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PHYSIOLOGY AND METABOLISM
Escherichia coli phnN, Encoding Ribose 1,5-Bisphosphokinase Activity (Phosphoribosyl Diphosphate Forming): Dual Role in Phosphonate Degradation and NAD Biosynthesis Pathways
Bjarne Hove-Jensen, Tina J. Rosenkrantz, Andreas Haldimann, Barry L. Wanner
Bjarne Hove-Jensen
Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark
For correspondence: hove@mermaid.molbio.ku.dk
Tina J. Rosenkrantz
Andreas Haldimann
Department of Biology, Purdue University, West Lafayette, Indiana 47907
Barry L. Wanner
DOI: 10.1128/JB.185.9.2793-2801.2003
An enzymatic pathway for synthesis of 5-phospho-d-ribosyl α-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate → ribose 1-phosphate → ribose 1,5-bisphosphate → PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + Pi → guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C—P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5′-XMP, and by cochromatography of the reaction product with authentic PRPP.
NAD biosynthesis in Escherichia coli usually proceeds by consumption of 5-phospho-d-ribosyl α-1-diphosphate (PRPP). NAD is synthesized from aspartate and dihydroxyacetone phosphate. A de novo pathway and a number of salvage pathways for the reutilization of nicotinamide mononucleotide and nicotinamide exist, as shown in Fig. 1 (32). Two of the enzymatic reactions, the reactions catalyzed by quinolinate and nicotinate phosphoribosyltransferases, require PRPP. PRPP-less mutants with a deletion of the prs gene, encoding PRPP synthase, consequently require NAD or nicotinamide mononucleotide. Δprs strains also require guanosine, uridine, histidine, and tryptophan, which are likewise synthesized with PRPP as an intermediate (14, 15). Nevertheless, mutants that suppress the NAD requirement are easily obtained by selecting for growth of Δprs cells on medium lacking NAD. These mutants still require guanosine, uridine, histidine, and tryptophan. All such NAD-suppressed mutants were previously shown to have lesions in the pst-phoU operon (17), which leads to high-level constitutive expression of genes belonging to the phosphate (Pho) regulon (39, 40). Furthermore, pst, phoU, or phoR also suppressed the NAD requirement of Δprs strains. Suppression by pst, phoU, or phoR mutations was eliminated by a phoB mutation, while suppression by a phoR mutation was also eliminated by a creC mutation. These data show that synthesis of a Pho regulon gene product is apparently responsible for suppression of the NAD requirement (17).
NAD metabolism of E. coli. The de novo and salvage pathways are shown. Abbreviations: Asp, aspartate; DHAP, dihydroxyacetone phosphate; Gln, glutamine; Glu, glutamate; NA, nicotinate; NAm, nicotinamide; NAMN, nicotinate mononucleotide; NAAD; nicotinate adenine dinucleotide; NMN, nicotinamide mononucleotide; QA, quinolinate; Rib-5P, ribose 5-phosphate. Relevant enzyme-catalyzed reactions are indicated by gene designations (nadA, quinolinate synthase; nadC, quinolinate phosphoribosyltransferase; pncB, nicotinate phosphoribosyltransferase).
We show here that suppression of the NAD requirement requires at least three polypeptides, one of which is specified by the phnN gene. The phnN gene lies within a 14-cistron operon, phnCDEFGHIJKLMNOP, which encodes proteins for uptake, breakdown, and regulation of phosphonate degradation by carbon-phosphorus (C-P) lyase (7, 29, 39). In this paper we describe the first detection of an in vitro activity associated with a component of a C-P lyase.
Bacterial strains and growth conditions.The E. coli K-12 strains used are listed in Table 1. Cells were grown in AB minimal medium (8) or in NZY medium (containing [per liter] 10 g of NZ-amin [Struers, Copenhagen, Denmark], 5 g of yeast extract [Oxoid], and 5 g of NaCl; pH adjusted to 7.5) (18). Carbon sources were used at a concentration of 0.2% (glucose, galactose, melibiose) or 0.1% (thymidine). Thiamine (1 mg/liter) was routinely added to minimal media. The following other compounds were added when necessary: guanosine (30 mg/liter), uridine (20 mg/liter), and amino acids and δ-aminolevulinate (40 mg/liter each). NAD was added to both NZY and AB media at a concentration of 25 mg/liter. Ampicillin was used at a concentration of 25 or 100 mg/liter, whereas kanamycin and tetracycline were used at concentrations of 30 and 10 mg/liter, respectively. Cell growth was monitored by determining the optical density at 436 nm (OD436) with an Eppendorf PCP6121 spectrophotometer. An OD436 of 1 (path length, 1 cm) corresponds to approximately 3 × 1011 cells per liter.
Genetic techniques.Previously described procedures were used for transduction (30) and transformation (26). Gene conversion by homologous recombination was performed by using a recD strain (35). Integration of pAH126 at attλ was performed as described elsewhere (10).
DNA technology and plasmids.Plasmid DNA was isolated by the procedure of Birnboim and Doly (4) or by commercial procedures (Qiagen). Restriction and ligation of DNA were performed as described by the vendors of restriction endonucleases (Amersham, Promega, and Roche) or T4 DNA ligase (Amersham). The plasmids containing deo cistrons were constructed as follows. Plasmid pBRdeo, provided by J. Neuhard (University of Copenhagen), consists of the entire deo operon (deoC+ deoA+ deoB+ deoD+) as a 5,660-bp EcoRI-PvuII-digested DNA fragment ligated to similarly digested DNA of pBR322 (5). To delete deoD, DNA of pBRdeo was digested by restriction endonucleases PvuII and HpaI, and this was followed by ligation, resulting in pHO365 (deoC+ deoA+ deoB+). To delete deoB and deoD, DNA of pBRdeo was digested by restriction endonucleases PvuII and HindIII, and this was followed by S1 nuclease treatment and ligation, resulting in pHO366 (deoC+ deoA+). To delete deoA, deoB, and deoD, DNA of pBRdeo was digested by restriction endonucleases PvuII and NcoI, and this was followed by S1 nuclease treatment and ligation, resulting in pHO367 (deoC+). The deo operon is expressed from the deoP2 promoter in these plasmids (37). To construct a plasmid containing only deoB, DNA of pBRdeo was digested by restriction endonuclease BclI. This treatment liberated a 1,551-bp DNA fragment containing deoB, as well as four other DNA fragments. This DNA was ligated to BamHI-digested DNA of pBR322. Recombinant plasmids containing deoB+ were obtained by transformation of the ligated DNA to strain HO1077 (deoB) and selection for deoB+ on medium with thymidine as the carbon source. The resulting plasmid, which presumably expressed deoB from the tet promoter, was pHO368 (deoB+). To replace deoB and deoD with a kanamycin resistance-encoding DNA fragment, DNA of pBRdeo was digested by restriction endonuclease HindIII, and this was followed by S1 nuclease treatment and digestion with HpaI. This DNA was ligated to DNA of pUC4-K (44) previously digested with HindII, which liberated the kanamycin resistance-encoding DNA fragment, as well as two other DNA fragments. The desired plasmid was obtained by transformation of the ligated DNA to strain HO1183 (deoA) and selection for deoA+ on medium containing kanamycin and thymidine as the carbon source. This procedure resulted in pHO370 (deoC+ deoA+ ΔdeoBD::Kanr). This plasmid contained approximately 1,000 bp downstream of the HpaI site to allow for homologous recombination.
Plasmids containing phnN were constructed as follows. Wild-type phnN was PCR amplified by using the oligodeoxyribonucleotides 5′-GAAGATCTCAT-ATGATGGGAAAAACTGATTTGG and 5′-GCGAGCTCT-ACAAGCAGGCATGGTGTTTC as the primers, DNA of pBW120 (41) as the template, the four deoxyribonucleotides, and Vent DNA polymerase (New England Biolabs) (the nucleotides preceding hyphens indicate noncomplementary extensions). The PCR product was cloned into pSK50-ΔuidA2 (9) by using NdeI and SacI, resulting in pCP-phnN. The insert was verified by automated DNA sequencing of both strands. The phnN gene was then subcloned into the conditional-replication and modular plasmid pCAH56 (10) by using NdeI and NheI, resulting in pAH126. This plasmid contained phnN under control of Ptac and could be readily integrated into the chromosome. In addition, phnN was amplified with the oligodeoxyribonucleotides 5′-GAGAATTCATTAAAGAGGAGAAATTAACT-ATGATGGGAAAACTGATTTGGTTAATGG (phnNupUHE) and 5′-TGGTTGGGATCCCGAGCCATGGTTATTACAAG-CAGGCATGGTGTTTCTCC as the primers, DNA of pAH126 as the template, the four deoxyribonucleotides, and DNA polymerase of Pyrococcus furiosum (Gibco-BRL). The resulting DNA was digested by restriction endonucleases EcoRI and NcoI, ligated to similarly digested DNA of pUHE23-2 (H. Bujard, personal communication), and transformed to strain MC1061, resulting in pHO500. A variant of phnN, specifying a polypeptide with a six-histidine tail at the carboxy-terminal end, was constructed by PCR with the oligodeoxyribonucleotides phnNupUHE and 5′-TGGTTGGGATCCCGAGCCATGGTTATTAATGGTGATGGTGATGGTGCAAG-CAGGCATGGTGTTTCTCC as the primers and DNA of pAH126 as the template, and this was followed by EcoRI and NcoI digestion, ligation, and transformation as described above for pHO500. The resulting plasmid was designated pTR553. The correct inserts of the plasmids were confirmed by automated nucleotide sequencing. Plasmid pGD248, a derivative of pSU18 (3), harbors a lac promoter-expressed xapAB operon, which encodes xanthosine phosphorylase and xanthosine permease (G. Dandanell, personal communication).
