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Grace Poe’s name to remain in official list of candidates ‘for now’ – Comelec
MANILA, Philippines — Despite the Commission on Elections (Comelec) en banc’s decision upholding Senator Grace Poe’s disqualification by the poll body’s two divisions, her name will remain in the ballot for now.
In a press briefing on Wednesday, Comelec Chair Andres Bautista said Poe’s name would still be included in the initial list of 23 presidential candidates to be uploaded in the Election Management System (EMS).
“Right now her name is still on the ballot as we speak,” Bautista said. The Comelec will not remove the senator’s name until the commission en banc’s decision to cancel her certificate of candidacy (COC) is final and executory, according to Bautista.
Rule 37 of the Omnibus Election Code, states that decisions in pre-proclamation cases and petitions to deny due course to or cancel COCs shall become final and executory after the lapse of five days from their promulgation.
The decisions of the en banc were promulgated on Wednesday. The lapse of the five-day period will be on Monday, December 28.
Asked until when Poe’s name would be on the initial list of candidates, Bautista said “it depends.”
On Poe’s chances that her name would make it to the final list to be printed on the ballot or on the possibility of her camp’s failure to secure a temporary restraining order from the high court, Bautista said: “We will cross that bridge when we get there… As I said, we’ll see. Let’s see what happens in the coming days.”
The poll body on Tuesday voted separately on the two disqualification cases filed against Poe — the petition of lawyer Estrella Elamparo to cancel Poe’s COC, alleging that the senator committed material misrepresentation with her claims of being a natural-born Filipino citizen and of having resided in the Philippines for the past 10 years (Second Division); and the consolidated petition of former University of the East College of Law Dean Amado Valdez, De La Salle University professor Antonio Contreras and former senator Kit Tatad (First Division).
Five commissioners have consistently voted in favor of petitions disqualifying Poe. On both motions for reconsideration filed by Poe’s camp, the five commissioners—Al Parreno, Luie Tito Guia, Arthur Lim, Rowena Guanzon and Sheriff Abas—ruled to uphold the earlier decisions of the first and second divisions canceling Poe’s COC, saying she has not been a resident of the Philippines for the past 10 years and she is not a natural-born Filipino.
“The majority, five commissioners in both cases, voted that there was deliberate attempt to mislead the electorate. The decision in the first division states that both—her statement that she is a natural-born citizen and that she had 10-year residency—are false and she intended and attempted to mislead the electorate,” Guanzon told reporters in an interview.
The members of the en banc voted 5-1 with one inhibition in favor of the decision of the Comelec Second Division. Bautista dissented with the majority while Commissioner Christian Lim inhibited from the case since he’s a former law partner of the petitioner, lawyer Estrella Elamparo.
On granting the petition, five commissioners voted in favor of canceling Poe’s COC while Bautista dissented.
On the other hand, the commission voted 5-2 in favor of the ruling of the Comelec First Division. Bautista and Lim gave dissenting votes to the majority decision.
Bautista presented a matrix on how the en banc voted. It showed that Comelec en banc unanimously voted in both cases, saying Poe is not a natural-born Filipino citizen.
“Members of the commission believe that Senator Poe is not a natural-born citizen,” said Bautista.
As for the issue on her lack of a residency of 10 years, the en banc voted 5-1 in the Second Division case with Commissioner Luie Guia dissenting while there was a vote of 5-2 in the First Division case with Guia and Lim dissenting.
“In the Second Division case, only Commissioner Guia believed that Senator Poe met the residency requirement. But if you look at the First Division it was 5-2, because both Commissioners Lim and Guia believe that Senator Poe met the residency requirement,” said Bautista.
The en banc said Poe had deliberate intent to mislead about her period of residency and her citizenship, with both votes on the matters coming in at 4-2.
Bautista was a dissenting vote on both items, saying he did not believe that there was a deliberate intent on Poe’s camp to mislead. Commissioner Al Parreno also dissented about the deliberate attempt to mislead about Poe’s natural-born status.
For residency, Bautista was joined by Guia.
Five commissioners voted to grant the consolidated petition.
The Comelec en banc also voted unanimously in both cases that they have clear jurisdiction on them.
