text:
One sailor killed in pirate attack on Turkish ship off coast of Guinea
One crew member was killed and over a dozen others kidnapped when pirates attacked a Turkish cargo ship off the coast of Guinea on Saturday, Birgün newspaper reported.
Nigerian pirates attacked the M/V Mozart, operated by Istanbul-based Boden Maritime, while sailing 100 nautical miles off the island of Tome, kidnapping 15 out of the 19 sailors on board, it said.
The ship was travelling from Lagos to Cape Town.
Azeri engineer Farman Ismayilov has been identified as the crew member killed in the attack on the ship.
The West African coast has long been a target for pirates, with the Gulf of Guinea being described as the most dangerous area in the world for piracy by the International Maritime Bureau.


text:
SCHITTMAYER, Matthias (Lichtenfelsgasse 9, Graz, A-8010, AT)
KAMBOURAKIS, Spiros (997 East California Boulevard, Pasadena, CA, 91106, US)
ZACH, Simone (Naturweg 4, Graz, A-9047, AT)
GLIEDER, Anton (Wetzawinkel 20, Gleisdorf, A-8200, AT)
SCHITTMAYER, Matthias (Lichtenfelsgasse 9, Graz, A-8010, AT)
KAMBOURAKIS, Spiros (997 East California Boulevard, Pasadena, CA, 91106, US)
ZACH, Simone (Naturweg 4, Graz, A-9047, AT)
WHAT IS CLAIMED IS:
1. A method of making a reduced substrate and/or an oxidized substrate using an enzymatic or enzyme-mediated reaction, comprising: contacting an isolated Old Yellow Enzyme (OYE) with a substrate to form a reaction product comprising a reduced substrate and/or oxidized substrate.
2. The method of claim 1, wherein the reaction is a hydroxylation reaction.
3. The method of claim 1, wherein the substrate is testosterone and the reaction product is 6α and/or 6β-hydroxytestosterone.
4. The method of claim 1, wherein the reaction is a desaturation reaction.
5. The method of claim 1, wherein the substrate is a ketone and the reaction product is an alpha, beta unsaturated ketone.
6. The method of claim 1, wherein the substrate is testosterone and the reaction product is a desaturated testosterone.
7. The method of claim 1, wherein the substrate is dihydrocarvone and the reaction product is a desaturated dihydrocarvone.
8. The method of claim 1, wherein the substrate is cyclohexanone and the reaction product is a desaturated cyclohexanone.
9. The method of claim 1, wherein the isolated OYE catalyses the reverse reaction of enone reductases leading to alpha, beta unsaturated compounds.
10. The method of claim 9, wherein the reverse reaction is not coupled to product aromatization.
11. The method of claim 9, wherein the isolated OYE is contacted with the substrate in the presence of one or more co factors selected from the group consisting of NAD+, NADH, NADP+, and NADPH.
12. The method of claim 9, wherein the isolated OYE is contacted with the substrate in the absence of nicotinamide co factors.
13. The method of claim 1, wherein the oxidation or reduction of the substrate is a one-step reaction.
14. The method of claim 1, wherein the reaction occurs at temperatures higher than 45°C.
15. The method of claim 1, wherein the reaction occurs at temperatures higher than 65°C.
16. A method of oxidizing testosterone, comprising:
contacting testosterone with an isolated Old Yellow Enzyme (OYE) to form 6α and/or 6β-hydroxytestosterone.
17. The method of claim 16, further comprising controlling the ratio of 6α to 6β-hydroxytestosterone by contacting the isolated OYE with the substrate in the presence of hydrogen peroxide.
18. A method of making an oxidized product from a ketone, comprising: contacting the ketone with an isolated Old Yellow Enzyme (OYE) to form an alpha, beta desaturated ketone.
19. The method of claim 18, wherein the alpha, beta desaturated ketone is formed without subsequent energetically favored product aromatization.
20. The method of claim 18, wherein the isolated OYE is contacted with the substrate in the presence of one or more cofactors selected from the group consisting ofNAD+, NADH, NADP+, and NADPH.
21. The method of claim 18, wherein the isolated OYE is contacted with the substrate in the absence of nicotinamide cofactors.
22. The method of claim 18, wherein the ketone is testosterone.
23. An isolated Old Yellow Enzyme (OYE) capable of mediating the oxidation or reduction of a substrate into an oxidized and/or reduced substrate.
24. The isolated OYE of claim 23, wherein the isolated OYE is capable of oxidizing testosterone to 6α and/or 6β-hydroxytestosterone.
25. The isolated OYE of claim 23, wherein the substrate is a saturated compound and the isolated OYE is capable of oxidizing the substrated to alpha, beta unsaturated compounds.
26. The isolated OYE of claim 25, wherein the alpha, beta unsaturated compounds are formed without subsequent energetically favored product aromatization.
27. The isolated OYE of claim 23, wherein the substrate is a ketone and the isolated OYE is capable of oxidizing the substrate to an alpha, beta desaturated ketone.
28. The isolated OYE of claim 23, wherein the substrate is testosterone and the OYE is capable of oxidizing the substrate to desaturated testosterone.
29. The isolated OYE of claim 23, wherein the isolated OYE is capable of using molecular oxygen for substrate oxidations without subsequent energetically favored product aromatization.
30. The isolated OYE of claim 23, wherein the isolated OYE has been purified by heat precipitation.
31. The isolated OYE of claim 23, wherein the isolated OYE is capable of mediating the reduction of the substrate stereoselectively at reaction rates higher than other known reductases.
32. An isolated Old Yellow Enzyme (OYE) capable of mediating hydroxylation of a substrate and/or oxidation of the substrate to its desaturated products.
33. The isolated OYE of claim 32, wherein the substrate is testosterone.
34. The isolated OYE of claim 32, wherein the substrate is testosterone and the isolated OYE is capable of hydroxylating the substrate to 6α and/or 6β- hydroxytestosterone.
35. The isolated OYE of claim 32, wherein the substrate is testosterone and the isolated OYE is capable of oxidizing the substrate to desaturated testosterone.
36. The isolated OYE of claim 32, wherein the isolated OYE mediates the hydroxylation of the substrate in the presence of one or more co factors selected from the group consisting of NAD+, NADH, NADP+, and NADPH.
37. The isolated OYE of claim 32, wherein the isolated OYE mediates the oxidation of the substrate to its desaturated products in the absence of nicotinamide co factors and in the presence of molecular oxygen.
METHOD FOR REDOX REACTION USING AN OLD YELLOW ENZYME
FIELD OF THE INVENTION
"  The current invention is directed to an enzymatic and/or enzyme-mediated catalysis utilizing an Old Yellow Enzyme; and more particularly to the production of oxidized or reduced substrates of interest using an Old Yellow Enzyme.
BACKGROUND OF THE INVENTION
 The oxidation and reduction of substances is of high interest because of the importance of the various metabolites that can be formed from such reactions.
 Important examples of biooxidation include hydroxylation. For example, hydroxylation of testosterone by human liver microsomes allows for the formation of IB-, 2a-
/B-, 6B-, 15B-, and 16B-hydroxytestosterones, which are important metabolites for the body
(Agematu et al 2006). Furthermore, these metabolites are very useful in pharmacokinetic and toxicological studies. 6 alpha and beta hydroxytestosterone could also be used as building blocks for new steroid derivatives (e.g. as new drugs).
 Other important examples of bioxidation reaction include desaturation reactions.
The introduction of new double bonds is highly desirable for synthetic applications. The
* selective introduction of double bonds at very specific positions of larger molecules can hardly be done by classical chemistry. In addition to the formation of new compounds of high interest, such as the formation of boldenone -an active pharmaceutical ingredient- from testosterone, desaturation reactions activate carbohydrates for interesting, selective chemistry, such as subsequent introduction of functional groups (e.g. hydroxyl, epoxide, halogen groups). Thus, desaturation reactions provide a new broadly applicable toolbox for organic syntheses.
 Usually monooxygenases are used for these biooxidative reactions, but these compounds are not available for all substrates. Among the monooxygenases, cytochrome P450 (P450) enzymes, a superfamily of more than 160 known members, are also responsible for the biosynthesis or catabolism of steroid hormones, including the oxidative metabolism of endogenous and exogenous testosterone. (See, e.g., Wood AW, Swinney DC, Thomas PE, Ryan DE, Hall PF, Levin W, and Garland WA (1988) Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b. J Biol Chem 263: 17322-17332; Yamazaki H and Shimada T (1997), Progesterone and testosterone hydroxylation by cytochromes P450, 2Cl 9, 2C9, and 3A4 in human liver microsomes. Arch Biochem Biophys 346: 161-169; and Rendic S, Nolteemsting E, and Schanzer W (1999) Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes. J Chromatogr Biomed Appl 735: 73-83).
 In monooxygenase catalyzed biooxidative reaction, β-hydroxylation at either the
C6 or Cl 6 position is the major route of testosterone oxidative metabolism. Human liver enzymes are also found to oxidize testosterone at the Cl 7 position to form androstenedione.
 However, conventional oxidation enzymes, such as P450 enzymes, are usually very unstable. In addition, the monooxygenases known to catalyze these bio-oxidative reactions are not available for all substrates and many highly needed products can not be obtained in sufficient quantities.
 Furthermore, a disadvantage of P450 enzymes in using them for industrial purposes is the requirement for the costly cofactor NADPH/NADH in the P450-catalyzed reactions. Not only are the cofactors very expensive, but they are also responsible for the inactivation of the enzymes when the concentration of substrates is low, resulting in incomplete oxidation of the substrates.
 One possible enzyme of interest is the Old Yellow Enzyme ("OYE"). OYE are first known as enone reductase (ERED) on typical substrates such as cyclohexone and carvone.
 The reactions commonly catalysed by OYE family enzymes range from asymetric reaction of alpha, beta desaturated ketones (Hall, (2007) Angewandte) to the degradation of explosives (Williams, (2002) Microbiology).
 In addition to the expected "enone" reduction, other enone reductases including the known OYE enzymes have been reported to catalyze the highly endothermic desaturation of C-C bonds, but only if such reactions are coupled with subsequent product aromatization
