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Harley riders raise funds for wounded warriors
At first, all Sgt. Kealoha Kuikahi saw was a couple of Harley-Davidsons rolling toward the Warrior Hope and Cure Center at Camp Pendleton on Friday morning.
Then the rumble grew louder. Soon there was what seemed to be an unending line of Harleys heading toward him along the Marine base's roads. In all, there were more than 400 motorcyclists.
They were part of the first Injured Warriors Appreciation Run from the Harley-Davidson dealership in Irvine to Camp Pendleton. The event, sponsored by the Orange Coast Chapter of the Harley Owners Group, raised more than $14,000 through registrations, sponsorships and online donations for the Warrior Foundation.
The foundation, based in San Diego, helps injured service members at Southern California military medical centers such as Camp Pendleton's Naval Hospital, said Sandy Lehmkuhler, who heads up the all-volunteer organization. Part of the money raised from the run will help more than 400 service members travel home for the holidays this year, she said.
Kuikahi, who was wounded in Iraq, is one of more than 400 Marines with the Wounded Warrior Battalion West, activated in 2007. The battalion provides recovery support to Marines, said Lt. Col Jim Fallwood, the battalion's commanding officer.
Kuikah said he plans to leave the battalion in January to pursue a law degree. He credits the battalion with preparing him to transition to civilian life.
He said he was humbled by what the Harley riders are doing to help.
"It's a blessing for us just to have these kinds of people," he said, downing a burger and salad at a barbecue that followed a presentation to the Harley Owners Group. "I'm already proud of being a Marine but to see this appreciation makes it even better."
Karen Davidson, great-granddaughter of the company co-founder William Davidson, led the procession to Pendleton.
"I'm thrilled to be a part of this," Davidson said. "Harley has always had a history with the military. We share a common bond and love for this country. You appreciate it on a different level when you're behind the handlebars and up close to nature. We have a deep appreciation for freedom. I can't help but appreciate what they do for freedom throughout the world."
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Working towards a sustainable and productive catchment
Koalas in the Gympie region
The Gympie Region Koala Action Group was established to encourage the conservation and protection of Koalas and their habitat in the Gympie region. The Koala Action Group seeks to ensure the conservation of the vulnerable koala and koala habitat through activities/actions and education that enhances their protection and sustainability and that … Continue reading →
Spring in the Mary Photo Competition winners announced
Saturday's Mary River Festival was intended as the place for the winners of the MRCCC's inaugural Spring in the Mary photo competition to be announced. The weather, unfortunately, had other ideas and everyone was sent packing by about 4 pm, when the rain and wind took over. The Festival did … Continue reading →
Mary River Festival – Kandanga, 14th November 2015
Mary River Festival 2015 The festival continues to capture the enormous sense relief of six years back when then-federal Environment Minister Peter Garrett vetoed the ill-conceived Traveston Crossing Dam. Mary River Catchment Coordinating Committee Chairman Ian Mackay, acting as one of the MCs for the day, told patrons the festival … Continue reading →


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Halloween genes and nuclear receptors in ecdysteroid biosynthesis and signalling in the pea aphid
Article first published online: 23 FEB 2010
DOI: 10.1111/j.1365-2583.2009.00957.x
© 2010 The Authors. Journal compilation © 2010 The Royal Entomological Society
Insect Molecular Biology
Special Issue: The Aphid Genome
Volume 19, Issue Supplement s2, pages 187–200, March 2010
Additional Information
How to Cite
Christiaens, O., Iga, M., Velarde, R. A., Rougé, P. and Smagghe, G. (2010), Halloween genes and nuclear receptors in ecdysteroid biosynthesis and signalling in the pea aphid. Insect Molecular Biology, 19: 187–200. doi: 10.1111/j.1365-2583.2009.00957.x
Publication History
- Issue published online: 23 FEB 2010
- Article first published online: 23 FEB 2010
- ecdysteroid biosynthesis;
- nuclear receptors;
- ecdysteroid cascade;
- pea aphid;
- development;
- moulting;
- metamorphosis
- Top of page
- Abstract
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
The pea aphid (Acyrthosiphon pisum) is the first whole genome sequenced insect with a hemimetabolic development and an emerging model organism for studies in ecology, evolution and development. The insect steroid moulting hormone 20-hydroxyecdysone (20E) controls and coordinates development in insects, especially the moulting/metamorphosis process. We, therefore present here a comprehensive characterization of the Halloween genes phantom, disembodied, shadow, shade, spook and spookiest, coding for the P450 enzymes that control the biosynthesis of 20E. Regarding the presence of nuclear receptors in the pea aphid genome, we found 19 genes, representing all of the seven known subfamilies. The annotation and phylogenetic analysis revealed a strong conservation in the class of Insecta. But compared with other sequenced insect genomes, three orthologues are missing in the Acyrthosiphon genome, namely HR96, PNR-like and Knirps. We also cloned the EcR, Usp, E75 and HR3. Finally, 3D-modelling of the ligand-binding domain of Ap-EcR exhibited the typical canonical structural scaffold with 12 α-helices associated with a short hairpin of two antiparallel β-strands. Upon docking, 20E was located in the hormone-binding groove, supporting the hypothesis that EcR has a role in 20E signalling.
- Top of page
- Abstract
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
The insect steroid hormone, 20-hydroxyecdysone (20E) controls and coordinates the development in insects. A peak titer of 20E triggers the moulting/metamorphosis process, allowing insects to lose their old exoskeleton and enter into the next developmental stage. To produce the steroid hormone 20E, the precursor hormone ecdysone (E) is synthesized in the prothoracic glands from dietary cholesterol or phytosterols as insects cannot synthesize the steroid precursor cholesterol de novo. This E is then secreted into the body cavity and converted into 20E in various peripheral tissues predominantly in the insect midgut and fat body (Petryk et al., 2003; Rewitz et al., 2006a; Iga & Smagghe, 2009). The whole trafficking mechanism of the ecdysteroid precursors has not been elucidated yet in insects, but it may use similar mechanisms as the steroidogenesis in vertebrates (Rees, 1995; Gilbert et al., 2002; Lafont et al., 2005; Huang et al., 2008).
Cytochrome P450 (CYP) enzymes, well known for their monooxygenase activity, constitute one of the largest families and are distributed throughout a wide variety of living organisms, from bacteria to mammals (Werck-Reichhart & Feyereisen, 2000). To date, four P450 enzymes, namely CYP306A1 (Phantom, Phm), CYP302A1 (Disembodied, Dib), CYP315A1 (Shadow, Sad) and CYP314A1 (Shade, Shd), involved in the ecdysteroid biosynthesis have been identified and characterized. As shown in Fig. 1, the products of phm, dib and sad sequentially convert the precursor of E, 2,22,25-trideoxyecdysone (ketodiol), into 22,2-dideoxyecdysone (ketotriol), 2-deoxyecdysone and E (Chavez et al., 2000; Warren et al., 2002, 2004; Niwa et al., 2004, 2005; Rewitz et al., 2006b). Further on, the product of shd mediates the last step of the conversion from E into 20E (Petryk et al., 2003; Rewitz et al., 2006a; Maeda et al., 2008). In addition, CYP307A1 (Spook, Spo), the paralogue gene of Spo, CYP307A2 (Spookier, Spok) and CYP307B1 (Spookiest, Spot), involved in the initial conversion process from 7-dehydrochoresterol into ketodiol are identified, but their biochemical functions are not well understood (Namiki et al., 2005; Ono et al., 2006). Spok has so far only been identified in Drosophila, while spot is identified in mosquitoes (Aedes aegypti and Anopheles gambiae), honey bees (Apis mellifera) and red flour beetles (Tribolium castaneum). Together, they are called the Halloween genes. The Halloween genes have been identified/predicted in multiple insect species (Niwa et al., 2004, 2005; Warren et al., 2004; Sieglaff et al., 2005; Rewitz et al., 2006a,b, 2007; Iga and Smagghe, 2009) and the function of these genes is characterized in the fruitfly (Drosophila melanogaster), the silkmoth (Bombyx mori) and the tobacco hornworm (Manduca sexta). In addition to insects, the Halloween genes are also identified in the crustacean genome of Daphnia pulex (Rewitz & Gilbert, 2008), suggesting a high conservation for ecdysteroid biosynthesis in the Arthropoda phylum.