Based on the 30-page decision on the First Division decision, the Comelec en banc said Poe did not offer any new evidence in her motion for reconsideration (MR) that would warrant a reversal of the earlier ruling.
“All other issues raised by Respondent are mere rehash of the arguments already addressed and ruled upon by the First Division,” said the Comelec en banc decision on MR against the First Division case.
The poll body agreed with the First Division ruling that Poe, being a senator, was deemed to have deliberately ignored her ineligibility as a presidential candidate and just opted to misrepresent herself as a natural-born citizen in her COC.
“We affirm the findings of the First Division that respondent deliberately misrepresented her citizenship so as to hide her ineligibility and mislead the electorate…clearly in order to serve her purpose and suit her intent of running for the presidency,” the en banc said in its decision.
The en banc added it found as unacceptable her claim that the inconsistency between her COC for senator in 2013 and her COC for the presidency was an honest mistake.
On the other hand, the 36-page ruling on the Second Division case, the commission en banc admitted that it would have been easier for the poll body to just adopt the vox populi mantra by allowing Poe to run for president and let the voters decide on her fate.
“But this, we cannot do. Not only because we are duty-bound by law to decide on cases brought before us. More so because no less than the people, through the Constitution, grants us this solemn duty. In applying the law, and disqualifying respondent, we are heeding the true voice of the people. From this task, we cannot desist,” said the Comelec en banc.
The commission said the Second Division was right in holding Poe accountable for her failure to prove her status as a natural-born citizen while being a foundling.
The decision also agreed with the Second Division that Poe was short of the 10-year residency requirement for presidential candidates even if they considered July 2006 as the earliest possible reckoning point of her residency in the Philippines. SFM
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domain: law


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Nielsen pollster John Stirton, like most statisticians, is always cautious about calling a trend based on one poll.
His caution was on show again yesterday when he was reluctant to say that the big jump in the approval rating of Prime Minister
But he is not at all cautious about pointing to what he says is the really significant trend emerging in his poll.
This trend suggests that, while today’s headlines will be about the Prime Minister, the much more important story is about
The signs are that the electorate’s anger over the carbon tax is now abating and, perhaps, also the angst over minority government. A majority of voters now want the government to serve its full term.
We can now start to assess the effectiveness and costs of the campaign run by Abbott against the carbon tax.
One compelling measure of the campaign’s impact is to look at the leaders’ approval figures before the carbon price announcement and now. The Prime Minister’s approval rating has fallen from 52 per cent in 2010 and early 2011 to 47 per cent.
The good news for Gillard is the 47 per cent figure is part of a four-month improving trend.
The Opposition Leader’s approval rating, by comparison, has slumped from 50 per cent to 37 per cent in the same period and is not improving.
It is hard not to come to the conclusion that the political cost of the carbon tax for Gillard has not been anywhere near as high as the cost to Abbott of attacking it, and through it, the Prime Minister.
The Coalition has tended to regard the Opposition Leader’s poor personal standing as collateral damage necessary to help bring down the government. But today’s figures show that a turning point really has been reached on this.
The Coalition now has all the collateral damage but confronts a Prime Minister whose personal approval ratings are now back to where they were before the whole carbon tax episode began.
More importantly, leaders’ personal approval ratings tend to be a lead indicator for party votes.
Labor’s primary vote remains at the lower edge of what some of its strategists call a “politically habitable" space.
That is, the primary vote is only at a level to allow Labor to think it might be heading towards being in contention at the next poll but is not there yet.
But voters, it seems, have moved on from the carbon tax.
The Nielsen poll suggests voters are starting to seriously assess both leaders, and the hostility to the Prime Minister is fading.
The Australian Financial Review

domain: news


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Cancelled. The reason was too many textures
I have a small map with landscape that has a few Paint layers. Edit layers on landscape are enabled (I mean Sculpt layers).
I have tried to paint whithout edit layers option enabled but it doesn’t matter.
So, the problem is when I try to paint with a new landscape blend layer on the landscape components it’s compiled but doesn’t drawing properly, always stay like whithout any material. Even if I relaunch the editor or change scalability it’s still looks empty. (After relaunching material shaders are recompiled again but nothing happened)
I can only clear all layers and paint again.