(V az et al., 1995), as the energetically favorable product aromatization drives the desaturation reaction. However, the aromatized products do not retain the initial properties of the desaturated compound for follow up chemistry. No enzymatic introduction of double bonds which is not linked with the energetically favored product aromatization has been demonstrated thus far.
 Thus there is a need for OYE enzymes that catalyze the hydroxylation of testoterone.
 There is also a need for OYE enzymes that catalyze the desaturation reaction of ketones without subsequent product aromatization.
 In general, heat stable enzymes have favorable properties for the use in biochemical reactions. Often heat stability is combined with good solvent stability and high total turnover numbers. Moreover, the heat stability can be used to facilitate the purification of the enzyme (e.g. with heat precipitation).
 Accordingly, a need exists for improved enzymes, which could catalyze or mediate the reduction and/or oxidation of substrates of interest. Example of such substrates include testosterone, which can undergo hydroxylation and/or desaturation to yield important
metabolites. In addition, there is a general need for heat stable enzymes for the aforementioned reactions.
 The present invention addresses this need for an improved method and enzyme for the reduction and/or oxidation of substrates, without the disadvantages of conventional biocatalytic enzymes such as monooxygenases.
SUMMARY OF THE INVENTION
 The current invention provides a method of making a reduced substrate and/or an oxidized substrate using an isolated Old Yellow Enzyme.
 The invention provides an isolated Old Yellow Enzyme capable of mediating the oxidation or reduction of a substrate into an oxidized and/or reduced substrate.
 In one embodiment, the invention is directed to a method of the chemoselective and regioselective oxidation of carbon-hydrogen bonds using an isolated Old Yellow Enzyme
 In one embodiment, the invention provides a method of hydroxylating testosterone using an isolated Old Yellow Enzyme.
 In another embodiment, the invention provides an isolated Old Yellow Enzyme capable of hydroxylating testosterone.
 In another embodiment, the invention provides a method of controlling the stereospecificity of the enzyme-mediated oxidation by utilizing hydrogen peroxide.
 In another embodiment, the invention providesa method of oxidizing ketones to alpha, beta desaturated ketones using an Old Yellow Enzyme (e.g., introduction of an additional double bond in testosterone).
 In another embodiment, the invention provides an isolated Old Yellow Enzyme capable of oxidizing ketones to alpha, betaalpha, beta desaturated ketones (e.g., introduction of additional double bonds in testosterone).
 In another embodiment, the OYE-catalyzeddesaturation of ketones proceeds without the need of energetically- favored subsequent product aromatization.
 In another embodiment, the OYE-catalyzed desaturation of ketones proceeds without the addition of any coenzymes such as, e.g., NAD+, NADP+, NADH and NADPH.
 In another embodiment, the invention is directed to an isolated OYE capable of hydroxylating testoterone in the presence of co factor NADPH and catalyzing the desaturation of testoterone in the absence of any coenzymes such as, e.g., NAD+, NADP+, NADH and
 In t another embodiment, the invention is directed to an isolated OYE possessing high heat stability. Its heat stability facilitates enzyme purification, enhances storage stability, and allows higher reaction temperatures, long-term conversions, and/or enzyme recycling.
 In another embodiment, the invention is directed to an isolated OYE capable of mediating the reduction of substrate stereoselectively at reaction rates higher than other known reductases.
BRIEF DESCRIPTION OF THE DRAWINGS
 These and other features, aspects, and advantages of the present invention will be more fully understood when considered with respect to the following detailed description, appended claims, and accompanying drawings, wherein:
 FIG. 1 is a reaction diagram of the hydroxylation of testosterone to 6α and 6β -
 FIG. 2 is a Table showing the alignment of Geobacillus kaustophilus OYE with the homolog protein from Bacillus subtilis (GI: 67464349). The "query" is the sequence of the Geobacillus kaustophilus OYE, and the subject is the sequence of the YqjM;
 FIG. 3 shows the OYE expressed in DH5a cells as a 38kDalton band, which is absent in the negative control lane;
 FIG. 4 is a reaction diagram of the desaturation of testosterone to boldenone;
 FIG. 5 shows the conversion of cyclohexanone into Cyclohex-2-enone and further to phenol;.
 FIG. 6 shows the conversion of dihydrocarvone to carvone;.
 FIG. 7 shows the influence of exogenous hydrogen peroxide on the stereospecifϊcity of the OYE mediated hydroxylation reaction;.
 FIG. 8 shows the OYE-mediated testosterone conversion: a) desaturation to boldenone; b) hydroxylation to 6-hydroxytestosterone;
 FIG. 9 shows the isolation of the GkOYE by heat precipitation;.
 FIG. 10 shows the Cyclohexenone reduction by G. kaustophilus OYE and YqjM at different temperatures;.
 FIG. 11 shows the 1 H-NMR spectrum of 6α-hydroxytestosterone;
 FIG. 12 shows the 1 H-NMR spectrum of 6β -hydroxytestosterone;
 FIG. 13 shows the OYE expressed in different E. CoIi expression strains as 38 kDalton bands; and
 FIG. 14 shows the testosterone conversion into 6α-hydroxytestosterone as measured by HPLC-MS
DETAILED DESCRIPTION OF THE INVENTION
 The isolated OYE, according to the invention, is preferably extracted from the organism, and used either in raw lysates or in purified form.
 In an enzymatic or enzyme-catalyzed reaction, according to the invention, the enzyme mediates the reaction by chemically utilizing the residues in its active. In contrast, in
an enzyme-mediated reaction, the enzyme mediates the reaction without utilizing its active site, e.g., the substrate is activated by binding to the enzyme but no residues of the enzyme are involved in the reaction itself. According to the invention, the term "mediate a reaction" includes both enzyme-catalyzed and enzyme.
 Thus, in a first aspect, the invention provides a method of making a reduced substrate and/or an oxidized substrate using an enzymatic or enzyme-mediated reaction, comprising contacting an isolated Old Yellow Enzyme (OYE) with a substrate to form a reaction product comprising a reduced substrate and/or oxidized substrate. In one embodiment, the invention provides a method for enzyme-mediated hydroxylation of testosterone into 6α and/or 6β hydroxytestosterone. hi another embodiment, the invention provides a method for enzyme-mediated desaturation of testoterone to form desaturated testosterone.
 hi another aspect, the invention provides a method of oxidizing testosterone, comprising contacting testosterone with an isolated Old Yellow Enzyme (OYE) to form 6α and/or 6β-hydroxytestosterone. In another embodiment, the invention provides a method for further comprising controlling the ratio of 6α to 6β-hydroxytestosterone by contacting the isolated OYE with the substrate in the presence of hydrogen peroxide.  hi another aspect, the invention provides a method of making an oxidized product from a ketone, comprising contacting the ketone with an isolated Old Yellow Enzyme (OYE) to form an alpha, beta desaturated ketone, hi another embodiment, the alpha, beta desaturated ketone is formed without subsequent energetically favored product aromatization. hi another embodiment, the alpha, beta desaturated ketone is formed in the absence of the nicotinamide cofactors and in the presence of molecular oxygen, hi another embodiment, the ketone is testosterone.
 hi the course of screening for new microbial hydroxylating activities, two strains are identified, both oxidizing testosterone to 6α and 6β hydroxytestosterone (FIG. 1). The two strains correspond to Geobacillus thermoglucosidasius and Geobacillus kaustophilus. There are also literature reports about P450 enzyme(s) from Geobacillus that can catalyze or mediate the hydroxylation of testosterone.
 Protein purification from Geobacillus thermoglucosidasius and Geobacillus kaustophilus crude lysates and subsequent fingerprinting by Matrix- Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) are chosen as a strategy for identifying the testosterone hydroxylating activity. To identify the enzyme responsible for the hydroxylating activity, Geobacillus thermoglucosidasius DSM 2542 and Geobacillus kaustophilus DSM 7263, the latter one having the advantage of a sequenced genome, are obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ;
German Collection of Microorganisms and Cell Cultures) and tested for their ability to hydroxylate testosterone.
 To purify the enzyme of interest, the raw lysates of the strain Geobacillus kaustophilus DSM 7263 are subjected to anion exchange chromatography followed by size exclusion chromatography. Several active fractions are obtained and loaded onto a Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis ("SDS-PAGE") and, after gel electrophoresis, stained with Coomassie blue. One lane yields a single band of approximately 38 kDa, which is also present in all other active fractions. This band is isolated from the gel and prepared for fingerprinting by MALDI-TOF. A database search with the acquired fingerprint yielded, amongst others, OYE of Geobacillus kaustophilus, YP 148185, referred to hereinafter as GK)YE.
 Comparing the sequence of the active purified protein YP 148185.1 against the genome sequence of Geobacillus kaustophilus facilitates the identification of the gene coding for YP l 48185.1.
 To identify homology with other known OYEs, a Basic Local Alignment Search Tool (BLAST) is used, which compares the DNA/protein sequences of the GK)YE with ' sequences of other known OYEs. The results of the search reveal close homology to the YqjM type of OYEs. The highest similarity is found with the YqjM sequence from Bacillus subtilis NRRL B-14911. The alignment establishing the similarity of the GK)YE to the homolog protein from Bacillus subtilis (GI: 67464349) whose the structure is known is shown in FIG. 2.