Expression of recombinant phnN and purification of histidine-tailed ribose 1,5-bisphosphokinase.Strain HO1088 (Δprs)/pTR553 (phnN) was grown in NZY broth supplemented with ampicillin and tetracycline. At an OD436 of approximately 1, isopropyl-1-thio-β-d-galactopyranoside (IPTG) was added at a concentration of 0.5 mM to induce expression of the phnN gene. After 2 h of incubation cells were harvested by centrifugation, washed with unsupplemented AB minimal medium, and broken by ultrasonic treatment. Debris was removed by centrifugation, and the supernatant fluid was applied to a Co2+-containing Talon metal affinity resin. After the column was washed, ribose 1,5-bisphosphokinase was eluted with imidazole as described by the resin supplier (Clontech).
Enzyme assays.The activity of ribose 1,5-bisphosphokinase was assayed as follows. A 100-μl reaction mixture contained 50 mM Tris-HCl (pH 7.5), 0.6 mM ribose 1,5-bisphosphate, 1 mM (120-GBq/mol) [γ-32P]ATP, and 2 mM magnesium chloride. The reaction was initiated by the addition of ribose 1,5-bisphosphokinase (0.8 nmol/min). Samples (10 μl) were removed after 1, 3, and 10 min of incubation at 30°C and mixed with 5 μl of 0.33 M formic acid. This 15 μl was applied to polyethyleneimine-coated cellulose thin-layer chromatographic plates (Baker-flex; J. T. Baker). After drying, the chromatogram was developed in 0.85 M KH2PO4 which had been previously adjusted to pH 3.4 with 0.85 M H3PO4 (20). Radioactivity was quantitated with an Instant Imager (Packard). Occasionally, 1 mM (120-GBq/mol) [α-32P]ATP or 1 mM (120-GBq/mol) [α-32P]GTP was used instead of [γ-32P]ATP. Production of PRPP with purified E. coli PRPP synthase was performed as previously described (42). When unlabeled ribose 1,5-bisphosphokinase reaction product or PRPP synthase reaction product was synthesized, the same procedures were employed, except that the labeled nucleotide was omitted, and heating at 90°C for 1 min terminated the reaction. Consumption of labeled ribose 1,5-bisphosphokinase reaction product or PRPP synthase reaction product by xanthine phosphoribosyltransferase (XPRTase) was performed as follows. A 50-μl reaction mixture contained 40 mM Tris-HCl (pH 7.5), 0.1 mM xanthine, 32P-labeled ribose 1,5-bisphosphokinase reaction product or 32P-labeled PRPP (approximately 0.1 kBq), 10 mM magnesium chloride, and XPRTase (30 nmol/min). A 10-μl sample was removed after 10 min of incubation at 37°C, mixed with formic acid, and examined by thin-layer chromatography as described above. The formation of 5′-XMP with unlabeled ribose 1,5-bisphosphokinase reaction product or PRPP synthase reaction product was assayed by a similar procedure. A 50-μl reaction mixture contained 40 mM Tris-HCl (pH 7.5), 0.1 mM (185-GBq/mol) xanthine, unlabeled ribose 1,5-bisphosphokinase reaction product or PRPP synthase reaction product (the same volume as described above), 10 mM magnesium chloride, and XPRTase (30 nmol/min). A 10-μl sample was removed after 10 min of incubation at 37°C and applied to a polyethyleneimine thin-layer plate. After drying, the chromatogram was developed in methanol (2 cm), followed by 1 M acetic acid (2 cm) and 0.9 M acetic acid-0.3 M lithium chloride (16 cm) (16, 34). Protein content was determined as previously described (23).
Materials.[α-32P]ATP (15 TBq/mmol) and [α-32P]GTP (110 TBq/mmol) were obtained from Amersham-Pharmacia Biotech. [γ-32P]ATP was prepared as previously described (20). [8-14C]xanthine was obtained from New England Nuclear. Unlabeled nucleotides were obtained from Roche. Xanthine, adenine, ribose, ribose 1-phosphate, ribose 5-phosphate, and PRPP were obtained from Sigma. Ribose 1,5-bisphosphate was supplied by H. Klenow (University of Copenhagen) (22). Purified recombinant Bacillus subtilis XPRTase was supplied by K. F. Jensen (University of Copenhagen).
Genes necessary for the NAD suppression phenotype.It has been shown previously that the NAD suppression phenotype requires a wild-type allele of the nadC gene, which encodes quinolinate phosphoribosyltransferase. Similarly, utilization of nicotinate by cells with the NAD suppression phenotype requires a wild-type allele of the pncB gene, which encodes nicotinate phosphoribosyltransferase (17).
Involvement of deoB.In general, conversion of purine or pyrimidine bases to the corresponding ribonucleoside 5′-monophosphates may occur by either of two pathways: (i) by phosphoribosylation or (ii) by ribosylation followed by phosphorylation. The former reaction, catalyzed by base-specific phosphoribosyltransferases, requires PRPP. The latter reaction occurs, with guanine as an example, as follows: guanine + ribose 1-phosphate → guanosine + Pi. The latter reaction is catalyzed by purine nucleoside phosphorylase. In a second reaction, guanosine is phosphorylated to GMP by guanosine kinase. Ribose 1-phosphate may be generated from ribose 5-phosphate in an isomerization reaction catalyzed by phosphoribomutase, which is encoded by the deoB gene. To test if the NAD suppression phenotype requires phosphoribomutase, several mutant strains and plasmids were constructed (Table 2). First, removal of the entire deo operon resulted in generation of an NAD requirement (compare strains HO861 and HO967). Although the extent of the deo deletion is unknown, introduction of a Δ(deoD-thr) allele (strain HO1156), a deoC allele (HO1359), or a deoA allele (HO1187) had no effect on the NAD phenotype, as these strains, like HO861, were NAD prototrophs. In contrast, the deoB strain HO1949 was an NAD auxotroph, indicating that deoB is required for the NAD suppression phenotype. Second, plasmids bearing various deo cistrons were introduced into strain HO967 [Δdeo(CABD)]. These results showed that either deoB or deoA restored the NAD suppression phenotype to strain HO967 (compare strains HO967, HO967/pHO365, HO967/pHO366, and HO967/pHO368). The phosphoribomutase (deoB) may provide ribose 1-phosphate by isomerization of ribose 5-phosphate. Thymidine phosphorylase (deoA) may also provide ribose 1-phosphate, although by a different mechanism, namely, by phosphorolysis of uridine: uridine + Pi → uracil + ribose 1-phosphate. It has been shown that thymidine phosphorylase has specificity for uridine (11). Finally, we showed that xanthosine phosphorylase (xapA) was able to restore the NAD suppression phenotype (compare strains HO967/pSU18 and HO967/pGD248). Xanthosine phosphorylase has specificity for guanosine, as well as for xanthosine, and it catalyzes the following reaction: guanosine + Pi → guanine + ribose 1-phosphate. Both uridine and guanosine were present in the growth medium, and the strains are wild type for deoA and xapA in the chromosome. However, apparently due to the low affinity of thymidine phosphorylase towards uridine, the deoA gene must be present in a high-copy-number plasmid to have an effect on the NAD suppression phenotype. While xanthosine phosphorylase is normally synthesized only in the presence of xanthosine (12), xapA gene expression is independent of xanthosine in pGD248, in which it is behind a lac promoter.
Effect of deo alleles on NAD suppression phenotypea
Involvement of phnN.The NAD suppression phenotype results from Pho regulon-controlling mutations (17). We therefore sought to identify which component of the Pho regulon is responsible for the suppression phenotype. First, several mutant phosphate starvation-inducible (psi) alleles were introduced into strain HO861, including psiC, psiD, psiE, and psiF alleles. One of these, psiD31, restored the NAD requirement (Table 3). The others had no effect. The psiD31 allele (also called phnD31) is a polar insertion at the phn locus (27), encoding enzymes involved in degradation of phosphonates (7, 41). As expected, deletion of the phn operon also restored the NAD requirement (strain HO1655). We therefore tested null alleles of every phn cistron, most of which are nonpolar (28), in the NAD suppression strain. Introduction of phnM28 or phnN45 eliminated suppression; i.e., both the Δprs pst-phoU phnM and Δprs pst-phoU phnN strains were NAD auxotrophs (Table 3). Among the entire collection of phn mutations analyzed, the phnM mutation was the only polar mutation. We therefore inferred that the effect of phnM was due to polarity on phnN. Furthermore, a mutation eliminating the phosphonate transport system (ΔphnCDE) had no effect on the NAD suppression phenotype (strain HO1496). This was also the case for a mutation in phnF (HO1565) (Table 3), mutations in phnGHIJKL, and mutations in phnO and phnP (data not shown).
Effect of phn mutations on NAD suppression phenotypea
To show that phnN was sufficient for establishing the NAD suppression phenotype, phnN was cloned into a plasmid (pAH126), and a single copy of this plasmid was subsequently integrated into the chromosome of the NAD auxotrophic strain HO1655 (Δprs pst-phoU Δphn). Introduction of phnN alone (strain HO1878) resulted in NAD prototrophy, whereas the vector had no effect (HO1877). Similarly, insertion of phnN into the NAD auxotrophic strain HO773 (Δprs [pst-phoU+]) resulted in NAD prototrophy. As expected, introduction of a mutant nad allele resulted in a pyridine requirement that could be satisfied by quinolinate or nicotinate in addition to NAD, whereas introduction of a Δdeo(CABD) allele resulted in a pyridine requirement that could be satisfied only by NAD (Table 3). Finally, a plasmid-harbored phnN allele, either a wild-type allele (pHO500) or a mutant allele specifying a histidine-tailed version of PhnN (pTR553), also suppressed the NAD requirement.