After secretion into the hemolymph, 20E will start the moulting/metamorphosis process by acting directly upon the transcriptional activity of specific target genes through chromosome puffing. Ashburner (1973) proposed a formal model to explain control of the transcription of the vast network of genes whose activity is induced by the hormone. As shown in Fig. 1, the first step in this cascade is the binding of 20E to a postulated receptor protein, a heterodimer formed by the ecdysone receptor (EcR) and Ultraspiracle (Usp), which are both members of the nuclear receptor (NR) superfamily (Henrich, 2005; Billas et al., 2009). Activation of this receptor complex initiates and mediates the transcription of a number of other NRs, in a cascade with at first expression of 'early', then 'early-late' and finally 'late' genes for a successful moulting/metamorphosis.
The NR superfamily is a group of ligand-activated transcription factors which are present in various animals. Studies of these NRs in both mammals and arthropods revealed seven distinct subfamilies (NR0-NR6) in which these NRs can be classified. All of them possess a highly-conserved DNA-binding domain (DBD), containing two C4-type zinc finger regions, that is responsible for binding of the transcription factor to the DNA. Except the NR0 superfamily, all NRs also contain a less-conserved ligand binding domain (LBD) with which the receptor is able to bind its ligand. Unlike most other transcription factors, NRs can be activated by binding of small lipophilic ligands such as hormones and fatty acids that are capable of going through the cell membrane. Besides moulting and metamorphosis, NRs are involved in, e.g. embryonic development (King-Jones & Thummel, 2005), cell differentiation (Siaussat et al., 2007), reproduction (Raikhel et al., 1999), and are therefore also considered as important novel targets in pest insect control (Palli et al., 2005; Billas et al., 2009).
Recent genome projects of both vertebrate and insect species contributed a lot in identifying the different NRs. In total, 48 NRs are known in humans (Robinson-Rechavi et al., 2001), over 284 are present in Caenorhabditis elegans (Gissendanner et al., 2004), 49 in mouse and 47 in rat (Zhang et al., 2004). In insects on the other hand, the number of NRs found is surprisingly lower. In Drosophila, only 21 NR genes have been identified, 20 in Anopheles, 22 in Apis, 19 in Bombyx and 21 in Tribolium (Adams et al., 2000; Holt et al., 2002; Velarde et al., 2006; Bonneton et al., 2008; Cheng et al., 2008). The latter studies also showed that these NRs, especially the DBD and LBD, are highly conserved in holometabolous insects. So far, however, no complete set of NRs from hemimetabolous insects, which are indirect developers that do not undergo a pupal metamorphosis stage, has been described. The recent genome project on the pea aphid, Acyrthosiphon pisum (International Aphid Genomic Consortium, 2010) gives us the unique opportunity to present a comprehensive identification and characterization of the NRs in this important hemipteran insect, which is an emerging model organism for ecological, developmental and evolutionary studies (Brisson & Stern, 2006; Stern, 2008).
In this paper, we will first focus on the Halloween genes that control the ecdysteroid biosynthesis pathway to build up a peak titer of 20E hormone. In a second part, we will unravel the presence of the NRs in the pea aphid with an emphasis on understanding and identifying the pathway of hormone signalling by 20E through a regulatory cascade of NRs – especially the functional receptor formed by the heterodimer EcR-Usp – and also on 'early', 'early/late' and 'late' genes. We performed a phylogenetic analysis to confirm the annotation and to investigate evolutionary traits of the pea aphid in the phylum of Arthropoda. Besides, we also cloned the EcR, Usp, E75 and HR3. Finally, we constructed a 3D-modelling of the LBD of Ap-EcR to evaluate if it exhibits the typical canonical structural scaffold with 12 α-helices, and then performed a ligand docking to support the theory that EcR has a role in 20E hormone signalling.
Results and discussion
- Top of page
- Abstract
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Phylogenetic analysis of Halloween genes
The candidates of the A. pisum Halloween genes were obtained from AphidBase (http://www.aphidbase.com/aphidbase/) by TBLASTN using the amino acid sequence of Apis mellifera and T. castaneum. We found three candidates of A. pisum spook (Ap-spo1, Ap-spo2 and Ap-spo3), one candidate for phantom (Ap-phm) and disembodied (Ap-dib) and also three candidates for shade (Ap-shd1, Ap-shd2 and Ap-shd3). The AphidBase ID and cross reference number are shown in Table 1. The expression of these predicted sequences was confirmed by reverse transcriptase-PCR (RT-PCR) (data not shown).
|D. melanogaster (Dm)
|T. castaneum (Tc)
|A. mellifera (Am)
|AphidBase ID
|identity %
|spook / spookier
The predicted sequences have an open reading frame (ORF) encoding the putative protein of Ap-Spo1 (518 amino acids), Ap-Spo2 (528 amino acids), Ap-Spo3 (507 amino acids), Ap-Phm (492 amino acids), Ap-Dib (493 amino acids), Ap-Sad (443 amino acids), Ap-Shd1 (518 amino acids), Ap-Shd2 (518 amino acids) and Ap-Shd3 (505 amino acids). The product size of these candidates is consistent with the character of the different CYP products (approx. 500 amino acids). Alignment of the different A. pisum Halloween genes candidates with those of other insect orders (Lepidoptera, Diptera, Hymenoptera and Coleoptera) show high conservation of insect P450 motifs (helix-C, helix-I, helix-K, PERF-motif and heme-binding domain) (Fig. S1A–E). For Spo, the helix-C and helix-I structures that are usually so typical for P450 proteins were not well conserved but this is consistent with the P450 proteins in other members of the class of Insecta. Only the heme-binding domain of Ap-Shd3 shows a significant difference compared with that of other insect orthologs. The Ap-Shd3 completely lacked the sequence of the heme-binding domain which means the protein may not be functional at all. RT-PCR showed however, that the protein is expressed in the pea aphid (data not shown). This leads to the hypothesis that the protein might have other functions or another role in the pea aphid than just the ones attributed to the Shd proteins so far.
The result of phylogenetic analysis shows two classes: the 2 Clan with Spo/Spok/Spot and Phm, and the Mito Clan with Dib, Sad and Shd (Fig. 2). In the A. pisum genome we detected two Spo-like products, Ap-Spo1 and Ap-Spo2. Both are on the same branch as Spo orthologues of other species, and sequence comparison shows that both Ap-Spo1 and Ap-Spo2 are quite similar to each other (85% identity). Ap-Spo3, however, is very different from Ap-Spo1 and Ap-Spo2, showing only 38% and 39% identity, respectively. When we compare with other Halloween genes, we notice that Ap-Spo3 shows 33–42% identity with Spo/Spok orthologues, and 44–47% identity with Spot orthologues, suggesting Ap-Spo3 might be a Spot orthologue rather than a Spo orthologue. In addition, phylogenetic analysis confirms this hypothesis since both Ap-Spo3 and Spot orthologues are branched together (Fig. 2). Three Shd candidates were identified in A. pisum. Two of them, Ap-Shd1 and Ap-Shd2 show a high conservation (94% identity), suggesting these could be duplicated genes, while another Shd candidate, Ap-Shd3, only shows 66% identity with both Ap-Shd1 and Ap-Shd2. Ap-Shd1 and Ap-Shd2 show 38–51% and 38–50% identity with Shd orthologues, respectively, while Ap-Shd3 exhibits 34–44% identity to those same Shd orthologues. As we described before, Ap-Shd3 will probably not function as a P450 enzyme since it lacks the necessary heme-binding domain which is important for P450 enzyme activity. We can therefore assume that only Ap-Shd1 and/or Ap-Shd2 are likely to be responsible for converting E into 20E.