But the same happens again, when I start to paint on landscape component that already have more than two paint layers, it’s compiling but staying empty after finished compiling or even after rebuilding all material layers.
And after relaunching editor, after recompiling all shaders, I see in log a few of this Warning Message, maybe it’s a reason why the materials not drawing, but I don’t how to fix that:
“LogShaderCompilers: Warning: Failed to compile Material /Game/Materials/M_Landscape_01.M_Landscape_01 (MI:/Game/Maps/Aworld.Aworld:PersistentLevel.Landscape_1.LandscapeMaterialInstanceConstant_15) for platform PCD3D_SM5, Default Material will be used in game.”

domain: artistic


Narada Michael Walden— I Shoulda Loved Ya from the album
CLICK HERE TO DONATE TO WIDR Fm.
Link will take you to WIDR’s MyWMU giving page.
Thank you for supporting Radio Evolution!
I was surprised and excited to see WIDR on that wikipedia.
I was even more surprised and excited to hear Bat Guano once again, thanks for playing the Cramps when I first tuned in.
Long live 89.1 WIDR!!!


Yes, it seems manufacturers are setting people's expectations for 2025 but it may come a little sooner. People are comparing the advancement of SSB with that of li-ion from the early 90's (spanning almost three decades with Sony/Goodenough being first to market) and are discounting the improvements in computing power, more collaboration, Toyota (and others) patents accumulated in the past decade, etc.I think latest on something production ready for SSB is 2025. So I would not count on it just yet.
Standard lithium chemistries get improved all the time too.
The Bolloré Group has perfected batteries and electrical storage solutions based on a unique technology, the LMP® (Lithium Metal Polymer) battery, as well as on supercapacitor technology.
SCiB™ is a rechargeable battery with outstanding safety performance that uses lithium titanium oxide for the anode. SCiB™ has been widely used for automobiles, buses, railway cars, and other vehicles; elevators and other industrial applications; and large-scale battery energy storage systems...
I have all the time to wait. Extended warranty on our 16 RAV4H doesn't end until early 2023 and what are the chances of the breaking down being too costly to repair?
If one of my three cars become too costly to repair, my household will be fine with two.
I don't mind li-ion battery BEVs but definitely not paying MSRP and will be willing to buy used, when they're like 40-60% off MSRP. There's even this one Audi e-tron with 11k km marked for $79k here when new it would have been a little over $100k.


Sir Elliot tells us that the 35-Band Mastering Vocal Equaliser is designed for Mastering Engineers who need to focus on midrange frequencies within the 440 Hz – 1760 kHz range. More bands enables more precision to pinpoint on bringing a particular vocal or instrument in the midrange region to the forefront.
Pricing and Availability:
I guess I've been trying to recreate a Korg Electribe all this time..
Beautifully designed analog summing mixers and pre-amps


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25 Jun Exciting Summer Table Tennis Training Camp 2018
Two weeks summer camp starts on Monday 30th July to Friday 10th August 2018
Participate in this brilliant Table Tennis camp to explore and develop your performance skills through guided training sessions. Everyone is welcome regardless of experience or standard. See flyer for details.
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domain: hobby


text:
If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
* This work was supported, in whole or in part, by National Institutes of Health (NIH) Grants 1R15GM101575-01 (to M. D.) and by the Intramural Research Program of the NIH (NICHD; to T. E. D.). This work was also supported by Research Growth Initiative (UW-Milwaukee Graduate School; to M. D.). The authors declare that they have no conflicts of interest with the contents of this article.