We conclude that the NAD suppression phenotype is caused by derepression of expression of the phn operon, which results in synthesis of the phnN gene product. This gene product, together with quinolinate phosphoribosyltransferase and phosphoribomutase, is necessary for the NAD suppression phenotype. The latter two enzymes were also present in the unsuppressed parental strain.
Reaction catalyzed by the phnN gene product.The ribose moiety of ribose 1-phosphate formed by phosphoribomutase eventually ends up as the ribose moiety of nicotinate mononucleotide (NAMN) in reactions which are catalyzed by the phnN gene product and by quinolinate phosphoribosyltransferase. One or more additional enzymes might be involved as well. We therefore considered the possibility that phnN encodes an enzyme with ribose 1-phosphokinase activity (ribose 1-phosphate + ATP → ribose 1,5-bisphosphate + ADP) or with ribose 1,5-bisphosphokinase activity (ribose 1,5-bisphosphate + ATP → PRPP + ADP). To do this, the phnN gene was altered to specify a version with a histidine tail, and this variant protein was purified as described in Materials and Methods. The activity of the enzyme was assayed by incubation with the ribose compounds of interest in the presence of [γ-32P]ATP, followed by separation of the reaction mixture by thin-layer chromatography and finally by identification of novel radioactive compounds. Indeed, incubation of PhnN with [γ-32P]ATP and ribose 1,5-bisphosphate resulted in the formation of a radioactive compound, which comigrated with PRPP in one-dimensional thin-layer chromatography (Fig. 2A). This compound also comigrated with PRPP in two-dimensional thin-layer chromatography (data not shown). In contrast, no novel radioactive compound could be detected by incubation of PhnN with [γ-32P]ATP and ribose, ribose 1-phosphate, or ribose 5-phosphate (Fig. 2A).
Identification of PRPP as the product of the PhnN-catalyzed reaction. Incubation and one-dimensional chromatography were performed as described in Materials and Methods. Abbreviations: Appl., application line; F, solvent front. (A) Incubation of PhnN with various ribose compounds and [γ-32P]ATP. All of the incubation mixtures contained [γ-32P]ATP. Lanes 1 and 6 to 9 contained PhnN, whereas PhnN was omitted from lanes 2 to 5. Other additions were as follows: lane 1, no ribose compound; lanes 2 and 6, ribose; lanes 3 and 7, ribose 1-phosphate; lanes 4 and 8, ribose 5-phosphate; and lanes 5 and 9, ribose 1,5-bisphosphate. The positions of ATP, PRPP, ADP, and Pi are indicated on the right. In the reaction mixture shown in lane 9 a small amount of ADP was formed in addition to PRPP, presumably due to the presence of traces of [β-32P]ATP in the [γ-32P]ATP preparation. (B) Consumption of 32P-labeled PhnN reaction product (lanes 1 to 5) or PRPP synthase-generated [32P]PRPP (lanes 6 to 10) by XPRTase. Production of labeled compounds and incubation with XPRTase were performed as described in Materials and Methods. The reaction mixture analyzed in each lane contained reaction cocktail with the following supplement(s): lanes 1 and 6, no supplement; lanes 2 and 7, xanthine; lanes 3 and 8, XPRTase; lanes 4 and 9, xanthine and XPRTase; lanes 5 and 10, xanthine, XPRTase, and unlabeled PRPP (5 mM). The positions of ATP, PRPP, and Pi are indicated on the right. Lanes 1 to 5 contained appreciable amounts of ATP, whereas no ATP was present in lanes 6 to 10. We were unable to identify conditions that allowed complete consumption of ATP by PhnN, whereas ATP was readily consumed by PRPP synthase. (C) Conversion of unlabeled PhnN reaction product or PRPP to 5′-[14C]XMP in the presence of XPRTase and [14C]xanthine. The assay conditions are described in Materials and Methods. The reaction mixture analyzed in each lane contained reaction cocktail consisting of [14C]xanthine with the following addition(s): lane 1, no addition; lane 2, XPRTase; lane 3, PhnN reaction product; lane 4, PRPP (synthesized by PRPP synthase); lane 5, XPRTase and PhnN reaction product; lane 6, XPRTase and PRPP (synthesized by PRPP synthase); lane 7, XPRTase and commercial PRPP (5 mM); lane 8, XPRTase and a sample of PhnN assay mixture to which no PhnN enzyme was added (the mixture contained ribose 1,5-bisphosphate and ATP); and lane 9, XPRTase and a sample of PRPP synthase assay to which no PRPP synthase was added (the mixture contained ribose 5-phosphate and ATP). The positions of xanthine and 5′-XMP are indicated on the right. Spots were visualized under a UV mineral lamp at 254 nm.
To confirm that the compound formed was PRPP, we used a purified preparation of B. subtilis XPRTase, which catalyzes the reaction xanthine + PRPP → 5′-XMP + PPi. Thus, if the compound synthesized by PhnN is PRPP, it should be consumed by XPRTase in the presence of xanthine. This is exactly what was observed (Fig. 2B, lanes 1 to 4). Furthermore, addition of unlabeled PRPP prevented consumption of the radiolabeled PhnN-synthesized compound by XPRTase (Fig. 2B, lane 5). Similar results were obtained by incubation of authentic PRPP synthesized by using highly purified E. coli PRPP synthase (Fig. 2B, lanes 5 to 10). In these experiments PRPP was presumably labeled in the β position of the diphosphoryl group. Upon reaction with XPRTase, this diphosphoryl group was released as PPi. Eventually, the formation of small amounts of PPi could be seen (Fig. 2B), although most of the PPi was presumably converted to Pi by a pyrophosphatase present in the XPRTase preparation.
To reinforce the finding that the compound synthesized by PhnN was a substrate for XPRTase, the compound was synthesized in unlabeled form and again used as a substrate, together with [8-14C]xanthine and XPRTase; in this way 5′-[14C]XMP was generated (Fig. 2C, lane 5). Similarly, 5′-[14C]XMP was generated by incubation of [8-14C]xanthine and XPRTase with PRPP synthesized by E. coli PRPP synthase or with commercial PRPP (Fig. 2C, lanes 6 and 7). The results of some additional control reactions are shown in Fig. 2C. Formation of 5′-XMP required PRPP and XPRTase (Fig. 2C, lanes 1 to 4). Including either ribose 1,5-bisphosphate and ATP or ribose 5-phosphate and ATP (i.e., the substrates of ribose 1,5-bisphosphokinase and PRPP synthase, respectively) in the XPRTase assay mixture resulted in abolishing 5′-XMP production at levels above background levels (Fig. 2C, compare lanes 8 and 9 to lane 1). Thus, the XPRTase preparation contained no PRPP-forming contaminant. In a similar way we showed that the compound synthesized by PhnN could be converted to [14C]UMP in the presence of highly purified uracil phosphoribosyltransferase and [14C]uracil (data not shown). When ribose 1,5-bisphosphate and PhnN were incubated with [α-32P]ATP, the labeled nucleotide product was ADP rather than AMP. This showed that the enzyme transferred a phosphoryl group rather than a diphosphoryl group. The PhnN protein was unable to use GTP as a phosphoryl donor, as measured with [α-32P]GTP, in which case no labeled compound besides GTP appeared on the thin-layer chromatogram (data not shown). We conclude from these experiments that the product of the PhnN-catalyzed reaction is PRPP and consequently, that PhnN is an enzyme with ribose 1,5-bisphosphokinase activity.
To further show that phosphorylation of ribose 1,5-bisphosphate was catalyzed by the phnN gene product, we compared the band intensity of the PhnN polypeptide during purification with the ribose 1,5-bisphosphokinase activity of the same fractions (Fig. 3). We were unable to determine the ribose 1,5-bisphosphokinase activity in a crude extract of strain HO1088/pTR553 grown in the absence of IPTG (i.e., the activity was less than 0.01 μmol/min/mg of protein), and similarly, no PhnN band was visible in the gel. In contrast, appreciable specific activity was observed in a crude extract of cells grown in the presence of IPTG, and there was a further increase in the specific activity of the purified enzyme. The relative intensity in the gel of the PhnN band increased from the crude extract to the purified enzyme. Thus, there was clear agreement between enzyme activity and band intensity of the PhnN polypeptide following gel electrophoresis in dodecyl-containing polyacrylamide. We therefore concluded that the band identified in Fig. 3 represents PhnN and that this polypeptide is responsible for ribose 1,5-bisphosphate phosphorylation. The relative molecular weight of this polypeptide was 23,500, which is in agreement with the value (21,408) calculated from the deduced nucleotide sequence of the allele specifying the histidine-tailed polypeptide. Lane 5 in Fig. 3 contained extract of a strain expressing wild-type phnN. As expected, the PhnN polypeptide was slightly smaller than the histidine-tailed polypeptide. The relative intensity of the PhnN band was somewhat less than that of the histidine-tailed PhnN (Fig. 3, lane 2). However, the ribose 1,5-bisphosphokinase activity of the former was significantly lower than that of the latter. We therefore concluded that the wild-type PhnN and the histidine-tailed PhnN have similar specific activities.