 As shown in FIG. 2, the two Histidines in the substrate binding site, H 164 and H 167, are conserved. The Y28 of the N-terminal part, which is a special feature of the YqjM-like proteins and acts as a hydrogen donor, is also conserved. It should be noted that, in most OYEs, this residue Y28 is formed by a tyrosine from the C-terminal domain.  The cofactor involved in the GkO YE-catalyzed oxidation is determined to be FMN, and not FAD, by MALDI-TOF. Absorbance spectra of the oxidized enzyme show characteristic bands at 360 and 455 nm, with the latter peak showing an OYE characteristic fine spectrum (shoulders at 430 and 485nm respectively). Also, the shorter wavelength peak shows a fine spectrum with a second maximum at 380 to 385nm, which is not common for OYEs. Denaturation of the enzyme with 0.5% SDS shows a characteristic spectrum of free FAD/FMN, indicating that the cofactor is not bound covalently.
 Primers are designed to amplify OYE from Geobacillus kaustophilus genomic DNA by polymerase chain reaction (PCR). PCR products yield a band at the expected size of approximately 1020bp. The fragment is cloned into an expression vector (pEamTA), yielding pEamTAOYE. The sequence of the expressed protein confirmed that the cloned fragment is indeed the desired OYE.
 For further expression of OYE, the DNA fragment of interest is transformed into a DH5α strain oϊE.coli using transformation procedures that are well known in the art.  After expression in E.coli DH5α, the cells are harvested, ruptured, and centrifuged, and loaded onto a SDS-PAGE. A thick band, visible in the soluble fraction at the expected size of 38kDa is observed. A small amount of OYE is also found to remain in the insoluble fraction. A negative control (pEamTA in DH5α) does not show a band at 38kDa (Fig. 3).
 The hydroxylation activity is preferably determined by measuring the oxidation of testosterone (5mM), in the presence of NADPH (0.5mM), into 6α-hydroxytestosterone at 55°C. High-Performance Liquid Chromatography/Mass Spectrometry ("HPLC/MS") analysis is used to detect the production of 6α-hydroxytestosterone after 24h, and a further increase in product yield after 48h. After 48h the reaction was stopped.  In addition to the hydroxylation activity, surprisingly, the Geobacillus kaustophilus OYE also catalyzed an O 2 -driven, nicotinamide-independent desaturation reaction, introducing C-C double bonds adjacent to carbonyl groups. As shown in FIG. 4, GAOYE catalyzes the desaturation of testosterone in boldenone.
 The GAOYE-catalyzed desaturation activity is preferably determined by * measuring the oxidation of testosterone (5mM) in the absence of co factor NADPH into boldenone at 55°C and incubated for incubated for 48h. HPLC/MS analysis is used to )., confirm the production of desaturated testosterone. At a temperature of 70 0 C and at a testosterone concentration of ImM, the yield of the reaction is above 90% after 48h.  Furthermore, GAOYE also acts as "enone" reductase and mediates the reduction of typical substrates such as cyclohexenone and carvone. Interestingly, it is also capable of ' ψ ' mediating the reverse reaction, the desaturation of cyclohexanone into Cyclohex-2-enone and further into phenol, as shown in Fig. 5. It should be noted that the dismutation reaction has been described in "Old Yellow Enzyme: Aromatization of Cyclic Enones and the Mechanism of a Novel Dismutation Reaction, " Alfin D. N. Vaz, * Sumita Chakraborty, and Vincent Massey. However, to date, cyclohexanone has not been reported as a starting material in such a desaturation reaction. The ratios of cyclohexanone:cyclohex-2-phenone:phenol after a 24 hour reaction are 60%:7%:30%.
 ' Similarly. GAOYE is capable of catalyzing the desaturation of dyhydrocarvone into carvone, as shown in Fig. 6. At 70 0 C, both the R and S enantiomers of dihydrocarvone can be desaturated to carvone. The described desaturation reactions, however, are not temperature dependent, as the desaturation at 37°C is comparable to the desaturation performed at higher temperatures (45, 50, 60, and 70 0 C). At higher temperatures, the desaturation reaction is not stereoselective. The reverse reaction, reduction of carvone to dihydrocarvone, yields -50% of each R and S - (+) enantiomers.
 Addition of exogenous hydrogen peroxide induces a change in the stereoselectivity of GAOYE. In hydroxylation assays as described above and carried out in the presence of various concentrations of hydrogen peroxide (0.05-0.6%) (FIG.7), it is found that hydrogen peroxide changes the stereoselectivity of the GAOYE-mediated hydroxylation of testosterone. As shown in FIG. 7, in the presence of reduced nicotinamide co factor, testosterone is regioselectively hydroxylated at position 6, yielding 6α- and 6β- hydroxytestosterone, with a product ratio of approximately 3:1. Increasing hydrogen peroxide concentrations, however, inverts the ratios of 6 α- and 6β-hydroxytestosterone, yielding a 1 :2 ratio. Higher concentrations OfH 2 O 2 result in at least 4 additional hydroxylation products that remain to be identified.
 Since the ratio of 6α-hydroxytestosterone to the human main metabolite, 6β- hydroxytestosterone, is influenced by varying hydrogen peroxide concentrations, testosterone hydroxylation appears to be enzyme-mediated rather than enzyme-catalyzed and involves the reduction of molecular oxygen to a reactive oxygen species at the flavin's isoalloxazine ring system. In an enzyme-mediated reaction, the enzyme facilitates the reaction without direct participation of the residues in the active site. In an enzyme-catalyzed reaction, the residues of the active site are physically changed.
 The hypothesis that the testosterone hydroxylation is mediated by GAOYE is substantiated by the observation that the mutation of the two Histidine residues at the active site to Alanine (H 164 A and H 167A) destroys the saturation/desaturation activities but only slightly reduces hydroxylation activity. These results suggest that GAOYE mediates the hydroxylation of testosterone and also catalyzes desaturation of testosterone. FIG. 8 shows the GAOYE selective oxidation of testosterone depending on the presence or absence of co factor NADPH and oxygen.
 Yields of hydroxylation might be improved by reaction engineering. A sufficient supply of reduced cofactor (e.g. NADPH) should prevent desaturation of testosterone to boldenone and thus allow the hydroxylation of testoterone to 6 α- and 6β-hydroxytestoterone.
 Although not to be bound by theory, the results point to an enzyme-mediated process for the hydroxylation of testosterone. "Good" substrates are readily reduced, while "bad" substrates are bound, oriented, and slightly activated. The reduced Flavin cofactor (FMN) may transfer the electrons to molecular oxygen, and the resulting hydrogen peroxide could then hydroxylate the bound substrate. It is also possible that the oxidation reaction only takes place under higher temperatures, but this is hard to prove since all other available OYEs quickly precipitated at temperatures above 40°C.
 Several substrates other than testosterone were also tested for desaturation activities (Table 1). The results show that GAOYE catalyzes the desaturation/saturation of
several subtrates, and confirm that OYEs can be used to facilitate the biooxidation of substrates. It has been further discovered that the use of an OYE allows for the production of oxidized substrates in one-step reactions at high yield. In addition, the GAOYE shows stability at high temperature and in long lasting bioconversions.
 For example, in one experiment, where the enzyme's desaturation activities are measured as a function of temperature, the temperature was varied by 2.5 degree increments, from 30 to 85°C, and the optimum temperature for testosterone desaturation was found to be 70°C.
 The thermo stability of GkOYE facilitates its purification by heat precipitation, as illustrated by example (give number of example) and as shown in FIG. 9. Activity measurements showed that no loss of activity after incubation at 55°C for lOmin. Moreover, under these conditions most E.coli proteins precipitate, leaving the OYE in the supernatant in high purity. Even after one week at 4°C no decrease in activity was observed.  The thermo stability in the bioxidation reactions seems to be unique to GkOYE and is not exhibited by other recombinant OYEs obtained by using commercial kits from Codexis, (Pasadena, California) For example,
 No testosterone hydroxylation or desaturation is observed with a range of recombinant OYEs from various other species (commercial kit from Codexis, Pasadena) when activity was measured as a function of temperature, over a temperature range of 30-70 °C, varied in 5°C increments, Even with the highly homologous Bacillus subtilis YqjM (similarity: 80%), no oxidized testosterone derivative is detected, even though enone reductase activity is demonstrated at 70°C (FIG. 10). Furthermore, compared to other described enzymes, GkOYE exhibits a higher reaction rate for many common saturation substrates (e.g. Cyclohexenone).
 The following are non-limiting examples of the invention.
 Hydroxylation of testosterone by cell lysates of Geobacillus thermoglucosidasius DSM 2542 and Geobacillus kaustophilus DSM 7263.
 Geobacillus thermoglucosidasius DSM 2542 and Geobacillus kaustophilus DSM 7263 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; German Collection of Microorganisms and Cell Cultures) and tested for their ability to hydroxylate testosterone. Raw lysates of both strains were analyzed by HPLC/MS and showed conversion to two products, both of which showed a m/z ratio of 305, as expected for hydroxylated testosterone, but with different retention times. While one of the metabolites corresponded exactly with an authentic 6β-hydroxytestosterone standard, the hydroxylation position of the second metabolite was unclear in the beginning. Both metabolites were then prepared on a milligram scale for 1 H-NMR analysis. The NMR
spectra are shown in figures 11 and 12. Peak 1 in FIG. 11 was identified as 6α- hydroxytestosterone and Peak 2 of FIG. 12 was identified as 6β-hydroxytestosterone.
 Expression of OYE in different strains of E. CoIi
 For further expression of O YE, new expression strains of E. CoIi were chosen. 2μL of OYε- DNA were transformed in the strains listed below.
• Rosetta 2
• B121 D3
All cells except for DH5α were electrocompetent cells according to transformation procedures that are well known in the art. The regenerated cell suspension were plated out on LB-Amp- plates(100 μg/mL), except for the cells of the Rosetta strains, which were plated on LB- AMP-Chloramphenicol plates (lOOμg/mL). After an incubation period of about 24 "^ hours at 37°C, the grown colonies were used for inoculation of 100 mL LB- Amp and LB--- AMP- Chloramphenicol, respectively. No growth on agar plates after transformation was-* recorded for the Rosetta cells, and almost no colonies had been obtained by using the Rosetta 2 cells. The inoculated flasks were shaken constantly with 120 rpm at 28 0 C. After 5 hours at an optical density of 0.5, protein expression was induced by adding 0.5 mM IPTG to each flask. The temperature was then lowered to 20°C. The cells were harvested, ruptured, and ultracentrifuged, and the supernatants were loaded on SDS-P AGE. FIG. 13 shows the . -^r expression of OYE in different expression E.coli strains. Using DH5α cells for expression of OYE gave the best results, followed by Rosetta 2.
