 4a (0.20 g, 0.66mmol) was converted to the target compound using general procedure C. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=130) to give 87% yield (179 mg) of 6c as a white powder. 1H NMR (400 MHz, CDCl3) δ 1.46 ppm (d, 6H), 5.04 (m, 1H), 7.54 (d, 2H), 7.78 (d, 2H), 8.25 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 21.71 ppm, 48.76, 82.22, 91.69, 120.34, 124.77, 128.77, 133.61, 134.11, 152.28, 156.21, 157.71. Anal. Calcd for C16H14ClN5 (311.09): C, 61.64; H, 4.53; N, 22.46. Found: C, 22.46; H, 4.53; N, 22.42.
Compound 4b (0.20 g, 0.66 mmol) was converted to the target compound using general procedure C. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=130) to give 81% yield (194 mg) of 6d as a slightly yellow powder. 1H NMR (400 MHz, CDCl3) δ 1.38–1.75 ppm (d, 6H), 3.41–3.52 (m, 1H), 3.62–3.70 (m, 1H) 3.91–3.99 (m, 1H), 4.14–4.22 (m, 1H), 4.56–4.71 (m, 3H), 5.93 (bs, 2H), 7.38–7.47 (m, 3H), 7.58–7.63 (m, 2H), 8.40 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 19.10 ppm, 25.48, 30.42. 47.47, 61.94, 65.00, 80.93, 94.26, 98.40, 101.85, 121.67, 126, 74, 128.78, 129.64, 131.94, 154.04, 156.60, 158.10. Anal. Calcd for C20H21N5O2 (363.17): C, 66.10; H, 5.82; N, 19.27. Found: C, 66.12; H, 5.83; N, 19.30.
Compound 4c (0.20 g, 0.66 mmol) was converted to the target compound using general procedure C. The crude product was purified by flash column chromatography on silica gel (MeOHCHCl3=120) to give 83% yield (191 mg) of 6e as a white powder. 1H NMR (400 MHz, CDCl3) δ 1.34 ppm (d, 3H), 1.40 (s, 3H), 3.94–4.00 (m, 1H), 4.06–4.11 (m, 1H) 4.43–4.52 (m, 1H), 4.58–4.69 (m, 2H), 6.20 (bs, 2H), 7.37–7.46 (m, 3H), 7.57–7.62 (m, 2H), 8.39 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 25.52 ppm, 27.01, 50,09, 67.42, 74.25, 80.80, 94.56, 101.90, 110.18, 121.60, 127.14, 128.85, 129.78, 132.01, 154.12, 156.93, 157.99. Anal. Calcd for C19H19N5O2 (349.15): C, 65.32; H, 5.48; N, 20.04. Found: C, 65.35; H, 5.50; N, 20.09.
The starting material (1 eq) was dissolved in 9 ml THF and 6 ml of 2N HCl was added. The mixture was stirred at 60°C for 2 h. The solvent was evaporated and the residue co-evaporated with toluene three times. The product was purified by crystallization from MeOH/Et2O.
Compound 6d (0.15 g, 0.48 mmol) was converted to the target compound using general procedure C and was obtained in 92% yield (120 mg) of 7a as a white powder. 1H NMR (400 MHz, DMSO) δ 3.84 ppm (t, 2H), 4.22 (t, 2H), 7.45–7.54 (m, 3H), 7.73–7.79 (m, 2H), 8.49 (s, 1H). 13C NMR (100 MHz, DMSO) δ 50.18 ppm, 59.01, 79.72, 94.50, 99.93, 120.98, 127.37, 128.68, 129.79, 132.06, 150.22, 151.82, 153.51. Anal. Calcd for C15H14ClN5O (315.09): C, 57.05; H, 4.47; N, 22.18. Found: C, 57.10; H, 4.51; N, 22.24.
Compound 6d (0.15 g, 0.43 mmol) was converted to the target compound using general procedure C and was obtained in 90% yield (133 mg) of 7b as a white powder. 1H NMR (400 MHz, DMSO) δ 3.37–3.50 ppm (m, 2H), 3.96–4.05 (m, 1H), 4.32–4.43 (m, 2H), 7.45–7.54 (m, 3H), 7.74–7.80 (m, 2H), 8.39 (s, 1H). 13C NMR (100 MHz, DMSO) δ 51.17 ppm, 63.61, 69.95, 79.57, 94.67, 99.82, 120.94, 127.55, 128.67, 129.82, 132.09, 149.35, 151.66, 152.99. Anal. Calcd for C16H16ClN5O2 (345.10): C, 55.58; H, 4.66; N, 20.25. Found: C, 55.65; H, 4.70; N, 20.28.
Strains used: W303 (MATa ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11 can1-100 GAL SUC2) and its derivatives, W303 hog1-as strain (MATa ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11 can1-100 GAL SUC2 hog1-as) and W303 pbs2-as strain (MATa ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11 can1-100 GAL SUC2 pbs2::URA pbs2-as:TRP). The pRS426TEG1 (pTEF1-GST, URA3+, 2 µm) vector was a gift from M. Takekawa. The pRS426TEG1-hog1as was created by cloning the hog1 mutant ORF plus 500 bp upstream and downstream, into pRS426TEG1 empty plasmid. To build the as mutant the hog1 orf was mutated by PCR in the amino acid 100 (T-ACG-→G-GGG- ). The pRS304-Pbs2as vector was created by cloning the pbs2 mutant ORF plus 500 bp upstream and downstream, into pRS304 empty plasmid. To build the as mutant the pbs2 orf was mutated by PCR in the amino acid 435 (M-ATG-→A-GCG-). ssk2ΔN, pbs2EE, HOG1, PBS2, pbs2-as and SIC1 ORFs were cloned into pGEX-4T1 (Pharmacia) to obtain GST fusion proteins. The STL1::LacZ reporter construct PEN05 was generated by cloning the STL1 promoter (base pairs −824 to +4) by PCR into YIp358R (CEN URA3).
GST fusion proteins were expressed in E.coli DH5α, and purified using glutathione-Sepharose beads (Pharmacia) in buffer B as described [28]. The beads were washed extensively with buffer B and finally proteins were eluted with kinase buffer plus glutathione (50 mM Tris-HCl pH 8.0, 2 mM DTT, 10 mM glutathione). GST-Hog1as was expressed in S. cerevisiae and purified using glutathione-Sepharose beads (Pharmacia) in buffer B, as described above.
One microgram of recombinant GST-Hog1 or GST-Pbs2 was activated by phosphorylation using 0.5 µg of either GST-Pbs2EE or GST-Ssk2ΔN in the presence of kinase buffer and ATP as described [28]. After 20 min at 30°C, substrate was added (GST-Sic1 for Hog1 or GST-Hog1 for Pbs2) to the previous mixture together with [γ-32P]-ATP (for wt kinases) or [γ-32P]-phenethyl-ATP (for as kinases) (5 µCi). The mixture was then incubated for 10 min at 30°C, and the reactions were terminated by the addition of 5× SDS loading buffer. Labeled proteins were resolved by SDS-PAGE and detected by autoradiography. Inhibitors were added to the first mixture at the indicated concentration. The assessment of the IC50 was performed by the quantification of phosphorylated substrates (Sic1 or Hog1) by using Quantity One software (BioRad).
Exponentially growing cells (OD660=0.8) were incubated with the specified inhibitor previous to be subjected to osmotic stress (0.4 M NaCl for 30 min). Cells were permeabilized by ethanol-toluene treatment, and β-galactosidase was measured as described [29]. Results are presented as mean values obtained from two independent transformants measured at least in triplicate.
For flow cytometry analyses, cells were fixed in ethanol, treated with RNAse A, stained with propidium iodide and analysed in a FACScan flow cytometer (Becton Dickinson) in the FL3 channel. A total of 10000 cells were analysed for each time point.
Yeast profiling was performed exactly as described by Ericson et al [30]. Gene set enrichment was carried out on the non-essential yeast genes in the chemogenomic profile using GSEA [31] and the results were visualized as a network using the enrichment map plugin [32] for cytoscape [33].
The initial model of Hog1 was created using the standard procedure built into the program ICM, based on the structure of human p38 (pdb code 1p38) [34]. The model was refined by regularization, which imposes ideal geometry onto the model, followed by geometry optimization by Monte Carlo simulated annealing in torsion space. (ICM manual, Molsoft, CA) Ligands were manually docked into the site and geometry optimized by minimization and Monte Carlo conformation sampling. In the early phase of optimization, ligand atoms were tethered to their original positions using harmonic potentials that were gradually decreased in strength.
Eight additional wild-type and mutant Hog1 models were created based on four structure templates (pdb codes 1cm8, 1m7q, 1p38 and 3erk) to provide a better understanding of the orthogonal ligands' affinities. Template selection was carried out using the MolIDE program [35], which is an interface to programs that performs psi-blast searches against the nr database and stores the generated alignment profiles, searches the pdb sequences using the profiles, constructs a backbone model for aligned residues, adds and geometry optimizes side-chains and builds loops. Pairwise sequence alignments were manually edited before model building.
Kinase models built by MolIDE were regularized by ICM before docking and quality assessment. The local quality of protein models was checked using ICM's calcEnergyStrain, which identifies bad regions by reporting the relative energy of each residue.
Docking of a small library of the designed and known kinase inhibitors was performed using the standard protocol in ICM (data not shown), and the representative mutant and wild-type Hog1 models were manually chosen based on the accuracy of ligand docking poses.
Gene deletion strains significantly sensitive to 500 uM 6a.
We gratefully thank Dr. Kavita Shah for providing the yeast strains and helpful discussions and M.A. Adrover for help with cytometry.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was financed by the European Commission (QUASI, contract LSHG-CT2003-503230 and CellComput, contract 043310). CN is supported by a grant from the Canadian Institutes for Health Research (#84305) and from the Canadian Cancer Society (grant # 020380) to GG. FP's laboratory is also supported by the FP7 UNICELLSYS grant (#201142) and the Fundación Marcelino Botín (FMB). FP is the recipient of an ICREA Acadèmia (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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.