Identification of the nuclear receptors in the genome of Acyrthosiphon pisum displays strong conservation in insects
All available Gene prediction sets (Gnomon, Augustus, Genscan, GeneID) and all available A. pisum sequence data were used to identify the NRs in the pea aphid genome. The in silico detection of NRs in the genome is greatly facilitated by the strongly conserved DBD and LBD regions that characterize these NRs (Table 2). Blast searches were performed using peptide sequences of all known NRs from D. melanogaster, Apis mellifera and T. castaneum. As a result, an initial 20 NR sequences were identified in the A. pisum genome, representing all of the seven NR subfamilies. Predicted mRNA sequences and gene models were also manually edited if necessary using the Apollo Genome Annotation Curation Tool (Lewis et al., 2002). After further analysis, two NR0 sequences turned out to be duplicated Knirps-like (Kni-like) genes, which bring the total set of different NR genes to 19.
|Drosophila melanogaster
|Tribolium castaneum
|Apis mellifera
|AphidBase ID
|Dm/Ap identity %
|Tc/Ap identity %
|Am/Ap identity %
|Ecdysone-induced protein 75
|Ecdysone-induced protein 78
|Hormone receptor like in 46
|Ecdysone receptor
|Hepatocyte nuclear factor 4
|Hormone receptor like in 78
|no orthologue
|Hormone receptor 51
|Hormone Receptor 83
|no orthologue
|Seven up
|Estrogen-related receptor
|Hormone receptor like in 38
|Fushi tarazu transcription factor 1
|no orthologue
|Hormone receptor like in 39
|Hormone receptor 4
|no LBD
|no LBD
|no LBD
|no orthologue
|no LBD
|no LBD
|no LBD
|Hormone receptor 96
|not present
|not present
|Photoreceptor specific NR
|no orthologue
|not present
|not present
|no orthologue
|no orthologue
|not present
|not present
RT-PCR was used to confirm the presence of the predicted NR mRNAs in the transcriptome of the pea aphid (data not shown). All NR mRNAs were picked up by RT-PCR, except for the HR83 gene. We did not manage to get a conclusive result for this gene despite using several different primer pairs. Some of them resulted in clear single bands, but the fragment size was not as would be predicted based on the annotated gene. This means that either we picked up a wrong fragment, or the exon/intron prediction of the gene is incorrect. Further sequencing of this fragment should give us more information about the identity of this fragment and about the transcription of this gene. These results prove that the complete set of NRs found in the A. pisum genome, except HR83, is transcribed and none of them are pseudogenes.
Table 2 presents all the pea aphid orthologues for each of the previously annotated D. melanogaster (Adams et al., 2000), Apis mellifera (Velarde et al., 2006), B. mori (Cheng et al., 2008) and T. castaneum (Bonneton et al., 2008) NRs. Similar numbers of NRs were found in these five insect genomes. All NRs are also structurally very similar to their orthologues. All of them possess a DBD and LBD, except for the NR0 subfamily, which only contains a DBD. As could be predicted from previous analyses of NRs, pairwise alignments of the conserved domains of D. melanogaster and A. pisum NRs show a very high (71–99%) convergence for DBDs while the LBDs are more divergent (26–97%; with 77% identity for HR39 being the second highest). The most divergent NRs are HR83 (NR2E5) and TLL (NR2E2), while SVP (NR2F3) shows the least divergence.
In general, these results prove that NRs have a very strong conservation among insects, also outside the holometabolous insect group. All pea aphid NRs show similar identity percentages for its orthologues as the identity percentages which were reported in earlier NR annotation publications, where the NRs in T. castaneum and Apis mellifera were compared with the NRs in D. melanogaster, even though we would expect bigger differences based on the evolutionary distances of these species. The NRs that are part of the 20E regulatory cascade, the 'early' gene E75 (NR1D3) and the 'early-late' genes HR3 (NR1F4), HR4 (NR2A4), HR38 (NR4A4), E78 (NR1E1) and FTZ-F1 (NR5A3) also show the same kind of convergence as reported with other species, demonstrating that all the main NR members of this cascade are present in the pea aphid. One remarkable observation was the extremely high conservation of the SVP-LBD among insects, much more than for the other NRs (97%, 96% and 99% compared with D. melanogaster, T. castaneum and Apis mellifera orthologues, respectively). The latter phenomenon may suggest that the structure of SVP, the insect orthologue of the vertebrate chicken ovalbumin upstream transcription factor (COUP-TF), is critical to its function and is under strong selective pressure against amino acid replacements in the LBD of the molecule. In D. melanogaster, where two isoforms of this protein are expressed, SVP has multiple reported functions. It is required for the development of four of the eight photoreceptors that develop in the ommatidia of the eye (Hiromi et al., 1993; Begemann et al., 1995; Kramer et al., 1995), it is a key component in the control of cell proliferation in Malpighian tubules (Kerber et al., 1998) and it also has an important role as a regulator in the development of neuroblasts by acting upon the Hunchback/Krüppel switch necessary for neuroblast differentiation (Kanai et al., 2005). In Ae. aegypti, this protein also has an effect on the vitellogenesis by acting as a negative regulator in the ecdysone receptor complex-mediated transactivation in the fat body (Miura et al., 2002).
Three NRs which were previously found in other insect species seem to be missing in the A. pisum genome: namely the NR1 subfamily member HR96, the NR0 subfamily member Knirps (Kni), and the NR2 family member PNR-like (NR2E6).
HR96 is an orphan receptor belonging to the NR1 subfamily (NR1J1). It is closely related to the EcR itself and is believed to be related to the vertebrate vitamin D-receptor (VDR), PXR and CAR, all of which bind a wide variety of xenobiotics (Laudet, 1997). HR96 is proven to play a role in the response of D. melanogaster to xenobiotics as reported by King-Jones et al. (2006), but a function regarding development or metamorphosis has not been reported yet. This NR is a part of the 20E signalling cascade and it is known that this ecdysteroid-induced NR can bind to the hp27 20E response element. This suggests that HR96 can compete with EcR-Usp for binding to a common set of target sequences (Fisk & Thummel, 1995), but since this NR has no known hormone ligand, it is difficult to speculate on its actual function regarding the ecdysteroid cascade. The absence of this gene suggests its role in this signalling pathway is redundant in aphids or being taken over by another protein or NR.
A member of the NR2 group that was initially identified in the honey bee, the NR2E6, and an orthologue for vertebrate photoreceptor-cell-specific nuclear receptors (PNRs), is missing in the A. pisum genome. This gene is also missing in the Drosophila genomes, although it has been identified in the T. castaneum genome. The absence of NR2E6 in the A. pisum and Drosophila genomes is a secondary loss in these lineages. A function for NR2E6 in the development of the compound eye has been proposed based on mRNA 'in situ' localizations in the Apis mellifera developing compound eyes (Velarde et al., 2006). The fact that A. pisum shares with D. melanogaster the vast majority of the genes involved in compound eye differentiation (Shigenobu et al., 2009) suggests this gene has been retained potentially to regulate lineage specific differences in compound eye architectures.
The third missing NR in Acyrthosiphon is Knirps (NR0A1), while we identified two paralogues of Knirps-like and one orthologue of Eagle. The T. castaneum, Apis mellifera, B. mori genomes also seem to lack a Knirps orthologue gene, as seen in the phylogenetic tree of the NR0 subfamily members. In Drosophila, Knirps has been characterized as encoding a transcriptional repressor important for the segmentation pathway (Nauber et al., 1988). The two other genes in the NR0 group Knirps-like (knrl) and Eagle (egon) are present in the A. pisum genome. Analysis of these genes in the honey bee suggested no direct involvement during segmentation (Dearden et al., 2006), as is the case in Drosophila. However, in the case of T. castaneum, Knirps-like has been characterized as having specific functions during head segmentation (Cerny et al., 2008). Our A. pisum analysis supports the notion that these genes have been independently duplicated in different insect lineages. At least in the case of Dipterans and Coleopterans, Knirps and Knirps-like have retained an ancestral role during segmentation, which has been likely lost from honey bees and pea aphids. Knirps-like and Eagle may also function as transcriptional repressors, but it remains to be determined in which pathways they participate.
Further phylogenetic analysis for nuclear receptors of pea aphid in phylum of Arthropoda
Besides the phylogenetic analysis of the NR0 subfamily (Fig. 3), NRs from the 6 other different subfamilies (NR1–NR6) were also examined by phylogenetic analysis and compared with NRs from several different species representing the major insect orders, such as Lepidoptera, Diptera, Hymenoptera, Coleoptera, and also from Crustacea and Arachnida (Fig. 4). This phylogenetic analysis showed that many of the NRs of A. pisum show close relationship with the NRs of the human louse (Pediculus humanus), as has been observed for genes throughout the genome. The T. castaneum NRs also showed very high convergence with both the A. pisum and P. humanus NRs for a number of NRs, even though the red flour beetle is a member of the Endopterygota, while the pea aphid and the human louse belong to the infraclass of the Paraneoptera.