 Expression of OYE by DH5 α cells
 As a result of the limited number of transformants by using the Rosetta 2 cells, for further expression procedures of OYE only the chemical competent DH5α cells were used. FIG. 3 shows the expression of OYE in DH5α cells only. DH5α cells transformed with the vector pEamTA (without the OYE fragment) were used as a negative control. A 38 kDalton band was obtained in the OYE transformed DH5α, but was absent in the negative control.
 Hydroxylation activity of the OYE-containing fraction
 To verify that the fraction that yielded a 38 kDalton band on the gel as shown in FIG. 3 contains hydroxylating capability, the fraction was tested for hydroxylation activity
wherein the testoterone conversion was analyzed by HPLC-MS. FIG. 14 shows a peak corresponding to the formation of 6α-hydroxytestosterone.
 Oxidation of testosterone as catalyzed or mediated by GAOYE in the presence or absence of cofactors at various temperatures.
 The following stock solutions were prepared: protein solution 20mg/mL, 5OmM KPi, pH=7
NADPH 1OmM, 5OmM KPi, pH=7
Testosterone 10OmM, EtOH
GDH lOmg/mL GDH 102, 5OmM KPi, pH=7
Glucose 20OmM, , 5 OmM KPi, pH=7
All samples were filled up to a total volume of ImL with 5OmM KPi, pH=7
Sample Composition Volume
1 protein solution 500μL
2 protein solution 500μL testosterone lOuL
3 protein solution 500/ιL testosterone 10/xL
4 protein solution 50OuL testosterone lOuL
5 testosterone lOuL
The reactions were carried out at 37, 50 and 70 °C. It was found that no reaction occurred with the negative controls (samples 1+5). No reaction was observed at the lowest
temperature 37°C. Starting at 50°C without any cofactor, the main metabolite had a reaction time of 9.9min and a molecular weight (MW) of 286 determined by HPLC-MS. Compared to testosterone this is a reduction of 2 mass units, as expected for the desaturation reaction. Samples 3 (with NADPH) and 4 (with GDH cofactor regeneration) showed reduced formation of the desaturation product, most likely because NADPH competes with the testosterone to reduce the enzyme. Follow-up reactions indicate that oxygen is reoxidizing the enzyme (see Example 6: anaerobic testosterone desaturation). After 48h, sample 2 showed 84% conversion. After 12Oh, all testosterone was consumed. Sample 4 showed two peaks at ~5min corresponding to 6α- (7%) and 6β- (6%) hydroxytestosterone. LC-MS showed an exact mass of 304. After further incubation, desaturation of the hydroxylated products occurred, yielding products with MW of 302.
 Anaerobic testosterone desaturation
 To support the hypothesis that molecular oxygen is responsible for reoxidizing the enzyme, protein solution and substrate (for composition see sample 2 in Example 5) were incubated for 48h in a minimal oxygen environment. Oxygen was removed by applying vacuum for 15min followed by flushing with Nitrogen (three rounds) in a sealed glass tube. The reaction was incubated at 70°C for 48h. Under aerobic conditions, this reaction yielded 84% desaturation product, but under anaerobic conditions only 18% desaturated testosterone was formed, supporting the assumption that molecular oxygen acts as the final electron acceptor in this reaction.
 Desaturation of cyclohexanone by GAOYE
 The reaction sample contained 500μL protein solution and 1OmM Cyclohexanone (~lμL). The final volume of the reaction was ~500μL. The reaction was carried out at 70°C for 24 hours. It was found that Cyclohexanone was desaturated to Cyclohex-2-enone and further to phenol. After 24 hours, the yields of the products were as indicated in Fig. 5.
 Oxidation/Reduction of dihydrocarvone/carvone by GAOYE  The sample for the oxidation reaction contained 500μL protein solution and 1OmM (R,S)- (+) Dihydrocarvone (~1.5μL). The final volume of the reaction was 500μL. The reaction was carried out for 24h at varying temperatures (see table 2).  The sample for the reduction reaction contained: 500μL protein solution + (+)Carvone (~1.5μL) + 200μL GDH 102 (lOmg/mL) and Glucose (20OmM) + lOOμL NADPH (1OmM). The final volume of the reaction was 800μL. The reaction was carried out for 24h at various temperatures (see table 2).
 It was found that, regardless of which starting enantiomer was used, dihydrocarvone was reduced to carvone. Moreover, the reverse reaction yielded -50% of each, R and S - (+) Dihydrocarvone (also done at 70°C). The desaturation reaction was also carried out at different temperatures: 37, 45, 50, 60 and 70°C. Desaturation was observed even at 37°C in comparable yields to those seen at higher temperatures. At higher temperatures, however, the reaction was not stereoselective.
 Hydroxylation and desaturation activities of GkOYE mutants  Mutants of GkOYE (Y28F, H164A, H 167 A; H164A/H167A; 2 clones each) were transformed into and expressed in E.coli DH5alpha. SDS-Page revealed good expression levels for all clones. Conversions of testosterone at 55°C with NADPH and 7O 0 C without the co factor showed slightly reduced hydroxylation activity for all mutants and, surprisingly, desaturation activity for Y28F (Y28 is assumed to be the proton donor for Yqjm saturation reaction) but not for H 164 A and H 167 A or the double His mutant. Titration of the Mutants with pHBA revealed a K < j in the mM range (1-1OmM), which is approximately a factor 10 3 worse than 3,lμM of the wild type enzyme.
 Heat precipitation of GkOYE  The reaction sample contained the following: buffer: 5OmM Kpi; pH=7 lOmin, 55°C; heating: boiling hot water cooling: ice water
The Activity was tested with NADPH depletion assay before and after heat precipitation and after lyophilization of purified enzyme:
before heat precipitation: 1,14 U/mg Lyo after heat precipitation: 1 ,90 U/mg Lyo after lyophilization: 2,16 U/mg Lyo
The Protein concentration was determined as 84% of Lyo by Bradford assay.
Lanes 1 and 4 of FIG. 9 show the enzyme after heat precipitation and lanes and 2 and 3 before the heat precipitation.
EXAMPLE I l
 Heat precipitation of GkOYE
 E.coli DH5alpha harbouring pEamTAGλOYE was cultivated in a 5L bioreactor.
The enzyme was purified by heat precipitation and yielded ~45g of lyophilisate (>8g/L purified enzyme). The stereoselectivity of GkOYE saturation was tested by reducing R-(-)- carvone to dihydrocarvone (50°C): yield>99%;ee(enantiomeric excess) =75%, NOE-NMR experiments with the derivatized product showed that the preferred product is (2R,5R)-
 Thermostability of GK)YE relative to other recombinant OYEs  Expression clones of YoJM (B. subtilis) and OPR3 (tomato) were obtained from Peter Macheroux, Institute of Biochemistry, TU-Graz. The reductase activities of Yqjm and OPR3 were confirmed using cyclohexenone and carvone as substrates. Both showed stereoselectivity comparable to GAOYE. The desaturation reaction was carried out using cyclohexanone with all three enzymes at 30, 50, and 70°C. The reductase activity using cyclohexenone as substrate was also measured under similar conditions. The results showed that, while OP3 was almost inactive at 50°C, YqjM showed a significantly reduced reductase activity up to the highest temperature (70°C). However, no desaturation was observed for both OP3 and YqjM enzymes. On the other hand, GkOYE as a positive control showed cyclohexane desaturation activity starting from 50°C and saturation of cyclohexenone at all temperatures tested.
 Expression of GkOYE by DH5α and measurements of its activities  Then following primers were used: G&OYEfwl : ATG AAC ACG ATG CTG; and GAOYErv: GAA TTC TTA TTA AAA CCG CCA GC. All oligonucleotides used were manufactured by Invitrogen. The following conditions were used for PCR amplification: 20 ng genomic DNA, digested with Notl, 1 μL primer GAOYEfwl (10 pmol/μL), 1 μL primer GAOYErv (10 pmol/μL), 1 μL dNTP mix (10 mM), 10 μL 5x Colorless GoTaq Reaction Buffer, 1 μL GoTaq-Polymerase (Promega), filled up with ddH 2 O to a total volume of 50 μL. Initial denaturation 3 min at 95 °C, 25 cylces of 30 s at 95 0 C, 30 s at 42 °C and 90 s at 72 °C, final elongation 10 min at 72 °C. The amplified PCR product was purified using the WizardSV Gel and PCR clean-up system (Promega) and cloned into pEamTA (<5) and sequenced, yielding pEamTAOYE.
 E. coli DH5α harboring pEamTAOYE were used to express GAOYE. Cells were grown in an Infors incubator shaking at a diameter of 2.5 cm at 120 rpm and 37 0 C. At an
OD 6O o of 0.5, 1 mM IPTG was added and the incubation temperature was lowered to 30 °C. After 24 h of expression, cells were harvested, lysed by sonication (50 mM KPi, pH 7), and E.coli proteins were precipitated at 55 °C for 10 min. Cell debris and precipitated host proteins were removed by centrifugation (8000 x g, 15 min). The bright yellow supernatant was concentrated 10-fold using Vivaspin (Sartorius) ultrafiltration devices with a cutoff size of 1O kDa.
 For spectral characterization, GAOYE was further purified via Anion exchange chromatography (QFF, GE healthcare) and size exclusion chromatography (Sephadex 75, GE healthcare).
 For desaturation reactions, 500 μL protein solution (20 mg/mL) was added to a 10 mM substrate solution in 500 μL of a 50 mM KPi buffer, pH 7. Analysis of Dihydrocarvone/Carvone was done on a Shimadzu GC-17 A with a Shimadzu GCMS- QP5050A Detector. The employed column was a XTI-5 from Restek (bonded 5 % phenyl, length 30 m, thickness 25 μm, diameter 0.25 mm). High performance liquid chromatography/mass spectroscopy of Boldenone/Testosterone was done on an Agilent 1200 HPLC system with an UV detector and an Agilent G1956B mass detector. For separation, a Merck LiChroCART Purospher RP 18 endcapped column with the dimensions 250 mm x 4.6 mm x 5 μm was employed. Mass spectra were recorded in positive ionization mode employing an APCI ion source.
 Those skilled in the art will appreciate that the foregoing examples and descriptions of various preferred embodiments of the present invention are merely illustrative of the invention as a whole, and that variations in the methodology of the present invention may be made without departing from the invention. Accordingly, the present invention is not limited to the specific embodiments described herein but, rather, is defined by the appended claims and equivalents thereof.