Purification and activity of histidine-tailed ribose 1,5-bisphosphokinase. Cells of strain HO1088 (Δprs)/pTR553 (specifying histidine-tailed PhnN) were grown in NZY medium, phnN gene expression was induced by IPTG, and ribose 1,5-bisphosphokinase was purified and assayed as described in Materials and Methods. In addition, a culture sample was removed before addition of IPTG. Furthermore, strain HO1088/pHO500 (specifying wild-type PhnN) was grown in parallel with strain HO1088/pTR553. Extracts and purified enzyme were subjected to gel electrophoresis in 17.5% polyacrylamide containing sodium dodecyl sulfate. Lane 1, crude extract (10 μg of protein) of HO1088/pTR553 cells before IPTG addition; lane 2, crude extract (10 μg) of HO1088/pTR553 cells after IPTG addition; lane 3, purified PhnN protein (1.3 μg); lane 4, molecular weight markers (positions indicated on the right, as follows: a, phosphorylase b [molecular weight, 97,400]; b, serum albumin [66,200]; c, ovalbumin [45,000]; d, carbonic anhydrase [31,000]; e, trypsin inhibitor [21,500]; f, lysozyme [14,400]); lane 5, crude extract (10 μg) of HO1088/pHO500 cells after IPTG addition. The arrow on the left indicates the position of the PhnN band. The ribose 1,5-bisphosphokinase activity of each fraction (in micromoles per minute per milligram of protein) is indicated at the bottom.
Formation in vivo of PRPP in NAD suppression mutants.It was shown previously that the PRPP content of an E. coli Δprs pst-phoU strain was less than 5% of that of a wild-type strain (i.e., PRPP was undetectable) (17). Strain HO1088 (Δprs)/pTR553 (phnN) is NAD prototrophic, presumably due to increased expression of phnN. However, in the presence of adenine (100 mg/liter) this strain became NAD auxotrophic. Adenine is a potent scavenger of PRPP in cells that contain a wild-type allele of the apt gene, which encodes adenine phosphoribosyltransferase (2). In addition, the strain formed colonies in the absence of tryptophan, although at a much lower rate than it formed colonies in the absence of NAD. These data suggest that PRPP indeed is synthesized in strain HO1088/pTR553. We were unable to cultivate strain HO1088/pTR553 in a low-phosphate medium, and thus, we were unable to determine the sizes of the intracellular pools of PRPP or nucleotides.
Pathway for NAMN synthesis in NAD suppression mutants.Our results show that PRPP can be synthesized from ribose 5-phosphate by the following pathway: ribose 5-phosphate → ribose 1-phosphate → ribose 1,5-bisphosphate → PRPP in reactions catalyzed by the sequential action of phosphoribomutase (deoB), ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase (phnN) (Fig. 4). The activity of the enzyme catalyzing the second step, ribose 1-phosphokinase, remains to be identified, along with the corresponding gene. However, our data leave no doubt that ribose 1-phosphate is indeed involved in the pathway. NAD suppression strictly requires the production of ribose 1-phosphate, an intermediate of cellular metabolism only under certain conditions, as follows: either (i) when ribose 5-phosphate and phosphoribomutase are present (ribose 5-phosphate → ribose 1-phosphate) or (ii) when a ribonucleoside can be phosphorolyzed (e.g., guanosine in the presence of xanthosine phosphorylase [guanosine + Pi → guanine + ribose 1-phosphate]). Both of these reactions are included in Fig. 4.
Proposed pathway for conversion of ribose 5-phosphate to the 5′-phosphoribosyl moiety of NAMN. Abbreviations: P, phosphate; RPK, ribose 1-phosphokinase. Other enzyme-catalyzed reactions are indicated by gene designations (deoB, phosphoribomutase; xapA, xanthosine phosphorylase; phnN, ribose 1,5-bisphosphokinase activity; nadC, quinolinate phosphoribosyltransferase; pncB, nicotinate phosphoribosyltransferase.)
The physiological role of ribose 1,5-bisphosphate is poorly understood. Its only known function is as a coenzyme for phosphopentomutase of E. coli and other organisms (22, 24). The pathway for ribose 1,5-bisphosphate synthesis has not been described previously, except that macrophages synthesize the compound by dephosphorylation of PRPP (21).
Role of the phnN gene product in phosphonate degradation by the C-P lyase pathway.We have shown that the PhnN protein is capable of catalyzing the phosphorylation of ribose 1,5-bisphosphate to PRPP; i.e., it is a ribose 1,5-bisphosphokinase. This is not only the first assignment of a chemical reaction to a specific polypeptide specified by the phn operon, but it is the first report of characterization of an enzyme with this activity. The specific activity of the enzyme with ribose 1,5-bisphosphate and ATP as substrates was rather low (i.e., undetectable) in an extract of uninduced cells. This low activity presumably provides an explanation for the retention in the NAD suppression mutants of requirements for purine and pyrimidine compounds. In wild-type E. coli cells approximately 80% of the PRPP formed is spent on purine and pyrimidine nucleotide synthesis, approximately 10% is spent on synthesis of the amino acid histidine, approximately 10% is spent on synthesis of the amino acid tryptophan, and approximately 1% is spent on NAD synthesis (19). Thus, only a small amount of PRPP may be required to satisfy the need for NAD and tryptophan biosynthesis. In addition, the elevated ribose 1,5-bisphosphokinase activity may not increase the PRPP pool to an extent that satisfies the amount required for nucleotide and histidine synthesis, as the level of the enzyme's substrate, ribose 1,5-bisphosphate, may not be elevated.
The biochemistry of phosphonate degradation by the E. coli C-P lyase pathway is poorly understood. Methyl phosphonate is converted to methane and some other unknown phosphorus-containing compound(s). Furthermore, methane formation appears to occur at the outside of the cell as the reaction may occur in cells which are deficient in the phosphonate ABC transporter specified by phnCDE (43; W. W. Metcalf and B. L. Wanner, unpublished data). It has also been shown that wild-type E. coli K-12 cells, which are unable to degrade phosphonates due to a transport defect (25), accumulate [32P]α-1-(ethylphosphono)ribose, when they are supplied with [32P]ethyl phosphonate; i.e., the ethyl phosphonate is attached to ribose at the C-1 hydroxyl as a phosphate ester. Most of this ribose derivative is found in the culture fluid. Phosphonate degradation-proficient cells fail to accumulate the compound, presumably because the compound is further degraded (1). A relationship between this phosphono-ribose compound and the activity of the phnN gene product, if any, remains to be established. However, it is possible that the α-1-(ethylphosphono)ribose is a dephosphorylation product of 5-phospho-α-1-(ethylphosphono)ribose. As noted above, α-1-(ethylphosphono)ribose was detected primarily in the culture fluid, possibly resulting from dephosphosphorylation of 5-phospho-α-1-(ethylphosphono)ribose by a nonspecific periplasmic phosphatase. Hypothetical reactions involving ribose 1,5-bisphosphokinase are shown in Fig. 5. We suggest that 5-phospho-α-1-(methylphosphono)ribose is an intermediate in methylphosphonate utilization. 5-Phospho-α-1-(methylphosphono)ribose may be demethylated by the C-P lyase, yielding methane and ribose 1,5-bisphosphate. The latter compound then may be phosphorylated to PRPP by PhnN, as was shown to occur in vitro in the present work. Alternatively, PhnN may phosphorylate 5-phos-pho-α-1-(methylphosphono)ribose to methylphosphono-PRPP, which in turn may be demethylated by the C-P lyase, yielding methane and PRPP.
Hypothetical reactions involved in the degradation of methylphosphonate by the C-P lyase pathway. An intermediate in the pathway is 5-phospho-α-1-(methylphosphono)ribose. This compound may be either demethylated to ribose 1,5-bisphosphate or phosphorylated to methylphosphono-PRPP. Ribose 1,5-bisphosphate or methylphosphono-PRPP may be phosphorylated or demethylated, which results in the formation of PRPP.
We are grateful to Hans Klenow for providing ribose 1,5-bisphosphate, to K. Frank Jensen for supplying purified XPRTase, to Carol Gross, Karin Hammer, and Tim Poulsen for supplying bacterial strains, and to Jan Neuhard and Gert Dandanell for providing DNA of pBRdeo and pGD248, respectively. We thank Tonny D. Hansen for expert technical assistance.
Financial support was provided by the Danish Natural Science Research Council to B.H.-J. and by the National Institutes of Health to B.L.W.
Received 15 July 2002.
American Society for Microbiology
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Journal of Bacteriology May 2003, 185 (9) 2793-2801; DOI: 10.1128/JB.185.9.2793-2801.2003
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Biografia
Nato e cresciuto nei pressi dello stadio di Wembley a Londra, figlio del produttore del Muppet Show e cugino di uno dei musicisti di Björk, fin da piccolo viene influenzato dagli scritti di Paul Auster e Arthur Miller e da figure musicali come David Bowie e Trent Reznor. Fin da piccolo dimostra abilità nella scrittura, già a otto anni scrive un originale testo incentrato su un piccolo villaggio ubicato all'interno di una pancia.