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Running Time: 148 minutes
Released: 12/2008
Studio Name: Sensational Video
Series: BBW Dreams
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Location: The hotel occupies a marvellous location, situated directly on the beach of Playa Cala Major. Numerous shopping and entertainment venues can be found at locations just a few minutes from the hotel in the town centre.
* Buffet breakfast
* Central internet access
* Laundry service
* Money exchange
* Outdoor pool
* Reception
* Terrace or solarium


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Julia Frauche (and friend) get up to all kinds of mischief in an airport terminal for the March issue of Numéro Magazine. But who can blame them? Like, really? Airport terminals are so boring so much of the time so any chance to break out and behave a little silly shouldn't be frowned upon wasps!!!
Photography by Greg Kadel


text: The Escambia County Sheriff's Office offers a training opportunity to help places of worship consider the security needs of their facilities and operations. The training is designed for anyone responsible for the safety of their place of worship and will help attendees to understand the challenges and needs of worship security. The goal is to make the process of creating a secure environment easier, less expensive and more effective. Keep checking back for future classes.


text:
Looking at apartments for Nashville TN residents was a really exciting deal for me. That is because I had never lived on my own before, and I was really looking forward to it. I am nearly 30 years old, but I have always lived with someone else. First it was my parents, and then my sorority sisters when I went off to college. After graduation, my best friend and I got an apartment together. Her boyfriend was always there though, and I started to feel like the odd one out. When he proposed to her last week, I suggested they keep the apartment and I would look for a new one.
I love her with all my heart, but there is no way I am ever going to have another roomie. Continue reading "I Am Getting a New Apartment Soon"