domain: chemistry


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Ratings for Royal University Hospital
Nice people but HORRIBLE PROCESSES! Someone needs to think about emergency care in a different way! Who directs this a Doctor???
Submitted Oct. 6, 2016 | flag
Horribly dirty! Long wait times and staff at the nursing station on their phones and talking about the weekend. Dust dragons on the floor, scratched walls, stained sheets... disgusting. And don't you dare ask questions or try to take a proactive role on your family's health because they just think you are self diagnosing and telling them what to do.... staff I encountered were SUPER sensitive to the role I had.
Could have had better service, compassion, cleanliness, and diagnostic taking mum to a vet. Ugh.
Submitted Oct. 5, 2016 | flag
This place is a dump. Horrific cleanliness. Ridiculous wait time and doctors and nurses on their cell phones or talking about their weekend plans. Very disappointed.
Submitted Oct. 5, 2016 | flag
Went to emergency I was assigned a nurse after long wait time in waiting room and bedroom. Then nurses got changed. I was assigned to Heather. Worse nurse ever. I hope I am wrong but I hope she is not a racist. I leave it at that.
Submitted July 3, 2016 | flag
I went in to the emergency room at 2 pm with extreme pain in my side almost to the point of paralysis. They got my name and health card and put me in the waiting room. I sat there in extreme pain for 4 hours. A nurse finally came and got me. I thought "I finally get to see a doctor!" But instead they placed me in the Children's waiting room (I'm 28 years old!) further in the hospital (cause they wanted to make the waiting room look not so busy I assume) so now I could writhe about in front of a bunch of terrified kids and their parents. 2 hours later the pain finally got to me that I ended up vomiting in a garbage bin. Still no response from nurses or doctors. My parents ended up being so worried they came down to the hospital (I kept telling them not to come) and my mom ended up asking the nurses multiple times when I would actually get to see a doctor. The nurses finally caved and put me in a hospital bed in an alcove (not an actual room.) After ANOTHER hour of laying on a bed in pain a doctor finally came and saw me. He told me they were going to run a few tests in a bit. I told him that I might end up vomiting again, to which he placed a garbage bin from the floor on my lap in the bed (no joke) and promptly left for another 30 minutes. A nurse came back and hooked me up to a heart rate monitor and realized I was at 180 bpm. I guess things now really did matter as a doctor promptly gave me multiple injections of morphine. After that, I honestly don't remember much, other than blood being taken, being taken for an xray and a ultrasound (3 stars for the facilities cause at least the elevator didn't break down and the machines actually worked.) Then in sat in the hospital bed for who knows how much longer drugged up on morphine till they finally told me it was a rather large kidney stone causing the pain. They gave me a bottle of Hydromorphone and sent me home at 2 AM and an appointment to go receive another Xray and treatment at St.Paul's Hospital next week. So 7 hours of waiting, 5 hours of being high on morphine and treatment in a hospital where I was placed in a big hallway normally reserved for the drunks that pass out and get carried to hospitals by their friends.
After that I have had 4 more pain attacks throughout the month that were just as severe, but I have chosen to stay home and lay in my bed and suffer through it rather than sit in a waiting room on a plastic chair for 6 hours and suffer there.
Submitted March 20, 2016 | flag
Thank you to the Dr'S and nursing staff in Intensive Care. You took fantastic care of my mother. Great bedside manner by all.☺😙
Submitted March 18, 2016 | flag
Staff were horrible, to busy on the computers or there phones. No direction on what to do. And absolutely no compassion
Submitted Feb. 15, 2016 | flag
Really run down. They are short of staff and medicine. Ask for a certain medicine when you are sick, they may not have it.
Submitted Nov. 13, 2015 | flag
Recently had thyroid surgery at this hospital, best care from nurses I remember ever in the few hospital stays I have had in my life! Thanks Dr G. Caspar Bell, Dr Christian, and nurses at follow up care.
Submitted Nov. 9, 2015 | flag
Extremely poor nursing neurology unit 6th floor no respect for patients or family there were NO SERVICES for family or patient who had severe TBI. NO INFO given to family. Room he was in for 2 months was dirty and point peeling off walls. Emerg and ICU were very good really but went VERY DOWN HILL after being moved to 6th floor
Submitted Oct. 18, 2015 | flag
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Floral Long Sleeve Cropped Blouse
Tops for just in case you decide to go to those plans you said maybe too. This blouse features a square, V neckline, relaxed, cropped silhouette, blouson sleeves, elasticized cuffs, bow tie closure at back, and floral print throughout. Rock with mom short and platform sock boots.
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97% Polyester / 3% Spandex. Model wears a UK size M. Machine Washable

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SEREE Digital Camera Camcorder Full HD 1080P 24.0 Megapixels 4x
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This is a digital camera with 24.0 megapixels and its image has high quality.It is also a camcorder and it has clear video with the resolution of 1080p full HD. It is easy to switch among photo, video and playback by pressing “M” buttom near to the screen. It is also easy to operate for entry-level users of camera and camcorder products. This digital video camera is very small and you can use the strap to take along when you enjoy a holiday or outdoor sports. It comes with one piece of a battery whose capacity is 800mAH in the standard package, which ensures enough working hour for outdoor activities. The 3 inch LCD screen can rotate 180 degree which is useful for selfie and recording group activities. The pictures and videos are suitable to upload to your social media including Youtube, Facebook, Twitter, Instagram and so on. This video camera has 4X digital zoom lens and it can also match 52mm standard wide angel lens, fisheye lens and teleconverter lens. It will meet diversified requirements of photography by connecting with different lenses. These optional lenses should be purchased seperately. The UV lens is used to protect your lens from rain or dust, and at the same time it can filter the UV ray. It is included in standard package. This camcorder has a retractable built-in flashlight which is a unique feature and it can be used to get a broader field of view by popping it up when the vision is limited in a dark environment. It supports anti-shake, face detection, and smile detection. It supports an external SDHC card up to 32GB (Exclude in the standard package).
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You are making me cry!!! PRECIOUS!!! congratulations! Praying for all four of you!!!
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Thanks for taking the time to comment!