Gary Go è autore dei suoi brani, che compone con l'utilizzo di piano, tastiere e chitarra. Si è fatto notare nel 2007 pubblicando l'EP The Diary of Rodney Harvey, ispirato dalla tragica morte del promettente attore. Successivamente crea una propria etichetta indipendente chiamata The Canvas Room, dove inizia a scrivere e registrare il suo album di debutto, le registrazioni hanno luogo tra Londra, Praga e New York. Messo sotto contratto con la Decca Records, inizia un tour promozionale proponendo i suoi lavori e facendo da supporto ad artisti come The Script, The Feeling e Amy Macdonald.

Il suo singolo di debutto si intitola Wonderful ed è stato pubblicato il 16 febbraio 2009. Il suo omonimo album è stato pubblicato nel maggio 2009, inoltre è stato annunciato che il cantante aprirà le date estive del tour dei Take That.

Nel 2011 ha pubblicato 2 brani con il dj italiano Benny Benassi: Cinema, che è diventato disco di platino, e Close to Me.

Il 16 gennaio 2012 ha annunciato sulla sua pagina ufficiale il duetto con Emma Marrone in occasione della 62ª edizione del Festival di Sanremo. I due infatti hanno poi cantato, in occasione della serata dedicata all'Italia nel mondo, If paradise is half as nice (Il paradiso resa famosa da Patty Pravo) e Wonderful.

Nel corso della sua carriera Gary Go ha inoltre collaborato come autore e produttore per artisti quali Rihanna, Robbie Williams, Take That, Steve Angello, Ruby Rose, Kylie Minogue, Simply Red, The 2 Bears, Ronan Keating, Joseph Arthur, Katharine McPhee, Carina Round, Laleh Pourkarim e Juliette Lewis.

Discografia

Album in studio
2009 - Gary Go
2020 - Love Lost Freedom Found

EP
2006 - So So... EP (2006)
2007 - The Diary of Rodney Harvey
2009 - Napster Live Sessions
2009 - The Heart Balloon
2012 - Now Was Once The Future

Singoli
2009 - Wonderful
2009 - Open Arms
2009 - Engines
2012 - Superfuture
2015 - Crying Sound
2018 - Cinema - Acoustic

Collaborazioni
2010 - Cinema - Benny Benassi feat. Gary Go
2011 - Control - Benny Benassi feat. Gary Go
2011 - Close to Me - Benny Benassi feat. Gary Go
2011 - Magic - The Knocks ft. Gary Go
2014 - Let This Last Forever - Benny Benassi feat. Gary Go
2015 - Prisoner - Steve Angello ft. Gary Go

Filmografia

Compositore
 Friend Request - La morte ha il tuo profilo (Friend Request), regia di Simon Verhoeven (2016)
 Benvenuto in Germania! (Willkommen bei den Hartmanns), regia di Simon Verhoeven (2016)

Note

Collegamenti esterni

 Gary Go su MySpace

Cantanti pop

redpajama_set_name: RedPajamaWikipedia


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est la forme neutre - ayant valeur de gentilé - de l'adjectif latin Complutensis-is-e qui qualifie ce qui est relatif à la ville d'Alcalá de Henares en Espagne, localité dont le nom se traduit  en effet Κόνπλουτον en grec (attesté chez Ptolémée), et  ''Complūtum en latin. Conformément à l'usage qui veut qu'on décline au neutre les mots latins utilisés dans une langue vulgaire, l'expression «  » peut désigner :
 L'université d'Alcalá de Henares, une ancienne université fondée en 1499 ;
 La Bible polyglotte d'Alcalá, une bible polyglotte établie dans cette même université et publiée en 1517 ;
 L'université complutense de Madrid, une université madrilène héritière de celle d'Alcalá.

 Complutense Cisneros, un club espagnol de rugby à XV

Notes et références

Voir aussi

Gentilé


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Part I original edition #
Introduction #
It is these boundary regions of science which offer the richest opportunities to the qualified investigator. They are at the same time the msot refractory to the accepted techniques of mass attack and the division of labor. If the difficulty of a physiological problem is mathematical in essence, ten physiologists ignorant of mathematics will get precisely as far as one physiologist ignorant of mathematics, and nor further. If a physiologist who knows no mathematics works together with a mathematician who knows no physiology, the one will be unable to state his problme in terms that the other can manipulate, and the second will be unable to put the answers in any form that the first can understand. Dr. Rosenblueth has always insisted that a proper exploration of these blank spaces on the map of science could only be made by a team of scientists, each a specialist in his own field but each possessing a thoroughly sound and trained acquantance with the fields of his neighbors; all in the habit of working together, of knowing one another's intellectual customs, and of recognizing the significance of a colleague's new suggestion before it has taken on a full formal expression. The mathematician need not have the skill to conduct a physiological experiment, but he must have the skill to understand one, to criticize one, and to suggest one. The physiologist need not be able to prove a certain mathematical theorem, but he must be able to grasp its physiological significance and to tell the mathematician for what he should look.
We have decided to call the entire field of control and communication theory, whether in the machine or in the animal, by the name Cybertnetics, which we form from the Greek κυβερνήτης or steersman. In choosing his term, we wish to recognize that the first significant paper on feedback mechanisms is an article on governors, which was published by Clerk Maxwell in 1868, and that governor is derived from a Latin corruption of κυβερνήτης. We also wish to refer to the fact that the steering engines of a ship are indeed one of the earliest and best-developed forms of feedback mechanisms.
Newtonian and Bergsonian Time #
... in that it puts side by side two sciences which have the one similarity of dealing with the heavens above us, but which in almost every other respect offer an extreme contrast. Astronomy is the oldest of the sciences, while meteorology is among the youngest to begin to deserve the name. The more familiar astronomical phenomena can be predicted for many centuries, while a precise prediction of tomorrow's weather is generally not easy and in many places very crude indeed.
Suggested Pages #
0.216 Home
0.025 Cybernetics
More suggestions...
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content-type text/x-markdown
schema Book

redpajama_set_name: RedPajamaCommonCrawl


Blended Scotch whisky constitutes about 90% of the whisky produced in Scotland. Blended Scotch whiskies contain both malt whisky and grain whisky. Producers combine the various malts and grain whiskies to produce a consistent brand style.


text:
Ten fun facts about Nigeria
Though the country has three dominant tribes, Hausa- Fulani, Yoruba and the Ibo (Igbo), there are over 250 ethnic groups.
Nigeria derives her name from the Niger River which is the largest and the longest river in West Africa and spans around 4,180 kms.
The Nigerian movie industry which produces between 150-200 movies very week is known as Nollywood is ranked as the world's second-largest producer of movies and trails just behind the Indian Bollywood industry.
It is believed that areas surrounding Cross State River and Calabar towards the south of Nigeria are home to the largest diversity of butterflies in the world.
The Niger and the Benue are Nigeria's main rivers. They converge empty their waters into the Niger Delta. The sheer size of these two rivers makes this delta one of the largest in the world.
Two of the world's rarest species of animals live in the mysterious Nigerian forests- the Drill monkey that lives in the Afi Mountain ranges and the lowland Gorilla.
This country is known for its 'twin genes". Nigeria has the highest twin birth-rate in the world.
After Egypt, this country is known to have one of the largest newspaper markets in the world. Several million copies of newsprint are sold here everyday.
Nigeria has always been among the top five cocoa bean producing countries in the world. In 2013 it stands in 5th place and produces around 210 thousand tons.
The 50,000 seater 'Faith Tabernacle" of Winners Chapel Churches is the largest church auditorium in the world. Its 2008 Guinness Book world record has not been broken even in 2013.
Go to more country facts ❯
MORE IN COUNTRIES
Short about Nigeria
Is a country situated in South Africa and officially known as the Federal Republic of Nigeria,
CIA The World Factbook


text:
Q: calling compute on a dask.dataframe converts datetime64[ns] to object I've attemped a MCVE below but it works. However, my parquet file is created in databricks and I am now reading it in locally so not sure if that is part of the issue. Therefore, open to suggestions of things to try. Works with calls using to_datetime.
Here's a screenshot of my 'real-world' problem:

and code here:
print(df.DATE.dtype)
print(df.compute().DATE.dtype)
print(dd.to_datetime(df.DATE).compute().dtype)
print(pd.to_datetime(df.compute().DATE).dtype)