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|Message view
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|Pier Fumagalli <p...@betaversion.org>
|Re: [OT] Eclipse on MacOS/X
|Mon, 10 Mar 2003 14:04:20 GMT
"Stefano Mazzocchi" <email@example.com> wrote: > Pier Fumagalli wrote: >> For those of you interested, a nice packaged up version of Eclipse RC1 for >> OS/X is available at http://nagoya.apache.org/~pier/macosx/ > > RC2 just came out :) Great, updated to it, rebundled, and with a few extra goodies in the distribution... If you're using OS/X, read the OS/X notes! :-) Is there anyone who knows someone in the Eclipse team who would possibly want to get that stuff incorporated in there? Pier
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text:
Associated Press on 40th anniversary of Beatles' ÂAbbey Road' photo shoot
Monday, August 10, 2009
LONDON -- Hundred of Beatles fans swarmed Abbey Road on Saturday, singing songs and snarling traffic to mark 40 years since John, Paul, George and Ringo strode across the leafy north London street and into the history books.
The famous photo of the Beatles graced the cover of "Abbey Road," the last album they recorded together, and shows the band mates walking across the zebra-striped asphalt.
It remains one of music's best-known album covers, endlessly imitated and parodied. Although the shoot itself took only a few minutes, so carefully studied was the cover for signs and symbolism that some fans came to the conclusion that Paul McCartney -- who appears barefoot and out of step with the rest -- had secretly died.
McCartney himself made fun of the bizarre conspiracy theory in the title of his 1993 concert album, "Paul Is Live."
The ease with which fans can imitate the scene has drawn throngs of tourists to the site every day, turning the street into "a shrine to the Beatles," said Richard Porter, who owns the nearby Beatles Coffee Shop and organized Saturday's event.
Crowds spilled into the street, cameramen jostled for angles and exasperated drivers honked their horns.
"I didn't expect so many people to be here," said German visitor Tschale Haas, 50, who was dressed in a Sgt. Pepper jacket.
Abbey Road, which cuts through London's well-to-do neighborhood of St. John's Wood, is home to the studios where the group recorded much of its work.
The group decided to shoot the photograph in August 1969. Photographer Iain Macmillan stood on a stepladder and police held up traffic while the Beatles walked back and forth across the street.
The enduring popularity of the site has caused headaches for local authorities, who have had to move the Abbey Road street sign up out of reach to prevent theft and repaint the wall every three months to hide fans' graffiti.


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Pearls of Greece
7 Days
SITA provides unparalleled quality service, as a preferred Tour Operator for exotic destinations. With internationally certified specialists ready to serve clients daily, customer care is elegantly handled from the peaks of Patagonia to the palaces of India.
Inclusions & Highlights:
Andreasen Travel & Tours24103 West Lockport Street Plainfield, Illinois 60544 815-436-7300


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sitting pretty
In loving memory of our Pupun who we love so much. Pupun will be greatly missed and be in our hearts forever.We were blessed to have Pupun in our lives for a long 16 and a half years.
This is your last picture before you left our world to go to Rainbow Bridge.I feel like a part of me is gone with you.She was my first and only cat I ever had. I remember when you would sleep in bed with me, every morning you would wake me up by nuzzleing my face, as if saying to me, 'its time to get up now", I guess you didnt want me to be late for work. I miss you so much pretty. My sweet babygirl, my little baby goose, those were a few nick names i had for her.She lived a long,beautiful life. She gave birth to three litters. So, she has a bunch of little Pupuns running around somewhere.I would tell her all the time how much i loved her and give her kisses on the nose, then she would shake her head.You were such a good cat, you never hissed at anyone, even when we got Max, you never hissed at him, all thoug you did do an occasional swat, but other wise you were very sweet. He just wanted to play with you. I know he made you mad, and when he would get too close to you, your eyes would get really small, like you were squinting at him, silly thing you are, my silly goose. You are the only cat I ever had. I got you when I was 17. And now you are gone. I never wanted to think of this day, all though I knew sooner or later, I knew It would come. You will be missed so much, my little baby girl, your are being missed. Mommy loves you, forever.The hardest thing I ever had to do was putting you to sleep.I cried so much, hoping that it was the right thing to do.I didnt want you to leave me yet, I wasnt ready for that. I wish I could of kept you with me forever, but I know thats impossible.It all hapeened so unexpectedly and fast. I know now your at peace and rested and no longer in pain, if you were.The vet thought it was the best thing to do. I didnt want you to suffer. We werent sure if you were, but we knew soon enough that you would be. You will forever be in my heart, and never be forgotten.I wonder if your missing me as much as i miss you. You are my pretty girl. I wish I could give you just one more kiss, and give you a great big hug. So here is my kiss and hug to you sweet baby girl.I wish I could scratch you under the chin, you loved that so much. I hope you enjoyed your life here with me, because I enjoyed mine with you.
12/20/08 It's been just over 3 weeks you've been gone. I still miss you like the day you left. Christmas will be here soon, and i miss seeing you lay under the tree. You always looked so pretty under it, with all the lights shining. Your such a pretty girl Pupun.I hope your running and playing and enjoying life at Rainbow Bridge. You deserve it. Max misses you too. H e still looks for you in the stairs to go downstairs. Your hangout was on the steps. Merry Christmas my pretty pets!! Mommy misses you and loves you, always will.
Pupun with her Max, that liked to chase her all the time
We got Max about 3 and half years before Pupun passed away. He always liked to chase her away, but she wouldnt always run, sometimes Max would get a nice swat across the nose. That would teach him to leave her alone, only for a while, till next time. They would have some nice times together, when they would share the same water dish. And they would touch noses. That was sweet. I know Max misses you ,too. He spends some time laying down ,looking down and waiting to see you at the stairs where you used to lay all the time. That was your spot. I still now , when I walk pass the stairs, expect to see you, and say hi to my pretty baby..
I know Im beautiful!!!
Here you at Memere and Peperes house, when we were still living there. Im sure they both miss you too.They loved you and cared for you too.
Getting ready to go for it!!
Here you are being a little silly, laying flat on the floor.This is what you would do right before you would go after something. I f you had your eye on something, you would lay flat, wiggle your butt, then go after it. Silly goose you are!!!
What a good mommy I am
Here she is being a good mommy and feeding her little babies. Pupun must of been about 2 or 3 years old here. She looks so young. She had 3 litters,but I dont remember which one this one is.
Taking care of my babies!!
Here she is taking care of her babies. What a good girl. She was so gentle with them. She was a good momma.
This is a good spot!!
She always liked to lay on the window sill. she was an indoor cat, so that was her place to watch all the birds, squirrels, and other cats in the neighborhood. we had alot of stray cats in the neighborhood. i kept her indoors when we moved next to a busy highway, i was afraid she would get hit by a car. and of all the stray cats,too.
Please sign the guestbook for Pupun by clicking here
This page has been visited 2484 times


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Here's Eagles coach Chip Kelly's statement with regard to the sanctions handed down by the NCAA Wednesday against him and his former team, the University of Oregon.
"Now that the NCAA has concluded their investigation and penalized the University of Oregon and its football program, I was to apologize to the University of Oregon, all of its current and former players, and fans. I accept my share of responsibility for the actions that led to the penalties. As I have stated before, the NCAA investigation and subsequent ruling and no impact on my decided to leave Oregon for Philadelphia. I have also maintained throughout that I had every intention to cooperate with the NCAA's investigation, which I did.
"I do expect the University of Oregon and its football program to continue to thrive at a high level. They are a talented and resilient group of coaches and players and I'm sure they will attempt to put today's news behind them very quickly and move forward as they prepare for the 2013 season."