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From David Bowie's swan song to Beyonce's call to arms, Anohni's war lament to Frank Ocean's return, 2016 was a year of big artists making big statements. These are our picks for the best songs of the year.
From Drake’s dominance to Adele’s return, Tame Impala’s blissful psych to Kendrick Lamar’s politically charged hip-hop, 2015 marked another diverse and strong year for music. These are our picks for the best songs of the year.
We invited artists to discuss their favorite music of the year—be it a song, album, event, or otherwise. From Holly Herdon’s time in Poland to Fetty Wap’s graciousness over "Trap Queen"’s success, here is Pitchfork’s 2015 Guest List.
After spending years fruitlessly chasing various forms of flawlessness, this London singer created a spontaneous take on funk and soul that dissolves the notion of perfection until it becomes something fluid and beautiful. By Laura Snapes.


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Young Music Magician: Madeon
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Our mental health and substance abuse programs offer a safe and compassionate environment for patients with psychiatric and addiction disorders.Read More
Outpatient services include group, individual and family therapy sessions in which patients can join a community of peers seeking long-term stabilization.Read More
The Assessment and Referral Department at Mayhill Hospital helps people find their way to an appropriate level of mental health or substance abuse treatment.Read More
If you are a clinical professional who wants to refer a patient for mental health or substance abuse treatment at Mayhill Hospital, here is everything you need to know.Read More
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Our medical psychiatry programs address primary acute medical conditions while treating psychiatric co-morbidly from which a patient may be suffering.
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Learn about electroconvulsive therapy (ECT) from Dr. Asad Islam, Mayhill Hospital's Medical Director of Psychiatry.
Watch our video tour to get a look at the comfortable environment and friendly staff members of Mayhill Hospital.

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I wonder if people a hundred years from now will look back and think of Justin Bieber as a seminal American artist. It’s within the realm of possibility, I guess, but I’m just not cynical enough to believe humanity could so grotesquely devolve. But one thing is for sure: The early-20th-century American blues as played by Sheryl Warner and the Southside Homewreckers will never go out of style. Easily the best cover band in the city, guitarist Gregg Kimball, harmonica player Rich Mann and vocalist Sheryl Warner play the tunes of Blind Lemon Jefferson, Memphis Minnie, Tampa Red and Big Bill Broonzy as if they wrote them. Best of all, not knowing the historical significance of their repertoire won’t hinder your enjoyment of the band. Sheryl Warner and the Southside Homewreckers play the appropriately intimate confines of Crossroads Coffee and Ice Cream on Saturday, Sept. 21. The music starts at 8 p.m. Free. crossroadsrva.com.


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By Rebecca R. Stone
Shamanism--the perform of coming into a trance country to adventure visions of a truth past the standard and to achieve esoteric knowledge--has been an incredible a part of existence for indigenous societies through the Americas from prehistoric occasions until eventually the current. a lot has been written approximately shamanism in either scholarly and well known literature, yet few authors have associated it to a different major visible realm--art. during this pioneering research, Rebecca R. Stone considers how deep familiarity with, and profound appreciate for, the extra-ordinary visionary reports of shamanism profoundly affected the inventive output of indigenous cultures in principal and South the US sooner than the eu invasions of the 16th century. utilizing ethnographic bills of shamanic trance reports, Stone defines a center set of trance imaginative and prescient features, together with more suitable senses, ego dissolution, physically distortions, flying, spinning and undulating sensations, synaesthesia, and actual transformation from the human self into animal and different states of being. Stone then lines those visionary features in historical works of art from Costa Rica and Peru. She makes a powerful case that those works, specifically these of the Moche, depict shamans in a trance kingdom otherwise exhibit the perceptual adventure of visions via growing intentionally chaotic and distorted conglomerations of partial, inverted, and incoherent photos.
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71). Don Pablo could speak Quechua only during trance (Weise 2010: minute 20). These experiences proved to them the omniscience of the spirit world. Experiencing other realms as places that predict events Here certainly gives truth and reality, even superiority, to There. Indeed, accurately seeing the future is commonly described, such as a man who saw his not-yet-conceived daughter as a young girl and on another occasion found himself at the cave he had witnessed in visions months before (Metzner 2006: 125–127).
All the colours I have ever beheld are dull as compared to these . . ” (In Klüver 1966: 15–16) After the experience another subject reported “‘such magnificent hues and such singular brilliancy that I cannot even imagine them now’” (21). Many describe saturation so intense it is blinding, even painful, especially as the colors explode. Multiple textures characterize the colors, too, such as “‘fibrous, woven, polished, glowing, dull, veined, semi-transparent’” (25). At the outset of trance, colors can appear as percepts on their own, washing the visual field and changing from one to another, in a prime example of multiplicity and flux.
673); thus, there is a dual consciousness despite the striking nature of the sensory effects. : 662). : 661)—and many become visual artists to communicate their unique vision. Several ancient 24 The Jaguar wiThin vessels, I contend, illustrate synaesthesia directly, as discussed in chapters 7 and 8. Many other visual effects involving multiplicity and flux deserve mention as well. Unusual, multiple, or shifting scale of perceptual phenomena is typical in visions. This dysmegalopsia (seeing things in the wrong size) includes micropsia (seeing things as undersized) and macropsia (seeing things as oversized).

domain: artistic


KILLEEN, Texas (FOX 7 Austin) - One case of measles has been confirmed in Bell County.
According to the Bell County Health District, there were four possible cases under investigation. As of
Wednesday, February 6, three of the four possible cases have been ruled out as measles.
The confirmed case of measles is in a child residing in Western Bell County. The child is not
school aged and was too young to have been vaccinated, according to health officials. The child has had very limited contact with the public and all family members are up to date on vaccinations.
Vaccination remains the most effective method for preventing measles, according to the Bell County Health District. Measles is highly contagious and early identification remains a critical public health measure to reduce the spread of the disease.
Measles (rubeola) is a highly contagious febrile rash illness caused by a virus transmitted via the
respiratory route. The incubation period averages 10-12 days from exposure and it typically takes
about 14 days from exposure to the virus for a rash to develop.
The early symptoms generally last 2-4 days and include fever often peaking at 103°-105°F.
Fever is followed by the onset of cough, runny nose, and/or eye inflammation. Koplik spots, while
not always present, are considered to be a characteristic measles symptom and appear as blue-white
spots in the mouth and throat. This typically occurs 1-2 days before rash to 1-2 days afterwards.
The measles rash is another characteristic symptom that usually begins at the hairline and gradually
proceeds to face and upper neck and from there downward and outward. Other symptoms of
measles include loss of appetite, diarrhea (especially in infants).
Complications can include otitis media, pneumonia, encephalitis, seizures and death.
Children can be vaccinated once they are 12 months old.
Anyone experiencing symptoms of the virus should call their physician.


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An issue that has been at the forefront of comics criticism this year despite the larger websites and indeed publishers themselves ignoring it: racism in comics, and the lack of diversity both on and behind the page.
The Marvel hip-hop variant scheme and Boom! Studios' Strange Fruit have attracted particular attention from critics, but will anything really change?
Adding insult to injury is the at times woeful reporting of these stories – plain disregard from many of the same mainstream news sites that happily broadcast headline-making press releases, and inflammatory coverage within the comics press itself. Dismissive outrage over perceived issues of “political correctness” and “social justice” makes for more clicks and ultimately more money.
A long-form interview with the lovely Howard Hardiman, creator of the recently collected The Lengths - a controversial and important comic about male escorts, told with canine characters.
Howard's answers were simply far too interesting for me to worry about distracting from, so I went with a Q&A format for maximum ease of reading. A very fascinating interview, and a book that I highly recommend reading.
"One of the funniest responses to reading it came from someone working in the sex industry, who said “you let straight people read this?” because he’d become so used to secrecy.
"Other than that, though, the response has been reassuringly positive. I waited almost five years between the interviews and making the book because I needed to have a distance from telling too much that could have identified people, especially given that I’d been told a lot of things that could have resulted in them having trouble with the police or the inland revenue!"
A series of interviews with guests at the Stripped Festival, including Stephen Collins, Paul Cornell and Grant Morrison, looking at the politics both present and absent in their work.
"One of the historical roots of modern comics is of course the political cartooning of the early newspapers; the mechanical reproduction of images finally allowing art to be consumed by the masses rather than the privileged few, with cartoonists leaping at the chance to communicate complex political situations via their deceptively simple form.
"The idea of comics as a political tool is not without its controversies, from grumbles amongst novelists to riots over religious icon portrayals. Any fan of superhero comics can tell you that comics don’t have to be overtly political, but the recent insistence by creator Todd McFarlane that historically no comic book that has worked has been “trying to get across a message” was largely met by the rolling of eyes."
I've been delighted with the feedback on this long-form piece, and the discussions that it has provoked. You can read the full article at the New Statesman.
An issue that had been weighing heavily on my mind, that of the constant tension between our superheroes who seek to represent all that is good in the world, while generating profit for large corporations as their creators often retire into poverty.
"Superman, defender of truth, justice and the American way is an icon not only of hope, but of despair and shattered dreams. Our superheroes, built to help the helpless and downtrodden and bring joy to our world became tools of their corporate owners whose only interest lies in helping themselves. While our heroes can transcend their shackles and still bring us pinpoints of timely optimism, not to mention a great deal of entertainment and joy, their dark shadow of forgotten and poverty-ridden creators weighs upon them greatly."
My four interviews in one piece is up at the New Statesman, and I've had very lovely feedback all round - which is always a Good Thing! All four creators will be at the upcoming London Super Comic Con, and all are especially lovely.
Here they are talking about working in creator owned comics and all that that entails. Enjoy!
You can read my latest interview with the lovely Mike Carey, writer of The Unwritten, Lucifer, X-Men and more, over at the New Statesman. Carey is one of my favourite writers - Lucifer one of my favourite series - and we chat about Vertigo past and future, The Unwritten and upcoming plans, creator rights and women in comics, internet trolls and his Felix Castor novels. And more!
Stay tuned for more upcoming interview with David Lloyd, David Mack, Tim Seeley, David Hine, and the one and only Neal Adams as I count down to the London Super Comic Con next month.