MCVE (but works):
import pandas as pd
df = pd.DataFrame(data={'col1': pd.date_range('2000', freq='D', periods=3)})
>>> df.col1.dtype
dtype('<M8[ns]')

import dask.dataframe as dd
ddf = dd.from_pandas(df, 1)
>>> ddf.col1.dtype
dtype('<M8[ns]')
>>> ddf.compute().col1.dtype
dtype('<M8[ns]')

redpajama_set_name: RedPajamaStackExchange


Another movie adapted from the pages of a YA novel is the romantic excursion Everything, Everything.
I've stopped treading so lightly to these Young Adult big screen offerings, having been thoroughly pulled in by the treatments of The Fault of Our Stars, Paper Towns and, episode by episode, the dystopian-ness of The Hunger Games and Divergent series.
So I was figured I was ready for just about anything, anything.
Taken from the pages of novelist Nicola Yoon and scripted by J. Mills Goodloe for director Stella Meghie, Everything, Everything differs from the most of the above offerings by going with a pair of main players with somewhat less immediate star power.
Amandla Stenberg plays Maddy, an 18-year-old who's spent her whole life battling a genetic disease that basically cripples her immune system. She lives daily at home with basically a blessing and curse. Mom, seriously portrayed by Anika Noni Rose, is a doctor who can provide her with a darn nice house from which she can't leave, a careful and caring nurse played with an open-heart by Ana de la Reguera who ventures into the friend zone, and endless advice about what she can't do if she wants to keep on living.
Enter new next-door neighbors, who appear a knockin' with a Bundt cake.
Maddy's eyes light up from her window seat at the sight – and thought – of Ollie, a hunky teen played by Nick Robinson with good, long hair and nice intentions that emerge more and more as the pair starts a texting relationship.
Teens being teens, they both want more. Especially Maddy, who longs to actually go to the ocean, which by the looks of things, can't be so far from this gorgeous and apparently SoCal neighborhood.
So far, not bad, as Goodloe uses some fantasy scenes to keep their texting from boring the heck out of every generation watching. Soon, though, not even Olly's great heart, Maddy's teen spirit and a shared sense of adventure are enough to keep the story rolling along in my soul.
A twist that turns mom from overprotective to something entirely different takes the tale totally off the rails.
It goes from cute to cumbersome for this no-longer-Y A.
Too bad. The kids really were all right until Too Much took over.
Everything, Everything' is part of the LPL's MediaBank collection.


Data-oriented design taught me something more than coding better for hardware. It taught me that it doesn't matter what you're doing, as long as you want to do it well, you need to be able to measure how well you're doing. If you aren't measuring your progress, how can you know you've had a success? How can you know you're making a difference?
This is something that a fair few older game designers often ignore, and it's because for a decent amount of the history of game development, design has been an airy fairy, hand waving, throw things around and see what sticks, kind of thing. It works to some extent, because a lot of the games we used to make were small enough that what we were actually designing was game mechanics. But that's not true any more. Now, games have a few mechanics they want to explore, and those mechanics might be refined over the development of the game, but in truth, the throwing stuff around and seeing what sticks stage of development is over pretty darned quick now. Plenty of companies that don't work with data have had a "great idea", implemented it, and then at publishing time, found that the thing on which they worked really hard is not received well because it's not very good. It's not even like the developers are likely to have thought it was really good while it was in development either, but for some reason, there's a blind spot for failure to make an engaging design. It's like they're frightened to measure how well the game is doing, in case they have to change something. It's almost like they're trying to win logical fallacy bingo. A bit of cognitive dissonance here, a bit of sunk-costs there, and a bit of gamblers to top it off.
Some companies make a big deal out of measuring their games, and they usually do pretty well. The spate of freemium games could not have landed such huge amounts of money without the constant attention to performance indicators. Measurements are key to a game that isn't actually designed to be "fun", but instead designed to extract cash from people with their consent. To this end, it appears that maybe the reason some people don't want to check to see if their design is good, might be that they want their game to be good without being polished by maths, or they want it to be a cult classic, warts and all. They don't want it to be as good as possible because they've sold their soul to the same people that make Cash of Beach, or Game of Candy. They want it to be as good as possible because they want to be proud that it's all their idea.
Using metrics saves you time thinking about stuff like "would the player find this too difficult" and allows a designer to concentrate on the actual inventing stuff. Being freed from having to do the laborious play-throughs and focus tests with hand written notes, gives the designers the mental space to come up with truly innovative game elements, and in house testing with feedback allows them to be able to see if they will work, or do work, without having to commit vast sums of money to them. Design, and art, love constraints, but hate the constraint of time. Give your designers and artists strict technical constraints and all the time in the world, and you end up with beautiful things. Stop them from working by not letting them utilise a universe of tools and metrics for seeing if their work is successful, and you end up with a big ball of ideas that only work in the heads of those that invented them. Nothing can be designed in a vacuum.
Graham McAllister on Molyneux's strange decision to ignore data.
"However, Peter is not alone in making such reckless statements. I was recently at a conference where a speaker was saying that all that was needed to make a great game was to hire smart people and have good ideas."
He's a different era of game designer. There's nothing inherently wrong with how he designs game mechanics, but my opinion is that he does need tempering, as without data, it's very hard for him to see what it is he does not know. Peter did found the cannery on the tagline "A great team with a great idea leads to great success".
This thinking is lovely, because it makes me sure that at the heart of it, Peter still believes in making a great game. The idea that numbers and metrics are something you don't bother with at the beginning, reminds me that at that desk, in that office, sits a game designer that really believes in striving to create something great, something unique, and something that will make a difference, all by sheer force of will, dedication, and persistence.
I don't think he can, which is why I don't work there any more, but I want to be proved wrong, because I think we'd be worse off without anyone like him doing it his way.


In this episode of Internet Business Mastery, we have an interview with Google Adwords master, Perry Marshall. And in the Quick Tip we reveal our latest favorite tool for managing Twitter and Facebook.
Have you ever used Adwords and what was your experience like?
Adwords was where I first started "internet business" and could NEVER get my landing page to comply. I ended up very frustrated and stopped trying. I want to give it another go (especially after listening to the interview) Hopefully I'll figure out the first step. I really want to begin to learn how to split test etc.
Split testing is definitely the next level of business and really helps you hone in your message.
The landing pages for adwords are really tricky. Perry has lots of great training to help you with that.
Thanks guys for the feedback. I'm balancing on the precipice of overwhelm and learning what I need as quickly as I can….It seems to be quite a balancing act for sure.
I use Adwords as the main driving force for my PAID advertising (word of mouth, and customers returning also play a big part of our business) but we've found print simply doesn't return the dollars like it used to – it's simply outdated. The great thing with Adwords is the fast, easy and detailed reporting options with testing and results. In the early days of Adwords I recall loosing around $3000 from simply being "stupid" with it. Its like any advertising it needs to be measured and tested to see the best results. I'm lucky because I have a high cost product/service, my beginner acting course is $297. Around 18 months ago I would spend around $40 to $55 per sale on Adwords advertising which is around 14% to 18% of each sale going on selling that product to the customer. Now those are not bad odds, as you guys say, I will always trade $55 to get $297 (and I guess that's the great thing about Adwords, you only pay for "quality" leads). However after 18 months or testing CPC rates, Ad Headlines, Ad Copy and the most important thing what the customers are actually using as search terms we have been able to reduce the per sale advertising rate to around $8.91 to $14.85 which is only 3% to 5% of each sale. I have also found it a great tool to use selective advertising times in the Adwords set-up options. As you guys know I split test everything. We had many customers late, and I mean late at night searching and clicking through to our site but would never and I mean never buy, they are simply time waster that I have to pay for, so I set specific times that our Adwords run (this data was obtained by Google Analytics). I also feel that a good Adwords campaign is also matched with a great SEO campaign. When our customers search the most popular search term for our niche (which will remain unknown in case anyone is looking to get into my niche) and they do so in the town the niche and business are "Perth, WA" we not only get a top Adwords listing, but we also get an organic ranking of #1 and #2 for our main site plus our landing page, plus an organic ranking of #3 and #4 for supporting feeder sites we own. It is interesting because I then tested (as I do) loosing the Adwords campaign because we have so many organic free listings, I thought that they will still find us, and something interesting happened. sales dropped 18%. I put Adwords back on, sales return 19% higher that week. I did some asking around of potential customers and found that, Adwords can act like a quality indicator or a type of social proof that your business is big and worthwhile. Our Google Analytics results show that Adwords only accounts for 42% of our traffic and organic search results in 51% of our traffic. So people don't always click on an Adword ad, but use the organic ranking, so this implies that the Adword Ad must be having some affect on them, because when you take it away, traffic reduces.
Interesting stuff, okay maybe I sound like a sad man for loving this type of stuff, but its great because testing shows you the results and allows you to build a better business.
I just bought Perry's 2011 Adwords Guide and hope to get some experience.
I've been thinking of starting some campaigns, running those people directly to a survey, just ask them what they want right there on the landing page, giving them a free e-book with an opt-in form.
The one thing I'm most frustrated about is that I really don't know what my potential market wants, and the content marketing seems slow to get to that point. Yes, they're "consuming" the content, but I want to know what they want as quickly as possible.
Has the extended Perry Marshall interview been added to the 'Grill the Guru' section within the IBM Academy yet?
It depends on what module you are on in the Academy. However, there is a thread in the mastermind community where we have posted the link.
Perry gave me some real food for thought on this whole organic v paid listing seo thing.
Perry Marshall in the "Grill the Guru" section of Academy 2 when it is published real soon ;-) .
Little bit of English understatement there (from an irishman!).