The Hac1 transcription factor in yeast up-regulates a collection of genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the 5′-untranslated region (5′ UTR) of the HAC1 mRNA represses Hac1 protein production under basal conditions, whereas cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under endoplasmic reticulum stress conditions, relieves the inhibition and enables Hac1 synthesis. Using a random mutational screen as well as site-directed mutagenesis, we identify point mutations within the 5′ UTR-intron interaction site that derepress translation of the unspliced HAC1 mRNA. We also show that insertion of an in-frame AUG start codon upstream of the interaction site releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. These results suggest that the major block of translation occurs at the initiation stage. Supporting this interpretation, the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes versus free ribosomal subunits. Thus, our results provide evidence that the 5′ UTR-intron interaction represses translation initiation on the unspliced HAC1 mRNA.
then travels along the 5′ mRNA leader in search of a translation start codon in a process referred to as ribosomal scanning. Typically, the first AUG codon encountered by the scanning PIC is selected as the translation start site in part due to base-pairing interactions between the anticodon loop of the Met-tRNAiMet and the AUG codon. After selection of the start codon, initiation factors dissociate from the PIC, and the 60S ribosomal subunit joins yielding an 80S ribosomal complex (
). In addition, nucleotide sequence context (the Kozak consensus motif) flanking an AUG or non-canonical start codon impacts the efficiency with which a scanning ribosome selects a translation start site (
). Additionally, out-of-frame AUG start codons and upstream open reading frames (ORFs) in the 5′ leader can restrict access of the ribosome to the main ORF. A classic example is the yeast GCN4 mRNA in which short upstream ORFs potentiate translation of the main GCN4 ORF and confer heightened dependence on translation factor activity (
). The intron (spanning nucleotides G661 to G913, with the adenine of AUG start codon assigned as 1) is not spliced in the nucleus by the spliceosome but instead is retained in the mRNA that is exported to the cytoplasm (
), resulting in an altered ORF with a new codon starting at nucleotide G661 and a UAG stop codon at nucleotide 963 (see Fig. 1A). Finally, the 3′ UTR has been reported to positively regulate translation of the spliced HAC1 mRNA (
), a key element in the UPR is repression of translation of the unspliced Hac1 mRNA.
Despite much progress in understanding the regulation of HAC1 mRNA translation, the molecular mechanism is not yet clear. To gain mechanistic insights into HAC1 mRNA translation, we conducted a genetic screen to identify intragenic mutations that enable a splicing-deficient and translationally inert HAC1 mRNA (i.e. HAC1-G661C) to produce a Hac1 protein capable of stimulating the ER stress response. From this screen we identified a single base mutation (i.e. G771A) in the intron that is predicted to disrupt the base-pairing interaction with the 5′ UTR (
), suggesting that disruption of the 5′ UTR-intron interaction leads to translation of the HAC1-G661C,G771A mRNA without splicing. Consistent with the notion that the 5′ UTR-intron interaction interferes with ribosomal scanning and translation initiation on the HAC1 mRNA, introduction of an upstream in-frame AUG codon before the 5′ UTR-intron base-pairing region relieved the translational block on the HAC1 mRNA. Taken together, in contrast to previously proposed models, our data reveal a unique mechanism of intron-mediated inhibition of ribosomal scanning.
Yeast Strains and Plasmids
The ire1Δ (MATa his3-Δ1 leu2-Δ0 met5-Δ0 ura3-Δ0 ire1::KanMX) and hac1Δ (MATa his3-Δ1 leu2-Δ0 met5-Δ0 ura3-Δ0 hac1::KanMX) yeast strains were obtained from the yeast genome deletion collection. Strain J751 (MATa his3-Δ1 leu2-Δ0 met5-Δ0 ura3-Δ0 ire1:NAT) was derived by replacing KanMX4 in the ire1Δ strain with the nourseothricin-resistance (NAT) marker gene. The hac1Δ ire1Δ (MATa his3-Δ1 leu2-Δ0 met5-Δ0 ura3-Δ0 ire1:NAT hac1::KanMX) double deletion strain J772 was constructed by replacing HAC1 with KanMX4 in strain J751. Strain J1167 (MATa his3-Δ1 leu2-Δ0 met5-Δ0 ura3-Δ0 ire1:NAT HAC1-G767C,G768C) was constructed by integration of plasmid pC4070 (linearized with AscI) into strain J751. After selection of segregants on 5-fluoroorotic acid, the replacement of the WT HAC1 allele with the HAC1-G767C,G768C intron mutant was confirmed by PCR. For the UPR assay, yeast growth was tested on SD medium containing the ER stress inducer tunicamycin (0.4 μg/ml). Mutations were created by site-directed mutagenesis or by fusion PCR. The plasmids used in this study are listed in Table 1.