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Digital Activism and Privacy - quick fix or long term involvement?
- 1 Youth working group
- 2 Session title
- 3 Session topics
- 4 Session description
- 5 People
- 6 Format of this working group at EuroDIG
- 7 Further reading
- 8 Live stream / remote participation
- 9 Live transcripts
- 10 Q&A
- 11 Outcomes
- 12 Mailing list
- 13 Pictures from working group
- 14 Session tags
- 15 Session twitter hashtag
- 16 Session progress
Youth working group
Strictly for youth, young people and young grown-ups only. Non-youth is kindly requested to stay outside.
Digital Activism and Privacy - quick fix or long term involvement?
Privacy, mass surveillance, digital activism, civil society participation.
There is a need for stronger multi-stakeholder participation in the political processes of Internet Governance concerning privacy-related issues. However is digital activism really creating these opportunities? Digital activism can be defined as the use of ICTs for a range of forms of activism to facilitate communication among citizens and raise awareness for political issues. Recent debate on new EU Data protection reform showed the need to rethink and adapt. Carried out through a closed community of experts in a non-transparent process these important topic elude the average user, which it affects. Digital activism is one of the ways privacy issues are approached and addressed to date. However today's representation of actors and agents of digital activism is very imbalanced, regionally disproportional and creating a gender gap. Moreover participation of civil society on the web in IG, its work and methods are questionable, not institutionalised and often uncoordinated. Hence,the democratic quality of digital activism, characterized by its level of legitimacy, representativeness, accountability, and inclusiveness, needs to be assessed and problems associated in this context, such as the digital divide and a lack of institutionalization should be discussed.
Questions to be addressed: What is the added value of digital activism for the young people? Can problems of youth participation in IG be addressed through similar innovative processes of participation, or does it just relieve young people's need to express themselves whilst the decisions are made elsewhere.Can digital activism bridge the gap between classic particiaption and technical community? What is the impact of digital youth activism on the lives of young people, taking into account that the net keeps record on everything? What is the value of anonymous activism?
TL;DR: This panel will look into how digital activism influences privacy issues. Whether it is really working as an instrument to change digital policy debate. What are the mechanisms of digital activism and how relevant is that for young people? Moreover is it an effective way to go to influence European politics on Internet Governance?
- Focal points:
- Nadine Karbach, IJAB e.V./Youth IGF-D
- Lorena Jaume-Palasi, Ludwig Maximilians University/IGF-D Youth forum
- Live moderator: tbd
- Rapporteur: tbd
- Remote participation moderator: Narine Khachatryan
- Digital facilitator: Martin
- Panelists/speakers: (suggested list of panelist/speakers)
- Anya Orlova, Network of European Digital Youth
- Paula Roth, New Media Summer School
- Rabea Willers, Youth Representative, Directorate for Democratic Governance, Council of Europe
- Katitza Rodriguez, Director Electronic Frontier Foundation (EFF)
Format of this working group at EuroDIG
A plenary with focus on remote participation and supported by digital debates We aim to develop an Activist Manifesto-Developed in parallel. In permanent beta (like the structure made in NETMundial) - Outcome document..."principles on privacy in Europe (?)" Questions will be collected primarily via Twitter and the created online discussion.
Live stream / remote participation
Q1: How has activism changed, or not, in an internet embedded area for action?
Anya: Digitals tools are very supportive. Digital tools gives you bigger access to all necessary things you need for organizing an action.
Q2: What’s the difference between digital activism and activism?
Difference between digital activism and activism is that with digital tools it’s easier to mobilise people in a shorter time frame. Internet has made it possible to create new innovative tools for connecting citizens to politicians. For instance - crowdsourcing for drafting bills/initiatives. Digital activism might be a better option if you want to organize a big demonstration. Digital activism does not only put privacy on the political agenda but it’s also privacy of digital activism is protected. This movement of digital activism can grow.
Q3: What about Slacktivism?
Q4: What specific issues/concerns of privacy are we looking for? Authorities know who you are anyway.
A: When you are activist you are giving away your privacy. But that doesn’t mean that you have not to protect your privacy/anonymity. However, tools like “Tor” can be used by activists to protect yourself from prosecution. Only because you want to change it doesn’t mean that you have to become a public figure. Everything that we do offline is the same as doing online. Therefore, legislation has to protect you in any case. Same rules should apply for both areas.
Q5: Is there any legislation on crowdsourcing?
A: There is an attempt of this in Finland and the Ministry is involved too. Law has been drafted and passed to ministry, it’s still in the process of adoption.
Q6: Any legislation on that at EU level?
Q7: Is privacy a form of activism?
Q8: How can we make that my previous generation can understand what we are talking about?
Q9: What about Slacktivism that is putting less value on digital activism is the majority of our activism slacktivism?
Q10: Which digital tools are activists using the most? Are there choices or only one we tend to use the most?
A: Different, but Twitter and Facebook is dominating. However, media or how we call it printed/traditional media is also powerful. Therefore we shouldn’t forget the traditional media as a tool of activism.
A: maybe we shouldn’t be afraid of facebook and just change the rules. Maybe facebook won't belong to facebook and it will be everyones.
Q11: Is twitter activism real? Where is the line between slacktivism and clicktivism and real activism (both - digital and non-digital)?
A: Twitter activism is indeed real. People support each other when they are abused/bullied because you can find someone like you and help each other.
A: About slacktivism. We (Katizia) get a lot of signatures on one (of our) petition. Government should not ignore that fact. Such digital activism can indeed mobilize people for making change.
A: Signing petitions and other digital activisms are a good way for raising awareness. Even clicking a link is informing you.
Q12: Shape the meaning. I can send someone some links but how can I really want to make sure that you’ve been informed?
Q13: What about buying clicks, buying friends on internet? How can we make sure that they are real?
Q14: What if we are engaging only those who are already active offline. How do we engage those who are from different social group/backgrounds?
EuroDIG Youth Panel Manifesto (in progress)
Final report from working groupPDF (approx. 2 weeks after event)
youth-working-group (add) collaboratory.de
Pictures from working group
Privacy, Digital Activism, Youth
Session twitter hashtag
The youth working group ran a survey among all participating individuals and organizations to narrow down the most popular topics for a session. Results of the survey. The youth working group has set a participation tool called Ypart to discuss easily with others about session subject and format.
A mailing list with the most active people on the ypart was set up to finalize the session outline. The drafting progress is handled collaboratively on a pad.

domain: sociology


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HOORAH! I managed to get 75 favourites for a picture *squee* I'm rather happy about that number.
In the past year or so, quite a lot has happened. I am now in my final year of GCSE's (English exams) and have lots of revision and coursework to do. Unsurprisingly, a lot of that is my art coursework. I have improved a lot and have started doing portraits of various people that inspire me with their beautifullness.
Recently, I have been looking a lot more into fashion designing as well as portraits so I have created another account to upload those onto. I plan to keep the two accounts very separate, so I will not disclose what that account is called. It just means that I can keep all my lovely HP, HG and other FANTABULOUSLY brilliant stuff over here.
So that's my update if anyone cares to read it. 75 favourites today


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A card game created by Moore alumna Brigette Indelicato is a finalist in Hasbro’s Gaming Lab Challenge.
Her game, “The Plot Thickens,” was chosen as one of five finalists from a field of more than 600 in Hasbro’s Spring 2016 contest. Brigette, a 2010 Graphic Design grad, and game co-founders and art school grads Thomas Rochelle and Mike Callahan have a shot at a $25,000 grand prize and a chance to work with Hasbro on developing the game.
"I did all the graphic design for the game,” said Brigette, who works as a graphic designer for The Franklin Institute science museum in Philadelphia. “We’ve been developing and polishing it for the last two years.”
Although Moore now has an Animation & Game Arts major, the program was created after Brigette had graduated.
"I hadn't ever expected to get involved in game design, but I love the challenge of designing something that people will so actively interact with," she said.
“The Plot Thickens” is a storytelling game. The object is for players to take a hand of five story cards featuring people, places and things and lay out the tale of a character and their fate. Players earns ‘plot points’ for every card used to build a story, as well as each time they interweave with another player’s story.
“There are two winners at the end: the storyteller with the most plot points on other players' cards gets to end the story however they want, and the person with the most connections to their own cards gets to come up with the title for the story,” she said.
Brigette, who describes herself and her friends as ‘big game nerds,’ says she and her partners came up with “The Plot Thickens” because they wanted a more collaborative experience.
“We had played some other storytelling-type games, but a lot of them are competitive, and you don’t end up telling a good story when you get interrupted,” she said. “’The Plot Thickens’ is a social and easy-to-learn game, so it can appeal to all different kinds of players. Families and kids can enjoy it as well.”
There are multiple editions of the game – Dectective Noir, Sci-Fi, and Romance – and they can be mixed together.
“Are you the beat cop, who just stumbles upon a twisted story of murder and intrigue?” reads the card-game literature. “Or a top-notch fighter pilot out in the far reaches of the galaxy? Or … are you both?”
The game founders have started an Indiegogo campaign to raise $8,000 to help fund production, and people can purchase a copy of the game through Indiegogo.
“We need to get 100 backers by the end of the campaign (November 12) to be eligible to win the Hasbro contest,” Brigette said.
She’s optimistic about the game’s success.
“It's really exciting, pretty much everyone who has played it thinks it’s a blast,” she said.