text:
Emotional Whitehead happy for Wighton's career turnaround
Scott Hazlewood NRL.com Reporter
Tue 28 May 2019, 08:11 PM
One is from Bradford in the north of England, the other from Orange in the central west of NSW, but Elliott Whitehead knows what makes Jack Wighton tick.
That's why he could not help but feel emotional when Wighton was named on the bench for the Blues for the Holden State of Origin series opener. That news capped a remarkable turnaround for the 26-year-old.
This time last year Wighton's career was at a crossroads, in trouble with the law over a drunken incident caught on CCTV.
Whitehead said the work Wighton has put into both his game and growing as a person since then means he is richly deserving of an Origin debut.
"He's worked really hard through the off-season all the way through and he's been one of our better performers," Wighton said.
"I nearly shed a tear when he told me he got in, I really like Jack and since I've been here I've been really close with him.
Every try from Round 11
"For him to get that call-up it means a lot to him and I hope he plays well and gets some good minutes."
With the Raiders having players in both NSW and Queensland camps for the first time in six years coming on top of a host of injuries, Saturday's clash with Canterbury takes on a totally different look.
On top of being without Wighton, Nick Cotric (NSW) and Josh Papalii (Queensland), the Raiders also now have skipper Josh Hodgson on the long-term injury list.
John Bateman (expected to return round 15-17), Jordan Rapana (round 13-14) and Joseph Leilua (out for the season) are also missing from Canberra's strongest 13.
A closer look at the Blues' debutants
Instead of lamenting what they will be without, Whitehead said the players stepping into the side have to make the most of the chance they will be given.
"It's also a great opportunity for the younger guys who are going to get their chance over the next couple of weeks," Whitehead said.
"They can really show people they want that NRL career so it's really exciting for them as well.
"The Origin guys really deserve their spots and I'm happy for them to be there."
Siliva Havili will likely be called upon to fill in for Hodsgon, having to reprise his role from last season when he was the first-choice hooker as the Englishman recovered from knee surgery.
"It's a big loss losing Hodgey with all of the experience and leadership he brings to the team," Havili said.
Canberra utility Siliva Havili. ©Keegan Carroll/NRL Photos
"I know my game is different to Hodgey's so all I can do is play a really simple game and have lots of clarity for the players around me."
Havili had been one of the Raiders' best bench players this season, with his powerful frame ideally suited to short, impact stints.
With the prospect of playing 80 minutes again starting this weekend against the Canterbury Bulldogs, Havili knows he will have to adjust his game.
"That's something I have to get my head around, not knowing what the team is this week, I'll just try and get my lungs ready and my mindset ready," he said.
Williams, Wighton are perfect halves match: Hodgson
Croker and Co. to go from hunters to hunted
Canberra Raiders: 2020 round 1 predicted team
Canberra Raiders: 2019 season by the numbers
Wed 18 Dec, 2019
Raiders v Broncos: Haas steps up for Lodge; Collins for Tapine


text:
Future of nuclear energy unclear
Despite a turbulent history, the allure of nuclear energy — electricity production on a massive scale with minimal emissions — remains attractive. Its low emission rate is why the United Nations International Panel on Climate Change recommends doubling the world's nuclear capacity by 2050. Yet the bulk of the 100 nuclear reactors currently operating in the U.S., which continue to produce about 20 percent of the nation's energy, are reaching retirement age, and energy market forces don't always favor nuclear.
Mystery of historic 1952 London killer fog, current Chinese haze solved
Few Americans may be aware of it, but in early December 1952 a killer fog that contained pollutants covered London for five days, causing breathing problems and killing more than 12,000 people of all ages, sending more than 150,000 to hospitals, and killing thousands of animals in the area. It is still considered the worst air pollution event in the European history. The exact chemical processes that led to the deadly mix of fog and pollution have not been fully understood over the past sixty years – until now. Scientists have now established that coal burning was the main culprit: sulfuric acid particles were formed from sulfur dioxide released by coal burning, and this process was facilitated by nitrogen dioxide, another co-product of coal burning. The study shows that similar chemistry occurs frequently in China, which has battled air pollution for decades.
Meeting global energy demands with nuclear power
An international team of scientists suggests that we must ramp up energy production by nuclear power if we are to succeed in warding off the worst effects of greenhouse gas emissions on climate change. The team suggests that beginning in 2020 we could achieve an annual electricity output of 20 terawatts without needing to develop carbon dioxide trapping and storage technology for the tens of billions of tons of emissions that would otherwise drive global warming to catastrophic levels.
Some early 20th century L.A. earthquakes might have been man-made
Some early twentieth century earthquakes in southern California might have been induced (man-made) by past practices that were used by the oil and gas industry. During the early decades of the oil boom, withdrawal of oil was not balanced by injection of fluids, in some cases leading to dramatic ground subsidence, and potentially perturbing the sub-surface stress field on nearby faults.
Wastewater disposal induced 2016 Magnitude 5.1 Oklahoma earthquake
Distant wastewater disposal wells likely induced the third largest earthquake in recent Oklahoma record, the 13 February 2016, magnitude 5.1 event roughly thirty-two kilometers northwest of Fairview, Oklahoma. at the time, the Fairview earthquake was the largest event in the central and eastern United States since a 2011 magnitude 5.7 struck Prague, Oklahoma.
Bolstering energy security with homegrown energy sources
The U.S. Department of Energy has a goal to develop and demonstrate transformative bioenergy technologies to fuel a more sustainable nation. Reaching that goal will require roughly a billion tons of biomass, so we will need to rely on a variety of resources to get the job done.
Coal's decline driven by technology, market forces – not policy
Many people – and many politicians — attribute coal's decline to the clean-air policies of the federal Environmental Protection Agency (EPA) through rules that the agency applied to electricity generation plants. A new study points out that largely because of court challenges, EPA clean-air regulations did not change until 2015 — twenty-five years after President G. H. W. Bush signed amendments to the Clean Air Act in 1980. For eighteen years following new EPA rules, coal continued to thrive — until 2008, when its production peaked and then declined 23 percent in the next seven years. The study found that the decline is correlated with the shale revolution that began to be fully felt in 2007-2008, after which cheap natural gas outcompeted coal markedly.
Abnormalities found in drinking water in Texas's Eagle Ford Shale region
Chemists studying well water quality in the Texas's Eagle Ford Shale region found some abnormal chloride/bromide ratios, alongside evidence of dissolved gases and sporadic episodes of volatile organic compounds, all indicative of some contamination from industrial or agricultural activities in the area.
Oklahoma shuts down 37 wells after Saturday's 5.6 magnitude earthquake
Oklahoma ordered the shutting down of 37 wells after Saturday's 5.6 magnitude earthquake. Experts note that the significant increase in the number earthquakes measuring 3.0 or higher in Oklahoma has been linked to the practice of underground disposal of wastewater from oil and natural gas production. Only three earthquakes 3.0 magnitude or higher were recorded in 2009. Last year, the state had 907 such quakes. So far this year, there have been more than 400.
Solar-powered Ring Garden combines desalination, agriculture for drought-stricken California
With roughly 80 percent of California's already-scarce water supply going to agriculture, it is crucial for the state to embrace new technologies that shrink the amount of water required to grow food. Alexandru Predonu has designed an elegant solution which uses solar energy to power a rotating desalination plant and farm that not only produces clean drinking water for the city of Santa Monica, but also food crops — including algae.
Preventing human-caused, fracking-related earthquakes
The 31 May 2014 earthquake that rumbled below Colorado's eastern plains surprised both local residents and local seismologists. The earthquake happened in an area that had seen no seismic activity in at least four decades. It was likely caused by the injection of industrial wastewater deep underground. New research shows actions taken by drillers and regulators can lessen risk in the case of earthquakes likely caused by the injection of industrial wastewater deep underground.
Wider temperature tolerance is based on ion-pair-coordinated polymers
A new class of fuel cells based on a newly discovered polymer-based material could bridge the gap between the operating temperature ranges of two existing types of polymer fuel cells, a breakthrough with the potential to accelerate the commercialization of low-cost fuel cells for automotive and stationary applications.
Pro-nuclear countries slowest to make progress on climate targets
A strong national commitment to nuclear energy goes hand in hand with weak performance on climate change targets, researchers found. A new study of European countries shows that the most progress toward reducing carbon emissions and increasing renewable energy sources has been made by nations without nuclear energy or with plans to reduce it. Conversely, pro-nuclear countries have been slower to implement wind, solar, and hydropower technologies and to tackle emissions.
NSF announces $55 million toward national research priorities
The National Science Foundation (NSF) has made eleven awards totaling $55 million aimed at building research capacity to address fundamental questions about the brain and develop new innovations at the intersection of food, energy, and water systems. These four-year awards support twenty-seven institutions in eighteen eligible jurisdictions.
Proposals would dam Little Colorado River for hydropower
UK energy firm says power cut was not caused by cyberattack
Proposed wind farm on Lake Erie panned as possible security threat
Oilfield wastewater may trigger earthquakes for 'decades'
PG&E could face murder charges for role in California wildfires
EPA: No new coal plants likely to be built despite relaxed rule
California moves towards 100% carbon-free electricity after landmark vote
Volcano Kilauea Destroys Hawaii's Only Geothermal Plant, a Top Source of Energy
Coal executive Bob Murray drafted 6 orders on coal, climate for Trump
Nuclear reactor restarts, but Japan's energy policy in flux
Hawking says he lost $100 bet over Higgs discovery
Kansas getting $500K in law enforcement grants
Bill widens Sacramento police, sheriff's contract security opportunities
DHS awards $97 million in port security grants
DHS awarding $1.3 billion in 2012 preparedness grants
Cellphone firms share location data with law enforcement, not users
Residents of Murrieta, California, will have to subscribe for emergency services
Ohio's Homeland Security funding drops sharply
Ports of L.A., Long Beach get Homeland Security grants
Homeland security gets involved with Indiana water conservation
LAPD embraces "predictive policing"
New GPS rival is hack-proof
German internal security service head quits over botched investigation
Americans favor Obama to defend against space aliens: poll
U.S. Coast Guard creates "protest-free zone" in Alaska oil drilling zone
Congress passes measure to enhance Israel security ties
Wickr enables encrypted, self-destructing iPhone messages
NASA explains Why clocks got an extra second on 30 June
Cybercrime disclosures rare despite new SEC rule
First nuclear reactor to go back online since Japan disaster met with protests
Israeli security fence architect: Why the barrier had to be built
DHS allocates nearly $10 million to Jewish nonprofits
Turkey deploys troops, tanks to Syrian border
Israel fears terror attacks on Syrian border
Ontario's emergency response protocols under review after Elliot Lake disaster
Colorado wildfires to raise insurance rates in future years
Colorado fires threaten IT businesses
Improve your disaster recovery preparedness for hurricane season
London 2012 business continuity plans must include protecting information from new risks
No stories