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Crowne Plaza Berlin City Centre, Schoneberg
Crowne Plaza Berlin City Centre Reviews
Add a review
16 December 2014
great hotel
E HAROLD, wicklow ireland
This hotel is a great hotel. Friendly & helpful staff. The rooms are a good size.Breakfast is good wide range of foods. The hotel is great if you want to go shopping. We have used this hotel for many years and never had a problem
The best thing about my stay was ...
the staff are very helpful
19 October 2010
r j sedgwick, chesterfield
the hotel was very good the only downside was the lobby ,the most uncomfortable i have ever been in
16 December 2008
Mid week break
Staff OK, but some not as helpful as expected. Rooms clean, compact but very warm. Hotel easy to get to by train and handy for shops, xmas markets, but local restaurants are poor. Friendly bar staff
12 January 2007
Review of 'CROWNE PLAZA BERLIN CITY CENTRE'
robert le breton, Kirkham, England
A well positioned hotel with a spacious foyer. Very helpful reception staff. The rooms were quiet and extremely comfortable, well appointed and a pleasure to be in.
The restaurant had a wide selection of breakfast fayre.


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Senior Accountant
- Recruiter
- Trace Recruit
- Location
- London (Central), London (Greater)
- Salary
- £46,000 - £54,000
- Posted
- 17 Mar 2017
- Closes
- 17 Apr 2017
- Ref
- Function
- Finance & Legal
- Sector
- Human Rights, International, Poverty Relief
- Hours
- Full Time
- Contract Type
- Contract
Trace are currently recruiting for a Senior Accountant to join an international charity based in central London. Leading a large finance function the successful candidate will have strong experience within international finance and become immediately influential in affecting decision making across key areas within the organisation.
Duties surrounding the role include the following
- Leading a large finance function in the delivery of financial support to overseas projects
- Business Partner to the International Department working closely with Senior Directors across the directorate.
- Acting as a key finance contact across the international division for staff within the UK and overseas
- Developing and coaching existing members of the existing finance function ensuring high standards are continually maintained
- Overseeing training for the international programmes ensuring programmes are developed and delivered successfully
- Proving insightful analysis to strengthen future funding opportunities.
- Working closely with other Senior Management Teams ensuring internal and external policy's are met.
The successful candidate will be/have
- A Qualified Accountant (CIPFA/ACA/ACCA/CIMA)
- Experience of leading an international finance function
- A strong background in high level business partnering
- The ability to develop and motivate staff
- Support and partake positively to a collaborative working environment.
- Immediate credibility to liaise with Heads of divisions
For further information on this new role please contact Jen McMurray on the attached details


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Rainbow Pride Mitts by Sarah Dennis
Welcome! You are browsing as a guestWould you like to see 6 projects made from this pattern and much more? create a free account What am I missing?
© Sarah Dennis
© Sarah Dennis
Rainbow Pride Mitts
This pattern is available as a free Ravelry download
These mitts feature a pride flag across the fingers and a small heart set into the thumb gusset – a great beginner intarsia project! The mitts are knit flat to make the intarsia easier and line the stripes up cleanly, but could easily be converted to knit in the round. Sized to fit an average-size woman's hand, they can easily be customised by adding a few extra rows or stitches.


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I love Coca Cola. And I love milk. But if I'm sitting at an outdoor restaurant enjoying an ice-cold Coca-Cola and the breeze on my face, and I accidentally reach down and pick up my son's glass of milk and send it down the hatch - in that moment, I hate milk. I am so surprised at getting something so different than I expected that I briefly hate something that I love.
We all love mysteries and we all love resolution. Each is great in the right moment, but if you fully expect one and get slapped in the face with the other - you may find yourself momentarily hating something you love.
I think that is why many people disliked Across the Sea so much. I think we all expected answers. We sat down thinking, "Ok. Finally. The answer to Jacob, MiB, and the Island."
But Across the Sea was not about answers. It was about back story and more mystery. And drinking down that episode while expecting answers made it something difficult to swallow.
We may have expected Coca-Cola, but don't forget, we love milk! We love an ice-cold glass of milk after a plate full of gooey, homemade chocolate-chip cookies.
Across the Sea was the milk of mystery. Once I stared at my glass and realized I had a different drink than I expected, I began to enjoy the episode so much more. It left me with mysteries in my head. Mysteries like...
- Was Mother really the protector of The Source and the island? Or was she lying?
- Did Mother induce Claudia's labor with whatever she gave her to eat?
- Was the entire episode the story of Mother in her elaborate plot to find a substitute to accomplish her own mysterious plan? Did she manipulate both of those boys so that Brother would kill her (she did say "Thank you,") and Jacob would throw him into The Source? She had to know that is exactly what would happen.
- Is the Black Smoke really Jacob's brother or the devil himself, released when some long-needed requirement was met by Mother's elaborate scheme?
- Was the point of revealing Adam & Eve really the most important part of showing the flashback of when Jack first discovered the bodies? Or was the real point to make us subtly remember the last time Jack saw Adam & Eve? In the episodeThe Lighthouse, Jack and Hurley stumble across the caves. Once they enter, Hurley's focus is on the two bodies. He stares at them, wondering who they are. He suggests that maybe the bodies are one of them - after they traveled back into "dinosaur times." But Jack does not care about Adam & Eve. He is staring at the empty coffin of Christian Shephard. When Hurley asks Jack why he smashed his father's coffin to pieces he said, "Because he wasn't in it." Jack wants to know where the body of Christian Shephard is. And so do I.
I'm not sure which drink we'll get tonight with What they Died For, but given the title, I'm hoping that when I pop the top on this episode I hear a little fizz.
42 minutes and counting... --Mystimus 00:19, May 19, 2010 (UTC)