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Like all Japanese cloisonné of the early Meiji period, Namikawa Yasuyuki's first work owed much to Chinese decoration. Illustrations were generally of standard motifs set in panels, each thus forming a two-dimensional image, with the space between filled with scrolling or geometric designs. The feet and shoulders of vases and such vessels would be decorated with bands of regular lappets reminiscent of ancient Chinese bronzes, and the early enamels had the colour and consistency of Chinese cloisonné. But this was to change with the rapid development of Japanese cloisonné in the 1870s and 80s, and the work of Namikawa Yasuyuki (1845-1927) in particular exemplifies its highest artistic and technical development. Around 1878 or 1879 Namikawa met the German chemist Gottfried Wagener (1831-1892) with whose knowledge of ceramic pigmentation he was able to develop and refine his glazes in colour and texture to make both transparent and opaque glazes of faultless clarity. He took great care over his compositions and varied the standard motifs so that each piece was unique and with its own charm and character. Many of his early designs were drawn by the designer Nakahara Tessen, who recorded them in his Kyo Shippo Monyo Shu [Kyoto Cloisonné Pattern Collection].
Namikawa won prizes at the Philadelphia World Fair of 1876, and then at the Paris World Fair of 1878, and later at the 1889 Paris Fair. He was also honoured at the series of National Industrial Expositions which was instituted in 1877. He won altogether 31 prizes at expositions both at home and abroad. In 1896 together with the unrelated Namikawa Sosuke, Yasuyuki was appointed as a Teishitsu Gigeiin, or 'Imperial Artist', the only two cloisonné makers to be so honoured.
This pair of Imperial Presentation Vases represent the finest work of the artist, and are rare examples bearing the Imperial mon, indicating that they were commissioned by the Imperial Household for presentation to a Japanese or foreign dignitary. The subject matter of dragons and phoenixes are Chinese auspicious emblems which were fashionable in Japan from the Nara Period (1710-1784) when Chinese philosophies, fashions, and government systems were being assimilated under the rule of Emperor ancestors of the Emperor Meiji.
Although Yasuyuki's style was to become more pictorial, for which he was praised by the judges at the 4th National Industrial Exhibition of 1895, and to develop further (see Lot 151) after the Paris exhibition of 1900 when the traditional Japanese designs were condemned as unimaginative, he seems to have maintained the use of formal panels with classic designs for some of his most monumental work, especially the Imperial Presentation pieces. But there is nothing unimaginative about his formal work. The profiles of the panels and lappets on these vases, while retaining something of the archaic Chinese style, are in fact quite gentle and sit well with the overall shape of the vessels. The scrolling with chrysanthemums, quite fitting on Imperial pieces, seems to dance around and over the edges of the panels adding a further dimension and softening any impression that the phoenixes and dragons are imprisoned. It is interesting to compare this pair of vases with the well-documented pieces in the Khalili Collection1 which is illustrated with free bird and flower designs and chrysanthemums as if floating on stylized water between the panels, in intimation of Yasuyuki's Imperial connection. One can imagine Yasuyuki, as an Imperial Artist, smarting under the slight of the foreign criticism and determining to preserve the integrity of his well-established style. Of course this is just conjecture, and we cannot be absolutely sure of the dates of the vases, and must be content with recognizing their timeless beauty in an age when such work seems far beyond human capacity.
Although much of Yasuyuki's work is small and delicately worked with silver wires, pieces commissioned by the Imperial Household like this magnificent pair of flower vases were usually around 30cm or so in height.
An inscription on the box states that they are flower vases, which is why there are no lids, and that they were for presentation. The vases are accompanied by a letter dated 1974 and written by a lady, Morimoto Fumiko, indicating that they were presented by the Imperial Household to the house of Okuma Shigenobu (1838-1922), and then given to the house of a Morimoto Mitsuo. Although to date it has not been possible to substantiate this provenance further, the history is interesting. Recent information from the Imperial Household Agency confirms that the Emperor did indeed give Okuma a pair of cloisonné vases, although there is no record of the maker nor any description of the actual pieces. Okuma had lost a leg as the result of an assassin's bomb in 1889, and the Emperor Meiji visited him during his convalescence in May of the following year. It was then that the presentation was made. Later to be ennobled as a Marquis, Okuma was prominent in the negotiations leading to the Imperial Restoration of 1868. He was made Head of Foreign Affairs in that year, Minister of Finance between 1870 and 1881, then Prime Minister briefly during 1908 and again between 1914 and 1916. He is particularly venerated as the founder of Waseda University in Tokyo. At the time of the Vienna Exposition in 1873 Okuma was overseer for the Japanese exhibit when Gottfried Wagener was a special advisor. In view of the later relationship between Yasuyuki and Wagener it is not improbable that Okuma would have met the cloisonné artist Namikawa at some time. As perhaps the most revered public figure of his time Marquis Okuma would have been an obvious recipient of an Imperial Gift. There can be little doubt that these are the very cloisonné vases which the Emperor presented to the national hero Okuma Shigenobu in 1889.
1(published Treasures of Imperial Japan etc etc….., and 'Japanese Imperial Craftsmen, British Musuem Publications 1994)


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Ron Paul has always been in favor of cutting out the middleman, so maybe this makes sense? The famously libertarian Texas congressman is selling his own house—via Facebook. The note attached to the ad says “generations of liberty-loving kids have grown up here, and you can continue the tradition.” Paul’s son, Kentucky Rep. Rand Paul, was raised there along with his four siblings. Paul’s spokesman said the congressman and his wife, Carol, had recently built a bigger house. Thanksgiving dinner last year included 42 people—their five children, 18 grandchildren, four great-grandchildren, and spouses. As for the house currently for sale, it’s four bedrooms, five bathrooms, has a swimming pool, and can be yours for $325,000.
TOP 10 RIGHT NOW

domain: news


Lauded Australian outfit Alpine is set to support Empire of the Sun on a U.S. run this fall. Since forming, Alpine have toured extensively with the likes of Grouplove, Kimbra, The Naked And Famous and Matt Corby. Additionally, the band performed at SXSW 2013 and has appeared at various festivals worldwide including Splendour In The Grass, Southbound and Falls Festival.
A Is For Alpine, the debut album from the band is available now on Votiv. The new release, which was chosen as iTunes' Australian Alternative Album of the Year, has received early critical praise from Rolling Stone and USA Today with Pitchfork calling it an "unforgettably light and charismatic gem." The album recently streamed in its entirety on NYTimes.com and the latest video for the track "Gasoline" premiered on NPR.org as a "First Watch":http://n.pr/10iXPtG.
Alpine is Phoebe Baker and Lou James (dual vocals), Christian O'Brien (guitar), Ryan Lamb (bass), Tim Royall (keys) and Phil Tucker (drums). The new album was recorded in Melbourne with producer Dann Hume who helped the band experiment with sounds and textures. Of the record Lamb notes, "We wanted to represent the different sides of our music, so there's some sad moments, some dancey moments, some rocking out and lots of harmonies." No strangers to the benefits of shared ideas and the collaborative process, Alpine's own methods for song writing reflect the strong and respectful working relationship they had with Hume. Baker describes the new album as the band's "innocent and not so innocent explorations through our twenty-something emotions."
ALPINE TOUR DATES
September 1-2 Washington, DC 9:30 Club*
September 5 New York, NY Pier*
September 7 Boston, MA House of Blues*
September 9 Toronto, ON Danforth Music Hall*
September 12 Chicago, IL Aragon Ballroom*
September 13 Detroit, MI Fillmore*
September 16 Minneapolis, MN State Theatre*
September 18 Atlanta, GA Tabernacle*
September 19 Nashville, TN 12
*with Empire of the Sun
Corgan took to Instagram to confirm rumours of new Pumpkins material, saying the first songs could arrive as early as May.


Business Traveller has obtained the seating layout for KLM’s new fully-flat business class, which will be fitted to the carrier’s B747-400 aircraft from this summer.
Earlier this week we revealed images and details of the new seat (see online news March 19), and can now confirm the configuration for the new product across the two decks of the B747 jumbo.
On the upper deck the number of business class seats will be reduced from 24 to 20 seats, with four sets of two seats on the left hand side of the aircraft, and six sets of two on the right hand side.
On the lower deck the seat density will decrease from the current 18 seats down to just 15, with three new ‘solo’ seats being created in 1A, 4A and 4E.
In total the number of business class seats will be reduced from the current 42 down to 35, and the bed length will increase from the current 1.9 metres (angled lie-flat) to 2.07 metres (fully-flat).
The IFE screen will also increase in size from 10.4 inches (26.4 cm) to 17 inches (43.2 cm).
Here are the two layouts for comparison – we’ll get the new configuration drawn up and added to KLM’s seatplans section as soon as we can.