When we look at these phylogenetic trees in Fig. 4 in detail, we notice that A. pisum NRs show a much higher resemblance to the T. castaneum and Apis mellifera orthologues than to the Diptera and Lepidoptera NRs, which often cluster together in a separate branch, even branching off before the Crustacea and Arachnida. This deviation from normal topology, as shown in the trees of Fig. 4A and B for EcR and E78, respectively, is due to a long branch attraction caused by an acceleration of evolutionary rate in the Mecopterida line (Diptera + Lepidoptera). This is consistent with the earlier findings of Bonneton et al. (2008) who have discovered that some NRs in Mecopterida species (Diptera + Lepidoptera), including EcR and E78, have undergone an increase in evolutionary rate. Other phylogenetic trees also confirm their results (data not shown).
In order to distinguish the different NR2 subfamily genes found in the genome, we also constructed phylogenetic trees for this entire subfamily. The NR2 subfamily tree (Fig. 5) clearly shows that the PNR-orthologue, found in T. castaneum and Apis mellifera is missing in the pea aphid. Genes are clustered together according to the group (A–F) they belong to.
Elements of the 20-hydroxyecdysone regulatory cascade
The 20E signalling cascade, as mentioned earlier in this work, is involved in moulting/metamorphosis and development. NRs play a very important role in this signalling pathway. Binding of 20E to the EcR-Usp heterodimer is the start of this signal. This complex, after binding to the hormone, will act as a transcription factor, immediately inducing expression of a number of 'early' genes, including the NRs E75 and HR96. These 'early' gene products will then be responsible for the upregulation of a set of 'early-late' genes, including the NRs HR3, HR4, E78 and HR39. Through FTZ-F1, this signal will then be passed on to induce expression of the 'late' genes (Fig. 1; Table 2). So far, most attention in this field has gone to holometabolous insects, which undergo a pupal metamorphosis stage. No extensive set of NRs for a hemimetabolous insect has been identified until now. Even though the pea aphid still undergoes several larval stages, it is possible that there are differences between the moulting processes of hemi- and holometabolous insects.
Most of the NRs involved in the 20E regulatory cascade proved to be present, not only in the genome of pea aphid, but also in its transcriptome, indicating they are expressed correctly. Only the HR96 gene, which was discussed above, is missing from this set of ecdysone-inducible NRs. And since its function in the moulting/metamorphosis processes is still unclear, speculation about the implications for the entire pathway are very difficult to make.
EcR and Usp, two NRs that are at the basis of the 20E signalling cascade, were cloned and sequenced in order to confirm the annotation and sequence of both genes (Fig. S2A, B). Primers used to pick up the fragment spanning most of the cDNA are listed in Table S1. The EcR in A. pisum has the typical DBD and LBD found in its orthologues in other insects. The EcR-DBD shows the typical C4 zinc finger domains in this protein, as is the case for the P-box, the D-box and the A/T-box. Regarding the EcR-LBD we score a strong conservation, with 63%, 75% and 74% identity compared with the Drosophila, Tribolium and Apis orthologues, respectively. Furthermore, the typical structure with 12 α-helices is well conserved. Both retrieved sequences confirmed the in silico analysis. Two other important NRs in the 20E signalling cascade, the so called 'early' genes, E75 and HR3 were also cloned and partially sequenced in order to confirm their presence in the transcriptome (Fig. S2C, D).
3D-modelling of the ligand binding pocket of Ap-EcR and ligand docking
The 3D model built for Ap-EcR-LBD exhibits the canonical structural scaffold of the EcR-LDBs, made of 12 α-helices associated with a short hairpin of two antiparallel β-strands (Fig. 6A). In addition, docking of 20E into the hormone-binding groove of Ap-EcR-LBD revealed a binding scheme similar to that found for other EcR-LBD (e.g. from the beetles Leptinotarsa decemlineata, Tenebrio molitor and Anthonomus grandis) (Billas et al., 2003; Soin et al., 2009). Upon docking, the alkyl chain of the hormone becomes inserted into one of the two pockets located at the bottom of the hormone-binding groove (Fig. 6B). A network of nine hydrogen bonds connects the hormone to residues Glu20, Met56, Thr57, Ala 112 and Tyr122, forming the binding groove (Fig. 6C). Stacking interactions with aromatic residues Phe111 and Trp238 help to complete the interaction.
Experimental procedures
- Top of page
- Abstract
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Annotation of Halloween and nuclear receptor genes
The 1.0 release of the A. pisum genome was used as a basis for the bioinformatic analysis. Putative Halloween genes sequences were searched and obtained by TBLASTN, using the known orthologues from Apis mellifera and T. castaneum against the complete scaffold collection of the pea aphid genome.
NR protein sequences from D. melanogaster, T. castaneum and Apis mellifera were first used in BlastP searches against the NCBI Gnomon version 1 predicted protein sequences to find putative A. pisum orthologues. In case no orthologues were found in the pea aphid Gnomon protein data set, we searched the Acyr 1.0 assembly of the pea aphid genome (International Aphid Genomic Consortium, 2010, main paper) for homologous sequences using TBLASTN. After identification and localization in the genome, genes were examined and manually edited if necessary using the Apollo Genome Annotation Curation Tool (Lewis et al., 2002). This editing was done based on alignments of D. melanogaster, T. castaneum and Apis mellifera orthologues together with several gene prediction programs (Gnomon, Augustus, Genscan and GeneID).
Phylogenetic analysis
Whole amino acid sequences for the Halloween gene orthologues in Apis mellifera, T. castaneum, D. melanogaster, Ae. aegypti, An. gambiae, M. sexta and B. mori and for the nuclear receptors of D. melanogaster, T. castaneum and Apis mellifera were collected from the GenBank database. The LBD and DBD sequences of B. mori, An. gambiae, Ae. aegypti, Culex quinquefasciatus, P. humanus corporis and Daphnia magna were retrieved by Blast searches of A. pisum LBD sequences against the GenBank database or against the species' sequenced genome if no GenBank entry was present. The chosen NR sequences were then aligned by CLUSTALW2/CLUSTALX2 (Larkin et al., 2007). The trees were made by the neighbour-joining method using MEGA4 software (Tamura et al., 2007). Bootstrap analysis with 1000 replicates for each branch position was used to assess support for nodes in the tree (Felsenstein, 1985).
Confirmation of transcription of the Halloween and NR genes
Presence of these transcripts in the A. pisum RNA was examined by RT-PCR. The pea aphids were taken from a continuous colony in the Laboratory of Agrozoology at Ghent University. A mixture of different stages of A. pisum and a collection of newborn aphids only was used to extract total RNA using the TRI Reagent (Sigma, Bornem, Belgium), based on the single-step liquid phase separation method reported by Chomczynski & Sacchi (1987). Then, cDNA was synthesized from 1 µg of this RNA in a 20 µl reaction using the First Strand cDNA synthesis kit (Roche, Berlin, Germany) according to the manufacturer's instructions. Both cDNA samples (newborn only and a mixture of stages) were used in these RT-PCR experiments. Primers were designed using Primer3 software (Rozen & Skaletsky, 2000) and are listed in Table S1.
Cloning and sequencing of EcR and USP, E75 and HR3 genes
Pea aphid EcR and Usp, E75 and HR3 were isolated by RT-PCR and afterwards sequenced. Same A. pisum cDNA as used in 2.3 was used for the initial PCR reactions. The PCR products were then purified using the Cycle Pure kit (Omega Bio-Tek, Norcross, GA, USA) and were ligated into a pGEM-T vector (Promega, Madison, WI, USA) according to the manufacturer's instructions. Afterwards, plasmids were transformed in competent Escherichia coli XL-1 Blue Cells by heat shock and then plated out on a carbenicillin-containing LB agar plate. After 16 h incubation, formed colonies were checked by colony PCR and several of these positive colonies were then purified using Plasmid mini prep kit (Omega Bio-Tek) and sent for sequencing (Agowa, Berlin, Germany).