). Yeast cells were grown in synthetic complete medium at 30 °C till the A600 reached ∼0.5–06. When desired, DTT (5 mm) was added to the medium to induce ER stress, and cells were grown for an additional 4 h. Total RNA was isolated using an RNeasy kit (Qiagen), and first strand cDNA was synthesized using Superscript-III reverse transcriptase (Invitrogen) and random primers. The synthetic cDNA was subjected to RT-PCR and Q-PCR analyses. For RT-PCR, the synthetic cDNA was amplified by PCR using primers specific for HAC1 (forward primer 5′-CGCAATCGAACTTGGCTATCCCTACC-3′, corresponding to nucleotides +35 to +60 of the HAC1 open reading frame (ORF), and reverse primer 5′-GGGTAGACTGTTTCCCGC-3′, corresponding to nucleotides +604 to +621) or ACT1 (forward primer 5′-CTGAAAGAGAAATTGTCCGTG-3′, corresponding to nucleotides +919 to +939 of the ACT1 ORF (numbering includes the intron), and reverse primer 5′-CTTGTGGTGAACGATAGATGG 3′, corresponding to nucleotides +1408 to +1428). For analysis of HAC1 mRNA splicing, the same forward primer was used along with a reverse primer (5′-CCCACCAACAGCGATAATAACGAG-3′) that corresponded to nucleotides +1002 to +1025. PCR products were then resolved by agarose gel electrophoresis. For Q-PCR, the synthetic cDNA was amplified on a real time PCR machine (Bio-Rad CFX 3.1) using primers specific for HAC1 (forward primer 5′-CGCAATCGAACTTGGCTATCCC-3′ and reverse primer 5′-CTCTCGAGATACTGCAGATG-3′) or ACT1 (forward primer 5′-CTGAAAGAGAAATTGTCCGTG-3′ and reverse primer5′-GTGATGACTTGACCATCTGG-3′). Experiments were repeated twice, and each sample was run in triplicate. Data were analyzed using CFX 3.1 software, and the threshold (Ct) value was calculated. The Ct value represents the average value of triplicate samples, and these average values were used to calculate the CtHAC1/CtACT1 ratio.
Western Blot Analysis
Yeast cells were grown in synthetic complete medium to A600 = 0.6. When desired, 5 mm DTT was then added to the medium to induce ER stress, and cells were incubated an additional 4 h before harvesting. Cells were harvested by centrifugation, mixed with 2 volumes of 20% (w/v) trichloroacetic acid, and then broken by agitation with glass beads. Proteins were extracted with SDS Loading Buffer (2% SDS, 2 mm EDTA, 50 mm Tris-HC1 (pH 6.8), 10% glycerol, 0.01% bromphenol blue), and after neutralization with 1 m Tris base, samples were boiled for 5 min and then subjected to SDS-PAGE and immunoblot analysis using antibodies raised against recombinant Hac1 protein or yeast eIF2α or using anti-HA antibodies (Cell Signaling Technology or Roche Applied Science).
Polysome and Northern Analysis
Cells were grown to mid-log phase, treated with cycloheximide (100 μg/ml), incubated for 5 min, and harvested. Cell pellets were resuspended in 5 ml of Polysome Buffer (20 mm Tris-HCl (pH 8.0), 140 mm KCl, 5 mm MgCl2, 100 μg/ml cycloheximide), pelleted, and then resuspended in 1 ml of Polysome Buffer containing 1% Triton X-100 (Sigma), 1 mm DTT, EDTA-free Protease Inhibitor tablet (Roche Applied Science) and 4 μl of RNaseOUT (Invitrogen). Cells were broken by vigorous mixing with glass beads at 4 °C using a vortex mixer. WCEs were clarified by centrifugation, and the equivalent of A600 = 25 units was layered on top of 7–47% (w/v) sucrose gradients made in Polysome Buffer containing 1 mm DTT and EDTA-free Protease Inhibitor tablets. Samples were sedimented at 39,000 rpm for 150 min at 4 °C in an SW41 rotor (Beckman Coulter). Gradients were fractionated while scanning at A254.