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Climate change adaptation: Where does global health fit in the agenda?
- First Online:
- Received:
- Accepted:
Human-induced climate change will affect the lives of most populations in the next decade and beyond. It will have greatest, and generally earliest, impact on the poorest and most disadvantaged populations on the planet. Changes in climatic conditions and increases in weather variability affect human wellbeing, safety, health and survival in many ways. Some impacts are direct-acting and immediate, such as impaired food yields and storm surges. Other health effects are less immediate and typically occur via more complex causal pathways that involve a range of underlying social conditions and sectors such as water and sanitation, agriculture and urban planning. Climate change adaptation is receiving much attention given the inevitability of climate change and its effects, particularly in developing contexts, where the effects of climate change will be experienced most strongly and the response mechanisms are weakest. Financial support towards adaptation activities from various actors including the World Bank, the European Union and the United Nations is increasing substantially. With this new global impetus and funding for adaptation action come challenges such as the importance of developing adaptation activities on a sound understanding of baseline community needs and vulnerabilities, and how these may alter with changes in climate. The global health community is paying heed to the strengthening focus on adaptation, albeit in a slow and unstructured manner. The aim of this paper is to provide an overview of adaptation and its relevance to global health, and highlight the opportunities to improve health and reduce health inequities via the new and additional funding that is available for climate change adaptation activities.
KeywordsGlobal health Climate change Adaptation Equity Sustainable development Adaptation funding Social determinants
Adapting to climate change is now seen as a core component of our climate change response arsenal. This is because, unfortunately, climate change mitigation strategies alone will not prevent adverse events resulting from existing climate change; we are now too far down the climate change road to avoid the repercussions of more severe weather events, changes to agricultural yields, conflict and displacement, and the health effects that arise from all of these impacts. Although adaptation has received much attention, both practically and in climate change research in the last decade or so, climate change adaptation for health has only recently begun to emerge in the public health literature and policy discourse. The objectives of this paper are to: i) outline the relevance of climate change adaptation to global health, ii) highlight the importance of linking the social determinants of health and sustainable development agendas with climate change adaptation measures, iii) provide an overview of the global health and climate change adaptation activities thus far, and present some examples of activities relevant to health, and iv) describe the (constantly evolving) main adaptation financial mechanisms for developing countries and implications of these for global health. The paper concludes by arguing that the global health community can use the opportunities provided by the increasing flow of funding to climate adaptation to address existing and future health burdens.
Adaptation – an overview and its relevance to global health
Adapting to environmental variability has been a focus of anthropological study since the early 1900s [1]. The term adaptation was first applied to the study of the consequences of human-induced climatic change in the 1990s. Adaptation is generally understood to mean an adjustment in social-ecological systems in response to environmental changes and their impacts. A central goal of adaptation is the development of adaptive capacity, without which, adaptation is less likely to occur. Adaptation strategies must be flexible and able to incorporate new hazard information, as well as information on socio-economic and environmental systems [2]. Theoretically, adaptation activities should be developed based on a sound assessment of a particular population's vulnerability (a 'vulnerability assessment' or VA) – i.e. who is most vulnerable, what are they most vulnerable to, and to what extent do they have the capacity to cope or adapt - but the actual conduct of a rigorous VA to inform adaptation planning and programs is not commonplace and often inadequate. Vulnerability assessments and resultant adaptation plans have been carried out at various scales and sectors by research institutions, non-governmental organisations, United Nations agencies and national and sub-national governments. For least-developed countries, the process of completing a National Adaptation Programme of Action (NAPA), which combines a vulnerability assessment and the development of adaptation activities, is a precursor to receiving adaptation financing through the United Nations Framework Convention on Climate Change (UNFCCC).
Examples of health-related adaptation activities and relevant sector involvement
Responsible sector
Partner sectors
Adaptation activity
Improved and increased surveillance for infectious diseases (particularly diarrhoeal disease, malaria and dengue)
Water Supply/Sanitation/Infrastructure
Developing community-based models of management of children with acute malnutrition
Agriculture/Community services
Develop sustainable methodologies for improving rice production practices
Health/Agriculture/Rural Development
Improve and strengthen the capacity of disaster management authorities to effectively coordinate and plan emergency response to droughts, floods and storms
Disaster Management
Provide water filters for household use
Rural Development
Water Supply/Sanitation/Infrastructure/Health/Finance
Climate change, social determinants of health and sustainable development
Changes in climatic conditions and increases in weather variability affect human well being, safety, health and survival in many ways [6]. Although some vector-borne diseases will expand their range and seasonality, and death tolls will increase because of heatwaves, the indirect effects of climate change on basic human needs such as food, water and shelter will be likely to have the biggest effect on global health [7]. A substantially increased fraction of the world's population is likely to face severe food shortages and water insecurity by the end of the century due to climate change. Climate change is likely to impair crops, herds and fishstocks through rising temperatures, water stress, extreme weather events, spreading plant and animal diseases, sea level rise and ocean acidification. The health of millions of people will be compromised through an increase in the frequency of intense hurricanes, cyclones, and storm surges causing flooding and direct injury, increasing the health risk among those living in urban slums and where shelter and human settlements are poor [8]. With this will come unemployment, homelessness, dislocation, migration and conflict. All of these may substantially increase levels of stress, anxiety and depression, impairing mental as well as physical health.
Much of the emerging global variation in health impacts of climate change is arguably due to existing economic, social and health inequities [9]. Key elements of our modern global world – the asymmetric distribution of power, income, goods, and services shaped by political, economic and social forces and norms and value in society, and the consequent unfairness in the immediate conditions of daily living (access to health care, access to nutritious foods, conditions of work and shelter, and the nature of communities and cities) have widened health inequities [10]. It is likely that inequities in these 'social determinants' of health also create inequities in climate change adaptive capacity. This is seen in the differing national and sub-national climate change vulnerabilities such as existing levels of heat and food stress, and exposure to disease vectors, and differing capacities to adapt to changing climatic conditions.
Take urbanisation for example. Urban living conditions already affect the health of more than half of the world's population. And urban living can amplify the health effects of climate change [11, 12]. Sea-level rise has profound implications for the 13% of the world's urban population living in the low elevation coastal zone [13]. Low-lying cities and towns near coasts face increased risks from more frequent and more intense hurricanes, cyclones, and storm surges causing flooding, direct injury and damage to infrastructure, including roads, housing, water and sanitation systems. Poorer urban households are usually at higher direct health risk due to weaker structures, less safe city locations and building sites, and the weaker resilience of infrastructure in poorer cities to withstand damage There may also be other health consequences [7]. Poorer urban households also often lack the economic resources to evacuate in the face of climate-related disasters, or to rebuild damaged structures. And they typically lack the political influence to secure protective policies [14].
Many cities in low and middle income countries are built in marginal lowlands, which are particularly hot, humid, and often plagued by disease, thereby exposing people who live there to a variety of heat-related health risks [15]. Poor neighbourhoods with weak infrastructure, buildings and unplanned settlement developments with little green spaces, and use of black corrugated steel panels for roofs and walls are likely to be more exposed to high temperatures compared to more affluent neighbourhoods, and will have less capacity to adapt to the impact [16]. Lower socio-economic groups are more likely to be those urban workers exposed to working conditions with excess heat and therefore at increased risk compared to higher social status groups [17].
Poor living conditions, particularly those among the one billion people living in urban slums are the breeding ground for climate-sensitive infectious diseases such as diarrhoea, malaria and dengue [18]. When basic infrastructure is inadequate, existing conditions of poor sanitation and drainage and impure drinking water are further stressed under conditions of extreme weather events and flooding, leading to the transmission of infectious diseases, which puts poor urban households at higher than usual risk. Around half of the urban population of Africa and Asia lacks provision for water and sanitation to a standard that is healthy and convenient. In Latin America and the Caribbean, more than a quarter lack such provision.
There is a significant risk that declining agricultural productivity growth, growing competition for land and water, and increasing food demands are creating a new era of increasing food prices.[19][20] Low-income urban populations are among the most vulnerable to food price increases, as they generally depend on the market for their food supplies and have limited budgets [21, 22]. As prices rise, urban poor groups will be increasingly unable to afford a diet of the quality necessary to maintain their health [23]. There may also be other health consequences if the price increases drive them deeper into poverty, as some recent research suggests, [24] while riots and social instability can result from food shortages among low income urban populations.
Strategies that address the social determinants of health and health inequities promote adaptive capacity. They also correlate with sustainable development [25][26] and include improved infrastructure, education, institutional capacity and fairer access to resources, reduction of poverty and lessened intergenerational inequities. If the ultimate ambition of climate change adaptation is to (preferably) improve human well-being, which partly involves environmental protection, then it means that a broader approach that encompasses sustainability principles needs to be considered [27]. Pathways to adaptation are indeed part of sustainable development pathways [27], and a better understanding of the determinants of adaptive capacity will assist the building of these pathways. The linking or 'mainstreaming' of adaptation (and mitigation) activities into sustainable development policies is one way to more effectively respond to climate change [28, 29, 30]. In fact, it has been argued that sustainable development will necessarily take centre stage if we are to realign the demands of humans on our planet [31]. The current influx of funding for adaptation activities can be used strategically to unite these often disparate pathways of sustainable development and adaptation, of which health is a key component.
Links between global health & climate change adaptation