3D-modelling of the ligand binding pocket of Ap-EcR and ligand docking
Multiple amino acid sequence alignments were carried out with CLUSTAL-X (Thompson et al., 1997) using the Risler's structural matrix for homologous amino acid residues (Risler et al., 1998). Molecular modelling of the EcR ligand-binding domain (EcR-LBD) from the pea aphid (Acces. NP_001152831.1), Ap-EcR-LBD, was performed on a Silicon Graphics O2 R10000 workstation, using the programs InsightII, Homology and Discover3 (Accelrys, San Diego, CA, USA). The atomic coordinates of Tribolium Tc-EcR-LBD in complex with ecdysone (RCSB Protein Data Bank code 2NXX) (Iwema et al., 2007) were used to build the 3D model of the receptor. The high percentages of both identity (∼75%) and similarity (∼90%) that ApEcR-LBD shares with the template Tc-EcR-LBD allowed us to build quite an accurate 3D model. Steric conflicts were corrected during the model building procedure using the rotamer library (Ponder & Richards, 1987) and the search algorithm of the Homology program (Mas et al., 1992) to maintain proper side-chain orientation. An energy minimization of the final model was carried out by 150 cycles of steepest descent using the cvff forcefield of Discover. PROCHECK (Laskowski et al., 1993) was used to assess the geometric quality of the 3D model. In this respect, about 87% of the residues of the modelled Ap-EcR-LBD were correctly assigned to the best-allowed regions of the Ramachandran plot. The remaining residues were located in the generously allowed regions of the plot except for three residues (Asn36, Glu39 and Glu42), which occur in the non-allowed region (result not shown). Molecular cartoons were drawn with PyMol (W.L. DeLano, http://pymol.sourceforge.net). The fold recognition program Phyre (http://www.sbg.bio.ic.ac.uk/phyre/html/index.html) (Bennett-Lovsey et al., 2008), which also used 2NXX and structurally related proteins as templates, yielded a readily superposable 3D model for Ap-EcR-LBD. However, some discrepancies that essentially deal with the shape of the loops, connecting the α-helical stretches, were observed with our lab-made, modelled structure. Importantly, these discrepancies occur far from the groove responsible for the binding of ecdysone.
Docking was performed with InsightII using Discover3 as a forcefield and we took TcEcR-LBD in complex with ecdysone as a template for docking. Clipping planes of Ap-EcR-LBD complexed to 20E were rendered with PyMol.
- Top of page
- Abstract
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
The authors are grateful for the support of the Special Research Fund of Ghent University and the Fund of Scientific Research (FWO-Vlaanderen, Belgium) to GS. PR acknowledges the financial support of Université Paul Sabatier and CNRS.
- Top of page
- Abstract
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
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Supporting Information
- Top of page
- Abstract
- Introduction
- Results and discussion
- Experimental procedures
- Acknowledgements
- References
- Supporting Information
Figure S1. Amino acid sequence alignment of Spo/Spok/Spot (A), Phm (B), Dib (C) Sad (D) and Shd (E). Residues in black are identities and in gray are similarities. The conserved P450 motifs are indicated: helix-C, helix-I, helix-K, PERF-motif and heme-binding domain.
Figure S2. Amino acid sequences of the cloned and sequenced fragments of EcR (A), Usp (B), E75 (C) and HR3 (D). For EcR and Usp, the entire DBD has been sequenced and the P box (yellow), D box (red) and T/A box (teal) are indicated. The eight zinc-coordinating cysteines in the DBD are boxed in the sequence. For E75, only a part of the DBD has been sequenced.
Table S1. Primers used for the RT-PCR detection in the transcriptome of the Halloween (upper part) and nuclear receptor genes and for the cloning of EcR, Usp, E75 and HR3 (lower part) in the pea aphid. The primers were designed to overlap at least one intron, except for the KNRL and EG genes, which mainly consist out of one big exon and only possess a very small second exon. Expected fragment sizes are also indicated in the table
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text:
Exodus from Facebook? Friends beg to differ
Wow, Facebook is so over -- really. Virginia Heffernan said so in The New York Times. She should know, she writes about these kinds of things. A lot. She wins awards, too.
Here's what she said last week: "Things fall apart; the center cannot hold. Facebook, the online social grid, could not command loyalty forever. If you ask around, as I did, you'll find quitters. One person shut down her account because she disliked how nosy it made her. Another thought the scene had turned desperate. A third feared stalkers. A fourth believed his privacy was compromised. A fifth disappeared without a word."
So I'm looking for some facts and figures. You know, data. She gets close. Or not. You be the judge: "The exodus is not evident from the site's overall numbers. According to comScore, Facebook attracted 87.7 million unique visitors in the United States in July. But while people are still joining Facebook and compulsively visiting the site, a small but noticeable group are fleeing -- some of them ostentatiously." Oy. "Small but noticeable."
Small? Can't tell -- did I mention there's no numbers? Noticeable, maybe, now that Heffernan has brought in the mighty Klieg lights from the folks who cover "all the news that's fit to print." Heffernan's Exhibit A is one Leif Harmsen, who "crusades" against Facebook. It's sucking his soul away. Or something. There's a slavery analogy in there, too. And then she talks with her friends. Her friends.
"Postings that seem private can scatter and slip," one says.
"I felt fairly detached from my Facebook buddies because I rarely directly contacted them," says another.
"I have noticed the exodus, and I kind of feel like it's kids getting tired of a new toy," another Facebooker says in an e-mail.
Exodus? A biblical kind of mass migration out of slavery? Here's what we know. Facebook boasts more than 250 million registered users. More than $600 million in investment capital has been sunk into the site, which now has an estimated value of $10 billion. ComScore ranked Facebook #5 in its Top 50 U.S. Web properties for July, behind only Google, Yahoo, Microsoft and AOL, but ahead of Fox Interactive (which includes MySpace), it's highest showing ever with almost 88 million unique visitors for the month. And Bloomberg News reported in late August that "Facebook may expand its staff by 40 percent to 50 percent this year as it benefits from a surplus of engineers amid the recession." That's according to Chief Executive Officer Mark Zuckerberg.
That doesn't sound like a company suffering from an exodus of customers. But in the spirit of Heffernan's piece, I should say that I do know people who have signed up, taken a look at postings from friends about soggy cereal and profound quotes from 80s hair bands, said "yikes" and bailed out. I also asked a few acquaintances if they thought Facebook was over, or if they would quit.
"No. How's that for an answer?" said Dusty Trice, a peripatetic blogger/tweeter/status updater who worked for Al Franken's Senate campaign. In fact, Trice figures he couldn't live -- or get noticed -- nearly as well without it. And the guy knows how to draw an audience. His "personal branding tool" is hosting his latest gimmick: He's challenged himself to eat 50 corn dogs in 12 days at the State Fair, and posting the results of every one on his Facebook page. (Current corn dog count is 28, for those of you keeping score at home.)
"I could easily live without Facebook, but it is something I enjoy in many ways," said Ed Timek, who works in radio ad sales in the Twin Cities. "First off, lots of my friends post interesting articles that I might not see if it wasn't for Facebook. Secondly, I've connected with so many people that I hadn't spoken to in years, and its been a lot of fun. Yes, there are some people I probably didn't need to reconnect with, but for the most part, its all positive."
"I'd say rumors of Facebook's demise are terribly exaggerated," said Chris Truscott, an assistant professor of political science at Brown College in Mendota Heights. "Yes, Facebook is commercial, but what isn't? My bank sends me things based on what I buy with my Visa. What Facebook's doing isn't any different. Like The New York Times, it's not going anywhere."
Besides, if we cancel our subscriptions, there might be a mass exodus.
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The Hershey volleyball team finished the regular season on Oct. 23 by splitting a pair of matches in Kearney.
Hershey was no match for Kearney Catholic in the first match and for good reason. The Stars are ranked No. 4 in the Omaha World-Heralds all-class state rankings and are the favorites to win its third straight championship in Class C1.
Panther coach Amy Beyer knew it was going to be a tough match for her team.
Kearney Catholic is the real deal, Beyer said.
After getting pounded by Kearney Catholic, Hershey finished the day on a good note by beating Broken Bow (11-18).
We played pretty well in the first set and then we had a huge let-up, mentally, in the second, said Beyer. We just cannot allow ourselves to lose focus like that. We are lucky that it just went two.
The Lady Panthers will head into the C2-11 subdistrict. In the C2-11, Hershey is the No. 2 seed and will play third-seed Dundy County-Stratton (18-10) in Ogallala on Oct. 30 at 6:30 p.m.
If the Panthers get past the Tigers, it is looking at a probable matchup with No. 1-seed North Platte St. Pats for the championship. The 26-1 Lady Irish have won both matchups with the Lady Panthers but Hershey cannot be counted out with the rivalry between the two schools.
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May 2, 2016
Savannah Bunn
Over the past four years, I have learned how to balance academics, athletics, volunteer work, and a part time job, all thanks to the help and support of my teachers, coaches, friends, and family that have been there for me every step of the way. In the fall, I will be attending the Honors College at East Carolina University . I plan to major in Nursing, then continue my education and become a Nurse Practitioner. Congratulations and good luck to the New Bern High School Class of 2013! Go Bears!