text:
Sexual Assault – No Priors
by Bill D · Updated November 22, 2017
This is case of Stalking and Sexual Assault involving an accused with no priors.
Our client was charged with Stalking, Behave in an Indecent Manner, and Sexual Assault. He in his early 20s at the time of the alleged offending and had no prior offences. All of the sexual assault offences involved inappropriate touching of women over their clothing on the buttocks area. One of the women was 16 years old.
One batch of offences occurred some 10 months after the first. As a result of the second and more recent batch of offences (Sexual Assault), our client was arrested and taken into custody. He did not apply for bail and pleaded guilty to all offences.
A psychological report was organised and this assessment occurred whilst our client was in custody. At the time of his plea of guilty, he had spent a period of 66 days in custody. The sexual assaults were not objectively at the upper end of the scale in terms of the seriousness, but an aggravating factor was that one of the women was 16 years old at the time of the offence. The court also noted the adverse impact each offence had on the women.
Shaun Pascoe acted on the client's behalf at the Heidelberg Magistrates' Court. The charges were:
Commit Indictable Offence Whilst on Bail
The Magistrate took note of the fact that the client was 24 at the time of the offences. It was especially noted that he was charged with Stalking and Sexual Assault but he had no priors. The contents of the psychological report that was tendered during the plea were also taken into account. This psychological report considered a number of factors and had diagnosed the client with a condition which had been hitherto undiagnosed.
Apart from the significance of our client's lack of prior history, the court also noted that he had lost his employment and what was otherwise a promising career, that he was remorseful, and that the psychological assessment placed him at low risk of future offending. The strong support from the client's parents who attended the court for his hearing was also remarkable. A letter was tendered to the court outlining their disappointment at their son's offending but also stating their resolve to ensure that he was successfully rehabilitated.
Further, the court noted that our client's plea of guilty saved the victims from further trauma which was possible through a contested process, and this fact was also determinative of the client's outcome. The court observed that our client had spent 66 days in custody following his arrest, and that for someone with no prior court history or experience in a prison, this experience was significant and would serve as a powerful deterrent against future offending.
In summary, our client's age, lack of priors, period of time already spent in custody, and the strong prospects for rehabilitation (his remorse, family support, and the matters identified in the psychological report) were factors which persuaded the Magistrate that a further period of imprisonment (beyond the 66 days he had already served) was not required having regard to all the purposes of sentencing.
It is unlikely a non-custodial outcome could have been achieved without these various factors coming together.
The client was ultimately sentenced with conviction to a Community Corrections Order for 18 months to comply with supervision and treatment as directed. An order for registration under the Sex Offenders Act for a period of 8 years was also made as it was mandatory for the court to do so. This was a good result for someone charged with Stalking and Sexual Assault but who has no priors.
Shaun Pascoe
Shaun is an Accredited Criminal Law Specialist and a partner of the firm. Shaun runs the Heidelberg branch of Doogue + George.
He is an experienced criminal law solicitor and works hard to achieve the best possible outcomes for his clients. Shaun handles indictable and summary criminal offences and is an expert at criminal defence for both contested and non-contested cases.
Visit Shaun's profile to read more about his background and experience.


text:
News & Insights / Press / Large-Scale Third-Party Addressability On The Open...
Large-Scale Third-Party Addressability On The Open Web Doesn't Work – Period
by Ruben Schreurs, Group Chief Product Officer
As appeared on AdExchanger on 17 February 2022: https://www.adexchanger.com/data-driven-thinking/large-scale-third-party-addressability-on-the-open-web-doesnt-work-period/
IAB Europe's Transparency & Consent Framework (TCF) is flawed and unfixable.
But let's back up.
On Feb. 2, the Belgian Data Protection Authority (APD) ruled that the TCF violates the GDPR on multiple levels. IAB Europe was given two months to submit a plan outlining a redesign of the TCF to bring it into compliance with the regulation. If approved, IAB Europe will have an additional six months to make the changes. On February 11, IAB Europe released a statement confirming its intention to appeal the APD's ruling.
After the ruling was published, debate ensued in the industry, and a clear division emerged between those who want to sustain the online advertising ecosystem in its current form (or something very similar), and those pushing for a radically reimagined ecosystem designed around privacy and data protection.
I'm part of the latter group and let me explain why.
The TCF is a framework that was designed in an effort to add "validity" to a system that is unethical at best and unlawful at worst. We need to acknowledge that the online ad ecosystem itself was not designed to protect consumers or their personal data.
Trying to redesign the TCF to make it compliant is therefore futile, because the underlying processes for which it seeks to gather consent will never be compliant unless the system itself is radically redesigned.
People who defend the TCF and OpenRTB often refer to those who seek to address this issue as privacy fanatics, alarmists or attention seekers. They opine ad nauseam about the supposed "rights" advertisers, publishers and tech companies have to operate a system that is out of control in order to sustain the open web (aka their own business models). They talk about how large-scale personalized advertising on the open web creates a meaningful value exchange with consumers.
One word: fugazi.
Industry folk often point to how Google and Facebook stand to benefit from tightened regulations and how the industry is doomed if regulators double down on privacy because supposedly only large walled garden platforms will survive. Yes, the increasing power and domination of advertising giants is concerning. But that's an area where antitrust regulators play a role, and it should not be addressed with regulatory lenience on privacy and data protection.
Why is there such an utter lack of imagination among leaders in the ad industry? Why try to desperately hold on to a system that is de facto unlawful under most contemporary regulatory frameworks?
I'd argue that advertising has thrived for nearly two centuries without large-scale third-party addressability and personalization. The industry won't collapse when adjustments are implemented that guarantee the safe and compliant processing of personal data.
At the IAB's Annual Leadership meeting this month, as reported by Digiday, Allan Thygesen, Google's president for the Americas and global partners, went so far as to say "our industry can either work together to reinvent the way data is used to deliver personalized ads in more privacy safe ways – or we can stop using personalization of scale altogether and walk away from the model that led to 30 years of global growth and prosperity for so many."
But the fundamental rights of people should always take precedence over the "prosperity" and "growth" of businesses – and it doesn't matter how many people are getting rich off the system.
And saying that the deprecation of third-party cookies without a viable alternative to preserve the status quo will "cost a lot of jobs" is also a moot point. Imagine the mafia getting publicly upset about the potential loss of employment for their mobsters because of increased enforcement against crime?
It's an extreme example to illustrate a point – but law is law. Anti-racketeering regulation is, at its core, no different from data protection regulation. We evolve as a society as does our usage of – and reliance on – technology, and so our regulatory frameworks must evolve as well.
The industry has had its fun, and Thygesen is right. The data-fueled ad tech ecosystem has printed money for corporations for decades. Now, however, it has run its course. Privacy and data protection needs to be given center stage, and uncontrolled access to troves of data by thousands of companies and vendors must be restricted as soon as possible.
Well-informed and compliant ways to add relevance to advertising can still exist, and good media owners will find a way to generate ad income while putting their audience's rights first. It's imperative that we are clear about exactly what part of the ecosystem is noncompliant, because no one is saying that all addressability must be removed from advertising. The debate is about the OpenRTB protocol and the large number of businesses that obtain and transact huge amounts of personal data in a highly opaque and, I'd argue, harmful environment.
Under the current regulations, it is impossible to simplify and streamline the way valid consent is obtained for data access and processing by thousands of vendors at once. The purpose limitation principle, for example, invalidates all the unhinged – and largely invisible – ways in which personal data is processed after a user innocently clicks "accept all' in a consent pop-up. Some people blame regulators for not providing clear guidance and supposedly leaving too much room for interpretation, but the law is clear – and so are the authorities as they ramp their enforcement.
One of the weirdest arguments made by people in the industry is that it would simply be too annoying and, therefore, essentially impossible to obtain valid consent in line with the regulations. And that is exactly right – but not for the reason they think it is.
It's impossible to implement a quick and dirty one-click solution for obtaining valid blanket consent, but that's because of the mechanics and unlawfulness of the underlying processing activities themselves, not because of an annoying user experience.
Regulators have no obligation to support businesses as they continue to grow through a reliance on unlawful models. There is no such thing as a fundamental right for corporations to process data, because otherwise their systems or commercial models won't work. That's not what legitimate interest is.
Regulators are paying attention and taking action, and now the industry must follow. This is a good thing.
It's time the industry understands this and moves on in a more constructive way.
Digiday also covered this topic with an in-depth article, including commentary from Ebiquity. Find it here.