The health field has attempted to align climate change adaptation to reduce health impacts in terms of the conventional public health categories of primary, secondary, and tertiary prevention and in some instances addresses the social determinants of health as described above [32, 33, 34]. Primary prevention in this context aims to intervene before disease or injury that may occur with climate change, such as rezoning coastal land to protect against rising sea levels and more intense extreme weather events such as coastal flooding. Primary prevention in this sense corresponds generally to anticipatory adaptation [35]. Secondary prevention involves the prevention of adverse health outcomes once disease has begun, but before it is symptomatic. Such approaches in the context of climate change include the strengthening of monitoring and surveillance of infectious diseases, and building public health and other infrastructure to better withstand extreme weather events and other likely incidents. Secondary prevention equates to reactive adaptation [35]. Tertiary prevention in public health aims to minimise the effects caused by existing disease. Tertiary prevention is the most reactive in this spectrum of adaptation and prevention actions, as the adverse health outcome is not prevented. An example of tertiary prevention is the improved treatment of manifest mental illness related to long-term drying. All of these prevention activities are beneficial and necessary for a healthy society; these measures constitute the basis of a "no-regrets" adaptation strategy [35]. This "no-regrets" approach to the health dimensions of climate change resonates with the fundamental humanitarian concerns underpinning much of the work on human vulnerability to 'natural' hazards and environmental change through its strong focus on minimise harm.
It could be argued that true primary prevention of the health effects of climate change involves averting climate change by the mitigation of greenhouse gas emissions, rather than responding to climate change events that are already occurring. The potential health co-benefits of mitigation activities have been well documented (e.g.[36, 37]). Primary prevention could be described instead as "primary adaptive intervention", as it is not genuine prevention. Adaptive strategies are perhaps better understood as secondary in nature as they recognise that climate change is occurring, and present options to reduce the potential health effects.
The World Health Organization's (WHO) World Health Assembly of 2008 adopted a resolution urging Member States to take decisive action to address health impacts from climate change. An objective of this resolution was to strengthen health systems to respond to climate change impacts, which included the conduct of vulnerability and adaptation assessments and the prioritisation of further support [38]. Since then, regional arms of the WHO have been conducting vulnerability assessments to identify the health effects of climate change at country levels, and develop subsequent adaptation activities. The WHO has provided technical support for vulnerability and adaptation assessments in over 30 countries. For example, the Western Pacific Regional Office (WPRO) commissioned work in 2009 in Samoa, Cambodia and Mongolia with cooperation from country WHO offices. Prior to this, there had not been a clear and specific focus on public health, and inclusion of public health in NAPAs had generally been fairly weak. For example, the NAPA produced by Cambodia in 2006 only identified six direct public health projects out of a total of thirty-nine, and indeed, all of these were very narrow in their scope, only focusing on malaria. Bangladesh is another example where public health receives only a brief mention in its NAPA; a project on awareness-raising of the health effects of climate change, particularly focusing on communicable diseases, although health is mentioned as a sector for mainstreaming climate change adaptation activities and capacity building. A review of Annex 1 countries (industrialised countries and countries in transition) reported that no countries recognised health vulnerability within their Fifth National Communications [39].
Current funding mechanisms for adaptation projects
The funding of adaptation activities to respond to unavoidable climate change has emerged as a major theme in climate change negotiations. There exist two maingroups–donors (generally developed countries) and recipients (developing countries). Again, equity is an important factor here–those most vulnerable to climate change are the ones that are least responsible for it, and this consequently underlines the responsibility of developed countries to provide substantial financial contributions. Donors' motivation to fund adaptation activities is partly in response to UNFCCC negotiations which include the funding of adaptation activities; however it is also feasible that donors may understand and concur with the logical links between funding climate change adaptation and supporting broader development activities. This link, though, can also complicate the need to demonstrate additional funding for adaptation, if sustainable development activities are potentially rebadged as adaptation. Given the complicated nature of funding for adaptation, an improved understanding of governance structures and processes that influence the development and financing of adaptation activities, particularly in relation to health, is required [40].
A broad overview of the main climate change adaptation funding mechanisms has recently been conducted, in light of definitional issues relating to country eligibility for the current influx of adaptation financing measures [41]. The three mechanisms identified by this overview are the World Bank's Pilot Program for Climate Resilience (PPCR), which sits within the Bank's Climate Investment Fund established in 2008; the European Commission's Global Climate Change Alliance of the European Union (GCCA), set up in 2007; and the Adaptation Fund (AF), operational since 2009 under the Kyoto Protocol to the UNFCCC. The different approaches used to identifying eligible countries for funding based on their levels of vulnerability has obvious implications for equitable resource allocation of adaptation funds. Although not included in Klein and Möhner's review, the Global Environmental Fund (GEF), which was established in 1991 under the UNFCCC, is also a major funder of adaptation (and mitigation) activities and responsible for supporting NAPA processes in countries; in this sense, the GEF supports the processes that produce the necessary baseline data around vulnerability and potential adaptation activities The GEF and the AF, while closely connected (e.g. with the GEF providing the secretariat to the AF), are separate.
All three funding bodies (PPCR, GCCA, AF) have identified the NAPA as a key basis for funding decisions in countries – this is either funding for implementing the NAPAs (e.g. PPCR), or funding for activities that are consistent with NAPAs (e.g. AF). It is unsurprising that the funding bodies link their programs with the NAPAs, however it is vital to bear in mind that the quality of these country-level assessments vary significantly, so although a consideration of their contents is important, it is most important to consider complementary country activities such as development plans, poverty reduction strategies, and in the case of public health, the consideration and where appropriate, the incorporation of health policies. Due to the NAPAs varying in their inclusion and analysis of the health effects of climate change, it may mean that the public health sector is not fully acknowledged when these funding bodies allocate adaptation resources. This is despite health being identified as one of the priority sectors identified by the NAPA guidelines. A recent WHO review found that less than one-quarter of the 41 NAPAs reviewed provided a comprehensive assessment of health vulnerability [42].
The Green Climate Fund (GCF), which sits within the UNFCCC, was agreed to in Cancun 2010 and is the latest in the iteration of climate change funding bodies. The purpose of this fund is to rapidly mobilise resources for adaptation and mitigation activities in developing countries. The stated objective of the GCF is to promote a paradigm shift towards low-emissions and climate-resilient development pathways. The fund has a financial goal of raising US$30billion by 2012 and US$100billion (from public and private sources) annually by 2020. An independent secretariat will support the fund's operations. The GCF was approved at CoP17 in 2011, however there are still outstanding issues such as which country will host the Fund's secretariat, selection of Board members and the nomination of a financial trustee. Financial arrangements will be finalised at CoP19. It would seem that it may become the most important international financial tool to respond to climate change, however the operation and funding structure of this fund is still unclear.
Currently, there is no specific focus on health within the funds described above, or the UNFCCC negotiation structure. Very little financial support is provided through the UNFCCC mechanisms to address the health impacts of climate change; less than 1% of the distribution of the Least Developed Countries Fund (LDCF), which finances the preparation and implementation of the NAPAs, has been allocated to health protection [43].
Global health plays an integral role when it comes to developing policies and actions to adapt to climate change as it intersects many sectors that will be directly affected by climate change. The massive influx of new and additional funding that is labelled for climate change adaptation can be used strategically by the global health field as a lever to address the fundamental social causes of health and health inequities. Climate change will bring nothing new per se to our global health status; however our current burden of disease and social inequities will be exacerbated.
There are a number of well-financed climate change adaptation mechanisms that will continue to grow, along with the confusion that comes with many different players and poorly organised governance structures. Although the WHO has identified climate change as an issue to be addressed, funding for rigorous vulnerability assessments that focus on the health effects of climate change remains minimal. The potential over-reliance on the NAPAs as a key source for the prioritisation of adaptation funding is a concerning prospect, given the historical lack of focus on global health within these plans. Currently the WHO needs to compete with other players in the adaptation scene, many of which can produce quick and visible adaptation outcomes for monetary investment (such as flood walls, bridges), which is generally not the case for public health. In addition, the allocation of funding for adaptation is often dependent on competing local priorities (which may have unrecognised synergies with adaptation activities) and effective lobbying of and outreach to bilateral and multilateral donors.
In terms of future research, a review is required for Non-Annex 1 countries (developing countries) to clarify to what extent health is being considered within adaptation responses.
The global health community cannot see climate change adaptation as a threat to its other work; this is an opportunity to improve coherence and synergies across disciplines and sectors that contribute to human health.
Copyright information
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Editors' Pick: Originally published Dec. 28.
China faces a "sudden stop" in its economy in 2016, as the government loses its ability to prop up state-subsidized industries and capital flight prompts a significant devaluation of the country's yuan, according to the analyst who accurately forecast Russia's stock-market crash in 1998.
JP Smith, who worked at Deutsche Bank before leaving last year to start independent research firm Ecstrat, says conditions in China look as ominous as those in the U.S. on the eve of the 2008 financial crisis, and in Korea just before the 1997 Asian crisis. As a result, Chinese stocks face greater downside potential than those of any other global market, he wrote in a Dec. 17 report.
The projection contrasts with the outlooks of many investors and analysts who are betting China's economy is on track for a gentle slowdown. Bank of America predicted in a report this month that the country will undergo a gradual currency devaluation as it shifts to a services-based economy.
Market forces probably will trump Chinese authorities' ability to control the economy and capital flows through official dictates, according to Smith.
"Companies across a number of key industries appear to be on the verge of a liquidity crisis, while the efficacy of repeated monetary and fiscal stimulus measures appears to be diminishing," Smith wrote. "It is clear that the underlying pressure for capital to leave China is likely to increase further over 2016."
A sharp downturn in Chinese stocks would mark a departure from this year, when the nation's equity market held up better than those of counterparts as authorities took steps to prop up share prices. The iShares MSCI China ETF (MCHI) is down 6.3% this year, compared with a decline of 13% for a broader gauge of emerging-market stocks.
Bank of America said in a report that its forecasts for China "suggest a soft landing toward slower growth." The economy will grow by 6.6% in 2016, slightly lower than this year's estimated growth of 6.9% growth, according to the bank.
So far, China has been deploying its foreign reserves to support the yuan, which is pegged to a basket of foreign currencies including the U.S. dollar. According to Trading Economics, China's foreign reserves fell by $87 billion in November to $3.43 trillion, the lowest in almost three years. They're down from $3.99 trillion in June 2014.