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NAME: Savannah Bunn
PLANS FOR NEXT YEAR: Honors College at East Carolina University
FAVORITE SCHOOL SUBJECT: Biology
FAVORITE EXTRA-CURRICULAR ACTIVITY: Lacrosse
FAVORITE BOOK: Life of Pi
FAVORITE SONG: Springsteen by Eric Church
FAVORITE BAND: Mumford & Sons
FAVORITE TV SHOW: Heroes
FAVORITE MOVIE: P.S. I Love You
FAVORITE ACTOR/ACTRESS: Johnny Depp
FAVORITE FOOD: Lasagna
FAVORITE THING TO DO ON WEEKENDS: Go to the beach
FAVORITE SEASON: Late summer
FUN FACT: I was a gymnast for nine years.
IF YOU WERE STRANDED ON A DESERTED ISLAND AND COULD ONLY TAKE ONE ITEM WITH YOU, WHAT WOULD IT BE?: A yacht with a full tank of gas and autopilot.
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Best of the Web: 2 July
10 tips to find happiness at work; Twitter musings in Syria elicit groans in Washington; Want to lose weight? Try sex and shopping
July 2, 2010 2:31 by Rasha Reslan
10 tips to find happiness at work
If "happiness at work" sounds like an oxymoron, then these tips are for you.
"Happiness is in vogue, and everyone – from psychologists and academics to career coaches –has advice on how to find it and how to keep it," Forbes says.
Up your tranquility quotient by judging less, moving on, and "quieting the chaos by redirecting your thoughts." Check out these common sense, Zen-inspired approaches for a kinder, gentler workplace.
Twitter musings in Syria elicit groans in Washington
With all the press Syria is getting these days, Mr. Assad and his administration defy an easy branding. Damascus is flush with tourist money, and you might just get elbowed in the face by all the EU-types bargaining in the souks and snapping photos in the Omayyad mosque.
But America's keeping its game face on – at least officially. So when two young State Department officials, part of a delegation to Syria, tweeted their light and favorable impressions of the Damascus experience, the powers that be were not amused.
Want to lose weight? Try sex and shopping
Why keep up that expensive gym membership, when you can you can just up the sex and shopping quotient and come out all the slimmer for it? So goes the wisdom when the calorie obsessed strap on the high tech Ki Fit lifestyle armband.
This pricy little gadget, which retails for around GBP 100, employs sensors that continuously monitor "motion, steps, body temperature, heat flux and galvanic skin response," the Independent reported. The happy findings? To stay slim, go heavy on the romance and retail therapy.
"During a manic shopping trip – I just had to have that jumpsuit – I was seeing off three-and-a-half calories a minute, the same rate as at my weekly line-dancing class. I always knew shopping was good for me. Sex sheds calories too: an extended session of rumpy-pumpy with my beloved waves goodbye to 3.7 calories per minute," the Independent's journalist found.
Fantastic presentation on how to analyze and think about gold prices
If turbulent financial winds have you thinking about tangible assets, you're not alone. Investors with shaky confidence are asking hard questions about the soft metal. As Reuters reported last week, gold prices are within sight of $1,260 – just $10 off their all-time high.
So get savvy about gold and find out when the experts expect gold prices to rise, whether gold is a reliable "fear gauge," and how the glittery stuff hedge's against inflation. It's all here, in this step by step guide to getting a handle on the investment potential of gold.
Gaza besieged
Despite Israel's blockade of the Gaza strip, Palestinians desperate for supplies have constructed an "intricate network of tunnels – circumventing Israel's stifling trade restrictions."
This audio-video story from the Economist features the photographs of Bruno Stevens.
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13 April 2016
AMERICA/MEXICO - An agreement has been signed that prohibits the employment of children in fields
Chihuahua (Agenzia Fides) - In the Mexican state of Chihuahua no farmer will be able to employ child workers as in all other States of the Country. Local authorities, along with 250 farm owners, have recently signed an agreement t ...
13 April 2016
AFRICA/SENEGAL - Migration: Caritas Africa and Europe at a seminar in Dakar
Dakar (Agenzia Fides) – To make proposals for migration policies that go beyond the current approach based solely on security, through a partnership between African and European countries. This is what will be discussed at the s ...
13 April 2016
AMERICA/BOLIVIA - Bishops: "drug trade threatens the peaceful and democratic coexistence of the country"
La Paz (Agenzia Fides) - The Bishops' Conference of Bolivia (CEB) has reiterated the seriousness of the problem of drug trafficking, which "threatens the peaceful and democratic coexistence of the Country". This is what was said y ...
13 April 2016
ASIA/PHILIPPINES - Appeal to the new leaders: a governance enriched by mercy
Manila (Agenzia Fides) - To ensure good governance and an action-oriented policy for the common good, the new leaders of the Philippines should adopt the approach of mercy: this is what Redemptorist Fr. Amado Picardal, executive S ...
13 April 2016
ASIA/PAKISTAN - Five new Franciscan priests in Lahore
Lahore (Agenzia Fides) - The Church in Pakistan has welcomed the gift of five new Capuchin Franciscan friars ordained priests: as reported to Fides by the Provincial of Pakistan, Fr. Francis Nadeem, the ordination was held on 8 Ap ...
13 April 2016
ASIA/MYANMAR - One child out of 5 goes to work instead of going to school
Yangon (Agenzia Fides) - One Burmese child out of 5, between 10 and 17 years of age, goes to work instead of going to school, compromising their personal and professional development as well as affecting the prospect for the futur ...
13 April 2016
AMERICA/CHILE - Church fires do not stop, the solution is in dialogue
Temuco (Agenzia Fides) - Another Catholic chapel was set on fire at dawn yesterday in the region of Antiquina, in the town of Cañete, Biobio region (Chile). According to police information, the arson completely destroyed the stru ...
12 April 2016
ASIA/SYRIA - Clarification on Christians killed in Qaryatayn, denial regarding the news about the "jihadist" massacre
Qaryatayn (Agenzia Fides) - In recent months, in the Syrian city of Qaryatayn, many Christians were killed in different circumstances, in different ways related to the fact that the region in which that city is situated - reconque ...
12 April 2016
AFRICA/KENYA - The Bishops denounce: "Corruption is killing the country"
Nairobi (Agenzia Fides) - "The cancer of corruption is killing our Country. Are we heading towards a society without God, plagued by tribalism, in which money has become the only God": is the sharp cry of alarm of the Bishops of K ...
12 April 2016
ASIA/PAKISTAN - The Bishops: need to remove the contents of hatred in school textbooks
Karachi (Agenzia Fides) - It is urgent to remove the contents of hatred and prejudice towards religious minorities in school textbooks in use in schools across the country: is the appeal launched by the National Commission "Justic ...
12 April 2016
AMERICA/PUERTO RICO - A growing crisis: teachers risk retirement
San Juan (Agenzia Fides) - While the Puerto Rican government cannot seem to recover from the deep economic crisis in which it has collapsed (see Fides 18/03/2016), now teachers in public and private schools risk retirement, since ...
12 April 2016
AMERICA/VENEZUELA - Cardinal Urosa's appeal in the face of lynchings and widespread violence: "Do not kill"
Caracas (Agenzia Fides) - The Archbishop of Caracas, Cardinal Jorge Urosa Savino, yesterday published a letter, also signed by the four auxiliary Bishops, in which he condemned the lynching of criminals as sin. "With deep sadness ...
12 April 2016
AMERICA/UNITED STATES - Poverty, abuse and family conflicts are forcing children to live on the streets
New York (Agenzia Fides) - A report by the National Center on Family Homelessness (NCFH) revealed that, in 2015, 2 and a half million children in the United States were living in shelters, on the streets, in cars, or in fields wit ...
12 April 2016
AMERICA/ARGENTINA - Inhumane conditions for children and workers found in a warehouse for packaging
Maipu (Agenzia Fides) - Following a Government inspection in a warehouse for packaging in the department of Maipú, province of Mendoza, workers and children were found living in inhuman conditions in these unhealthy environments. ...
12 April 2016
ASIA/INDIA - The South Asian Franciscans are committed to the poor and marginalized
Bangalore (Agenzia Fides) - The Franciscans of the South Asian countries reaffirm their commitment to serve the poor and marginalized, helping, in particular, to respond to the most pressing social issues that occur in South Asia: ...
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Prince's Sister Tyka Nelson Storms Out of Family Meeting. Why?