To isolate RNA, glycogen was added to the sucrose gradient fractions; the fractions were then mixed with ethanol and stored at −20 °C overnight. RNA pellets were collected by sedimentation, resuspended, and then extracted twice with phenol:chloroform. The RNA was then precipitated in ethanol, washed with 70% ethanol, and resuspended in 30 μl of Loading Buffer (21 μl of H2O, 3 μl of formaldehyde, 3 μl of 10× MOPS buffer, 3 μl of 10× RNA loading dye). Equal volumes (25 μl) were used for Northern analysis.
To visualize HAC1 by Northern analysis, RNA samples were separated on a 1.2% agarose, 5% formaldehyde gel in MOPS buffer, transferred to a NyTran membrane (Whatman) overnight in 20× SSC buffer, and then UV cross-linked to the membrane in a Stratalinker (Stratagene). After prehybridization in 12 ml UltraHYB buffer (Ambion) for 2 h at 42 °C, the membrane was incubated with randomly labeled [32P]dCTP probes for HAC1 and ADH1 overnight at 42 °C while rotating. The HAC1 (primers oCB1 (5′-GTCTACGGCAGGTAGCGTCGT-3′) and oCB25 (5′-AATTCGCAATCGAACTTGGCTATCCC-3′)) and ADH1 (primers oCB105 (5′-ATGTCTATCCCAGAAAC-3′) and oCB106 (5′-ACCCAAGACTCTGTAACC-3′)) probes were generated by PCR followed by random-labeling with [32P]dCTP probes (Lofstrand). Blots were washed at 42 °C for 2 × 10 min in 2× SSC, 0.1% SDS buffer followed by 2 × 15 min in 0.1× SSC, 0.1% SDS buffer. After the final wash, the RNA was imaged and quantitated using phosphorimaging.
Genetic Screen Identifies a Single Mutation in the HAC1 Intron That Bypasses the Requirement for mRNA Splicing to Derepress HAC1 Production
Under conditions of ER stress, the endonuclease Ire1 cleaves the phosphodiester bonds after nucleotides G661 and G913 (Fig. 1A) to remove the intron from the HAC1 mRNA (
), also known as the Hac1i isoform (“i” for induced; produced from the ER stress-induced, spliced mRNA). In the absence of splicing, the first in-frame stop codon (UAG) after G661 is located at nucleotide positions 691–693 (see Fig. 1A). This alternate 693-nucleotide ORF in the unspliced mRNA can be translated into a functional Hac1 protein of 230 amino acids (
), known as the Hac1u isoform (“u” for uninduced; produced under non-stress conditions). However, translation of the unspliced mRNA and, thus, production of Hac1u is repressed by the intron (Fig. 1A, orange), which interacts with the 5′ UTR and somehow blocks translation of the HAC1 ORF (
). To gain mechanistic insights into the intron-mediated translational repression, we screened for intragenic suppressor mutations in a splicing-deficient and translationally inert HAC1-G661C allele (see below), expecting that the second-site mutations would allow ribosomes to bypass the 5′ UTR-intron interaction and produce a Hac1 protein from the unspliced mRNA.
Whereas a hac1Δ yeast strain carrying an empty vector grew as well on synthetic dextrose (SD) medium as the same strain expressing HAC1 from a low copy number plasmid (Fig. 1B, rows 1 and 2), growth on SD medium containing the ER stress inducer tunicamycin required HAC1 expression (Fig. 1B, rows 1 and 2). These results are consistent with the previous reports showing that Hac1 function is essential for the ER stress response (
). Mutation of the first Ire1-cleavage site (i.e. G661C) impaired yeast cell growth on tunicamycin medium (Fig. 1B, row 3), suggesting that the G661C mutation reduced the HAC1 mRNA level, mRNA splicing, and/or translation.