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You might be thinking about the WileyFox Swift, or the BLU Life Mark 4G, as both phones feature a 5inch screen, and good specifications for the price (both on offer for around £99 on Amazon UK). Lets have a look at how they compare:
We recently had a look at the WileyFox Swift, and have been impressed by the smartphone, considering the price and features available, and the BLU Life Mark 4G could be a competitor.
|WileyFox Swift 4G
13mp main camera, f/2.5, Samsung BSI CMOS
Dual LED flash
5mp selfie camera
FullHD video recording (rear and front camera)
5inch screen, 1280×720 (Corning Gorilla Glass 3)
1.2GHz Quadcore (Snapdragon 410, 64-bit)
2GB / 16GB built in
MicroSD slot
Dual SIM slots
Removable 2500mAh battery
141.15mm x 71mm x 9.37mm
Available in black or white
Bundled with USB cable, screen protector, battery, and very little else.
|BLU Life Mark 4G
13mp f/2.0 aperture main camera
LED-flash (single)
5mp selfie camera
FullHD video
5inch screen, 1280×720
1.3GHz Quadcore (MediaTek 6735)
2GB / 16GB built in
MicroSD slot
Dual SIM slot
2300mAh battery (Removable)
142.4mm x 72mm x 9.5mm
Grey, White or Gold
Fingerprint sensor
Bundled with case, screen protector, USB, battery, charger, ear buds.
Looking at the specifications of both phones, they are quite closely matched in a number of ways, and the BLU Life Mark also offers a Fingerprint sensor for additional security, however the battery life is likely to be quite poor due to the lower battery rating. The Blue Life Mark is also larger and heavier, and it's not clear what kind of protection is built in to the screen, with the WileyFox Swift offering Gorilla Glass 3.