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Broughtupcy - is the act of bringing up a child in the right way; proper home training including but not limited to manners, respect and situational awareness
Those wild children have no broughtupcy
by krazie242 November 21, 2013
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|By Marketwired .
Article Rating:
|January 14, 2013 08:00 AM EST
REDWOOD SHORES, CA -- (Marketwire) -- 01/14/13 -- Oracle (NASDAQ: ORCL)
News Facts
- To improve business performance and transform global business processes, Natus Medical Incorporated has adopted the Oracle E-Business Suite 12.1 using Oracle Business Accelerators.
- Oracle Business Accelerators are powerful, easy-to-use, cloud-based rapid implementation tools developed by Oracle to get customers up and running on a wide range of Oracle Applications, both quickly and reliably.
- Natus is a leading provider of healthcare products used for the screening, detection, treatment, monitoring and tracking of common medical ailments. The company sells its products worldwide through a direct sales force and distributors in the U.S. and in more than 100 other countries.
- With an increasing volume of customer orders coming from around the globe, Natus needed to replace its existing ERP system with a modern, flexible and scalable suite of business applications that could help it lower total cost of ownership by standardizing and automating processes.
- Working with NTT DATA, a Platinum level member in Oracle PartnerNetwork (OPN), and leveraging out-of-the-box, industry-specific Oracle Business Accelerators, Natus implemented the Oracle E-Business Suite 12.1, including Oracle Advanced Supply Chain Planning, Oracle's Demantra, Oracle E-Business Suite Financials, Oracle Order Management, Oracle Inventory Management, Oracle Advanced Procurement, Oracle Discrete Manufacturing and Oracle Service Management. Natus also implemented Oracle Customer Relationship Management, Oracle's Agile Product Lifecycle Management and Oracle Hyperion Performance Management, in an aggressive 10-month implementation timeframe.
- In addition, to provide a common infrastructure for producing, delivering and managing enterprise reports, scorecards, dashboards, ad-hoc analysis, and OLAP analysis, Natus selected Oracle Business Intelligence.
- With this integrated suite of Oracle Applications, Natus has been able to significantly reduce end of month reporting times, effectively meet increasing customer demand and establish a flexible and scalable platform to support future growth.
- Natus is live in the US and Canada. Europe and South America are planned for deployment in early 2013.
- Implementation partner NTT DATA provides modern, rapid implementation tools and implementation services that can help midsize customers achieve a quick return on their Oracle technology investment.
Supporting Quotes
- "To support our growth and acquisition strategy in both the U.S. and international markets, we needed a modern, cost-effective, scalable and reliable business platform that would enable us to standardize operations and lower total cost of ownership in the long term," said Bharat Poria, senior director of information technology, Natus Medical. "We were able to standardize processes and go live on the Oracle E-Business Suite 12.1, using the leading industry practices in the Oracle Business Accelerators for Oracle E-Business Suite 12.1, in ten months. Ninety percent of the functionality we needed was available out-of-the-box."
- "NTT DATA is committed to our relationship with both Oracle and our client Natus Medical," said Hiren Bhatt, senior vice president of NTT DATA's Oracle practice. "We are able to deliver this industry-specific solution for midsize companies to speed the deployment of Oracle solutions, resulting in the out-of-the-box experience that our clients expect."
- "Midsize manufacturers and distributors today seek solutions that will facilitate future growth," said Steve Cox, vice president, Oracle Accelerate for Midsize Companies. "Oracle Accelerate solutions, implemented using Oracle Business Accelerators, help rapidly growing organizations like Natus Medical to utilize best-in-class enterprise software solutions rapidly, so that the benefits of use are realized as quickly as possible."
Supporting Resources
- Oracle Accelerate for Midsize Companies
- Oracle Business Accelerators
- Oracle E-Business Suite
- Oracle Advanced Supply Chain Planning
- Oracle's Demantra
- Oracle CRM Applications
- Oracle's Agile Product Lifecycle Management Solutions
- Oracle Hyperion Performance Management
- Oracle Business Intelligence Enterprise Edition
- Oracle Midsize Customer Success Stories
- Video: Grow Your Business with Oracle Accelerate
- Natus Medical Incorporated
About Oracle Accelerate
Oracle Accelerate provides simple to deploy, packaged, enterprise-class software solutions to growing midsize organizations through Oracle's network of expert partners. Created by Oracle's expert partners, Oracle Accelerate solutions are simple to deploy, industry-specific packaged solutions, designed for fast time to benefit, which means getting the right solution in place quickly, inexpensively and with a controlled scope and predictable returns. The best Oracle Accelerate Solutions are based on Oracle Business Accelerators, which are powerful, cloud-based rapid implementation tools developed and maintained by Oracle to get you up and running on a wide range of Oracle Applications, both quickly and reliably. Oracle has more than 290,000 midsize customers who buy Oracle to grow. To find out more, visit www.oracle.com/accelerate.
About Oracle
Oracle engineers hardware and software to work together in the cloud and in your data center. For more information about Oracle (NASDAQ: ORCL), visit www.oracle.com.
Oracle and Java are registered trademarks of Oracle and/or its affiliates. Other names may be trademarks of their respective owners.
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SYS-CON Events announced today that DatacenterDynamics has been named "Media Sponsor" of SYS-CON's 18th International Cloud Expo, which will take place on June 7–9, 2016, at the Javits Center in New York City, NY. DatacenterDynamics is a brand of DCD Group, a global B2B media and publishing company that develops products to help senior professionals in the world's most ICT dependent organizations make risk-based infrastructure and capacity decisions.
May. 2, 2016 06:30 AM EDT Reads: 2,508
So, you bought into the current machine learning craze and went on to collect millions/billions of records from this promising new data source. Now, what do you do with them? Too often, the abundance of data quickly turns into an abundance of problems. How do you extract that "magic essence" from your data without falling into the common pitfalls? In her session at @ThingsExpo, Natalia Ponomareva, Software Engineer at Google, will provide tips on how to be successful in large scale machine lear...
May. 2, 2016 06:00 AM EDT Reads: 1,269
The IoT has the potential to create a renaissance of manufacturing in the US and elsewhere. In his session at 18th Cloud Expo, Florent Solt, CTO and chief architect of Netvibes, will discuss how the expected exponential increase in the amount of data that will be processed, transported, stored, and accessed means there will be a huge demand for smart technologies to deliver it. Florent Solt is the CTO and chief architect of Netvibes. Prior to joining Netvibes in 2007, he co-founded Rift Technol...
May. 1, 2016 11:00 PM EDT Reads: 1,593
The increasing popularity of the Internet of Things necessitates that our physical and cognitive relationship with wearable technology will change rapidly in the near future. This advent means logging has become a thing of the past. Before, it was on us to track our own data, but now that data is automatically available. What does this mean for mHealth and the "connected" body? In her session at @ThingsExpo, Lisa Calkins, CEO and co-founder of Amadeus Consulting, will discuss the impact of wea...
May. 1, 2016 07:00 PM EDT Reads: 764
SYS-CON Events announced today that Peak 10, Inc., a national IT infrastructure and cloud services provider, will exhibit at SYS-CON's 18th International Cloud Expo®, which will take place on June 7-9, 2016, at the Javits Center in New York City, NY. Peak 10 provides reliable, tailored data center and network services, cloud and managed services. Its solutions are designed to scale and adapt to customers' changing business needs, enabling them to lower costs, improve performance and focus inter...
May. 1, 2016 06:00 PM EDT Reads: 1,169
We've worked with dozens of early adopters across numerous industries and will debunk common misperceptions, which starts with understanding that many of the connected products we'll use over the next 5 years are already products, they're just not yet connected. With an IoT product, time-in-market provides much more essential feedback than ever before. Innovation comes from what you do with the data that the connected product provides in order to enhance the customer experience and optimize busi...
May. 1, 2016 05:00 PM EDT Reads: 1,035
In his session at @ThingsExpo, Chris Klein, CEO and Co-founder of Rachio, will discuss next generation communities that are using IoT to create more sustainable, intelligent communities. One example is Sterling Ranch, a 10,000 home development that – with the help of Siemens – will integrate IoT technology into the community to provide residents with energy and water savings as well as intelligent security. Everything from stop lights to sprinkler systems to building infrastructures will run ef...
May. 1, 2016 04:00 PM EDT Reads: 957
Digital payments using wearable devices such as smart watches, fitness trackers, and payment wristbands are an increasing area of focus for industry participants, and consumer acceptance from early trials and deployments has encouraged some of the biggest names in technology and banking to continue their push to drive growth in this nascent market. Wearable payment systems may utilize near field communication (NFC), radio frequency identification (RFID), or quick response (QR) codes and barcodes...
May. 1, 2016 01:30 PM EDT Reads: 822
SYS-CON Events announced today that Stratoscale, the software company developing the next generation data center operating system, will exhibit at SYS-CON's 18th International Cloud Expo®, which will take place on June 7-9, 2016, at the Javits Center in New York City, NY. Stratoscale is revolutionizing the data center with a zero-to-cloud-in-minutes solution. With Stratoscale's hardware-agnostic, Software Defined Data Center (SDDC) solution to store everything, run anything and scale everywhere...
May. 1, 2016 01:30 PM EDT Reads: 1,582
Angular 2 is a complete re-write of the popular framework AngularJS. Programming in Angular 2 is greatly simplified – now it's a component-based well-performing framework. This immersive one-day workshop at 18th Cloud Expo, led by Yakov Fain, a Java Champion and a co-founder of the IT consultancy Farata Systems and the product company SuranceBay, will provide you with everything you wanted to know about Angular 2.
May. 1, 2016 01:00 PM EDT Reads: 1,754
You think you know what's in your data. But do you? Most organizations are now aware of the business intelligence represented by their data. Data science stands to take this to a level you never thought of – literally. The techniques of data science, when used with the capabilities of Big Data technologies, can make connections you had not yet imagined, helping you discover new insights and ask new questions of your data. In his session at @ThingsExpo, Sarbjit Sarkaria, data science team lead ...
May. 1, 2016 12:45 PM EDT Reads: 835
SYS-CON Events announced today that Men & Mice, the leading global provider of DNS, DHCP and IP address management overlay solutions, will exhibit at SYS-CON's 18th International Cloud Expo®, which will take place on June 7-9, 2016, at the Javits Center in New York City, NY. The Men & Mice Suite overlay solution is already known for its powerful application in heterogeneous operating environments, enabling enterprises to scale without fuss. Building on a solid range of diverse platform support,...
May. 1, 2016 12:15 PM EDT Reads: 2,333
You deployed your app with the Bluemix PaaS and it's gaining some serious traction, so it's time to make some tweaks. Did you design your application in a way that it can scale in the cloud? Were you even thinking about the cloud when you built the app? If not, chances are your app is going to break. Check out this webcast to learn various techniques for designing applications that will scale successfully in Bluemix, for the confidence you need to take your apps to the next level and beyond.
May. 1, 2016 11:45 AM EDT Reads: 1,500
Whether your IoT service is connecting cars, homes, appliances, wearable, cameras or other devices, one question hangs in the balance – how do you actually make money from this service? The ability to turn your IoT service into profit requires the ability to create a monetization strategy that is flexible, scalable and working for you in real-time. It must be a transparent, smoothly implemented strategy that all stakeholders – from customers to the board – will be able to understand and comprehe...
May. 1, 2016 11:00 AM EDT Reads: 1,097
Increasing IoT connectivity is forcing enterprises to find elegant solutions to organize and visualize all incoming data from these connected devices with re-configurable dashboard widgets to effectively allow rapid decision-making for everything from immediate actions in tactical situations to strategic analysis and reporting. In his session at 18th Cloud Expo, Shikhir Singh, Senior Developer Relations Manager at Sencha, will discuss how to create HTML5 dashboards that interact with IoT devic...
May. 1, 2016 09:45 AM EDT Reads: 1,039
Artificial Intelligence has the potential to massively disrupt IoT. In his session at 18th Cloud Expo, AJ Abdallat, CEO of Beyond AI, will discuss what the five main drivers are in Artificial Intelligence that could shape the future of the Internet of Things. AJ Abdallat is CEO of Beyond AI. He has over 20 years of management experience in the fields of artificial intelligence, sensors, instruments, devices and software for telecommunications, life sciences, environmental monitoring, process...
May. 1, 2016 09:45 AM EDT Reads: 932
SYS-CON Events announced today that Ericsson has been named "Gold Sponsor" of SYS-CON's @ThingsExpo, which will take place on June 7-9, 2016, at the Javits Center in New York, New York. Ericsson is a world leader in the rapidly changing environment of communications technology – providing equipment, software and services to enable transformation through mobility. Some 40 percent of global mobile traffic runs through networks we have supplied. More than 1 billion subscribers around the world re...
May. 1, 2016 07:30 AM EDT Reads: 1,003
There is an ever-growing explosion of new devices that are connected to the Internet using "cloud" solutions. This rapid growth is creating a massive new demand for efficient access to data. And it's not just about connecting to that data anymore. This new demand is bringing new issues and challenges and it is important for companies to scale for the coming growth. And with that scaling comes the need for greater security, gathering and data analysis, storage, connectivity and, of course, the...
May. 1, 2016 07:00 AM EDT Reads: 884
Machine Learning helps make complex systems more efficient. By applying advanced Machine Learning techniques such as Cognitive Fingerprinting, wind project operators can utilize these tools to learn from collected data, detect regular patterns, and optimize their own operations. In his session at 18th Cloud Expo, Stuart Gillen, Director of Business Development at SparkCognition, will discuss how research has demonstrated the value of Machine Learning in delivering next generation analytics to im...
Apr. 30, 2016 04:15 PM EDT Reads: 1,710
This is not a small hotel event. It is also not a big vendor party where politicians and entertainers are more important than real content. This is Cloud Expo, the world's longest-running conference and exhibition focused on Cloud Computing and all that it entails. If you want serious presentations and valuable insight about Cloud Computing for three straight days, then register now for Cloud Expo.
Apr. 30, 2016 02:30 PM EDT Reads: 1,741


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Adolor Corporation To Present At The William Blair & Company 2011 Life Sciences Conference
www.adolor.com and click on "Investor Insights," then "Webcast." Click on the link and follow the prompts for registration and access.About Adolor Adolor Corporation is a biopharmaceutical company specializing in the discovery, development and commercialization of novel prescription pain and pain management products. Adolor's first approved product in the United States is ENTEREG® (alvimopan), which is indicated to accelerate the time to upper and lower gastrointestinal recovery following partial large or small bowel resection surgery with primary anastomosis. ENTEREG is available for short-term use in hospitals registered under the E.A.S.E.® Program. For more information on ENTEREG, including its full prescribing information, visit www.ENTEREG.com. The Company's lead development program compound is ADL5945, a novel mu opioid receptor antagonist being developed for chronic OIC that demonstrated positive results in Phase 2 trials. The Company also has several earlier-stage compounds under development for the management of pain and CNS disorders. For more information, visit www.adolor.com.
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Screw that, it's too damn long!
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June 17th, 2013 (8:43 AM).
Kikaito plush
Angeline plushxKikaito plush
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the number ep is a turn off for me, the longest Anime I have ever watch was Toyko Mew Mew with 52 eps. Anything longer that than I will not even try as I get bored easily with them. I did try watching "One Piece" but after 4 eps I gave up as it was boring.
so yeah 52 eps max for me.
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post #1 of 3 Old 04-20-2012, 03:44 PM - Thread Starter
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Should I contact them and tell to bring along an M-Card or would that be too confusing for them once they see my PC? Anybody with Comcast experience is the SF Bay Area?
post #2 of 3 Old 04-21-2012, 04:57 AM
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In the past, the recommended method was to tell your cable provider that you had a Tivo. I did that and they guy showed up and said "That's not a Tivo". I just replied "It's like a Tivo that hooks up to your computer." He stated that he's never seen one before, but he plugged in the M-Card and continued through the usual process.
Since the Ceton and Prime have been on the streets for some time, perhaps it's now ok to tell them your actual device.
The Prime forums have lots of good advice on getting setup with the various cable providers.
post #3 of 3 Old 04-23-2012, 12:34 PM
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Don't worry about it. They are all M cards these days.
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text:
Issue #50 / Jan 2005
Cover Theme: Firewalls
DVD: Fedora Core 3
DVD Inlay
Article Code
Insider Tips: Locks
SYSADMIN: Competitive Thinking
Remote control with a Jabber Bot
PROGRAMMING: Inside
Setting up SuxPanel
LINUXUSER: Desktop Sideboard
Configuring KDM and GDM
LINUXUSER: The First Display
« Previous 1 2 3 Next »
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