text:
This course on managing international business through co-operative strategies discusses the nature of co-operative arrangements between otherwise independent firms in the pursuit of sustainable competitive advantage that they hope they may achieve together. It includes joint ventures, and other forms of strategic alliances. It is particularly valuable in a globalising world where all firms are concerned to increase their global reach.
An increasingly large number of firms today engage in co-operative strategies. A co-operative strategy is an attempt by a firm to realise its objectives through co-operation with other firms, in alliances, rather than competition with them.It focuses on the benefits that can be gained through co-operation and how to manage the co-operation so as to realise them. A co-operative strategy can offer significant advantages for companies that are lacking in particular competencies or resources to secure these through links to others possessing complementary skills or assets; it may also offer easier access to new markets, and opportunities for mutual synergy and learning. The most common form of co-operative strategy is the joint venture. However there are many other forms including networks and the virtual corporation. In a globalised world co-operative strategy is the means by which most nationally oriented companies attempt to achieve global reach.
There are ten sessions in total and you should plan to spend around one hour on each session.
There is no formal assessment for this course, but there are self-assessment opportunities throughout the course.
The course consists of nine substantive sessions and one revision session:
The nature of co-operative strategy
Different perspectives of alliances
Partner selection and alliance negotiation
Alliance forms, networks and virtual corporations
Alliance structure: ownership and control
General management in alliances
HRM in alliances
Culture in alliances
Alliance performance
|Data de início
The course is intended for directors and senior or middle managers of private, not for profit and public sector organisations.Quais são os objetivos do curso?
By the end of the course you should be able to:
Understand the role of co-operative strategy, and its expression through strategic alliances, in the modern business context.
Appreciate the perspectives from different disciplines that contribute to an understanding of co-operative strategy and strategic alliances.
Understand the considerations involved in establishing alliances, selecting partners and choosing an alliance form.
Appreciate the issues arising in the management of strategic alliances, with special reference to cross-border alliances.
Understand the difficulties involved in the role of a General Manager of a joint venture
Be able to identify the requirements for performance and evolution in alliances
There are no entry requirements, although you should have a reasonable level of English and Mathematics.O que torna especial este curso?
There are nine substantive sessions and one revision session, all recorded in the form of audio lectures. You listen to these first before you do any reading, and then once more after you have read the recommended texts. You will be able to calibrate your progress by carrying out the self assessed activities. Feedback to these activities is provided in the 'Activity Feedback' folder under each session.
The course is delivered through our Teaching Zone so you also gain free access to our Manager's tool kit and effective learning skills sections.Como faço para pagar este curso?
Transferência, Paypal


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NEOSPORIN® MOISTURE ESSENTIALS™ Daily Body Wash. The Neosporin® Essentials™ products have RELIPID™ formulas that contain a blend of ingredients to help retain moisture for healthy-looking skin.
The Neosporin® Moisture Essentials™ Daily Body Wash contains sodium laureth sulfate and dipropylene glycol. These ingredients are potential irritants at concentration levels thought to be insignificant for most patients.
For more information, please visit www.neosporin.com