To determine whether the G661C mutation caused a reduction in HAC1 mRNA levels and/or mRNA splicing, total RNA was extracted from these cells and then used as a template to amplify the HAC1 mRNA as well as an endogenous ACT1 mRNA (housekeeping control) by RT-PCR as described under “Experimental Procedures.” As shown in Fig. 1D, similar amounts of ACT1 mRNA were detected in each RNA sample (lanes 1–3). As expected, no HAC1 mRNA was detected in the hac1Δ strain transformed with an empty vector (Fig. 1D, lane 1), whereas similar amounts of HAC1 mRNA were detected in the strains expressing WT HAC1 (lane 2) or the HAC1-G661C mutant (lane 3). Thus, the tunicamycin-sensitive (TmS) phenotype of the strain expressing the HAC1-G661C mutant is likely not due to reduced HAC1 mRNA levels.
To determine whether the G661C mutation impaired mRNA splicing, RT-PCR was performed using primers that readily distinguish the unspliced (HAC1u) and spliced (HAC1s) transcripts. Again, as expected, no HAC1 mRNA was detected in the RNA sample prepared from the hac1Δ strain carrying an empty vector (Fig. 1E, upper panel, lane 1). Consistent with the Ire1 endonuclease functioning at basal levels in unstressedcells, both HAC1u and HAC1s transcripts were amplified from cells expressing WT HAC1 (Fig. 1E, upper panel, lane 2). In cells expressing the HAC1-G661C allele, the vast majority of mRNA was unspliced (HAC1u; Fig. 1E, lane 3), consistent with the idea that the G661C mutation in the first cleavage site impaired HAC1 mRNA splicing and consequently reduced the ER stress response. The defective splicing of the HAC1-G661C mRNA is supported by previous studies showing that mutation of this guanine residue at the cleavage site impairs Ire1 cleavage of the HAC1 mRNA both in vivo and in vitro (
To confirm that the G661C mutation eliminated Hac1 protein expression, WCEs were prepared from the cells exposed to DTT to induce ER stress (“Experimental Procedures”) and then subjected to immunoblot analysis using an antibody against the Hac1 protein. As expected, the Hac1 protein was detected in the extract obtained from cells expressing the WT HAC1 allele (Fig. 1C, lane 2). In contrast, no Hac1 protein was detected in cells expressing the HAC1-G661C mutant (lane 3). These results are consistent with the model (
) that translation of the unspliced HAC1 mRNA is blocked.
To screen for intragenic suppressors of the splicing- and translation-defective HAC1-G661C allele, a plasmid bearing the HAC1-G661C variant was amplified in Escherichia coli XL-1 red cells (Stratagene) that are deficient in three DNA repair pathways. The mutagenized pool of plasmids was purified and used to transform a hac1Δ yeast strain. Transformants were screened for the ability to grow on medium containing tunicamycin. The HAC1 plasmid was isolated from the tunicamycin-resistant (TmR) colonies, re-tested in a hac1Δ strain to confirm that the TmR phenotype was associated with the plasmid, and then sequenced to detect the mutation. A single second-site suppressor mutation, G771A, was found to restore the ability of the HAC1-G661C variant to activate the ER stress response (Fig. 1B, row 4).
The base G771 is located in the region of the intron that is predicted to form base-pairing interactions with the 5′ UTR of the HAC1 mRNA (
). In the alignment shown in Fig. 1A, the G771 base in the intron is base-paired with C−27 in the 5′ UTR. Given the location of this suppressor mutation, we hypothesized that the G771A mutation disrupted the 5′ UTR-intron interaction leading to derepression of HAC1 mRNA translation in the absence of splicing. To test the possibility that the G771A mutation enabled translation of unspliced HAC1 mRNA, plasmids encoding WT HAC1, the splice-site mutant HAC1-G661C, or the suppressor mutant HAC1-G661C,G771A were introduced into a yeast strain lacking the Ire1 endonuclease. The transformants were then tested for the ability to activate the UPR as detected by growth on medium containing tunicamycin. As shown in the Fig. 1B (rows 5 and 6), the ire1Δ strain expressing WT HAC1 or HAC1-G661C failed to grow on the medium containing tunicamycin, consistent with the notion that Ire1-mediated splicing of HAC1 mRNA is essential for the ER stress response (
). In contrast, the HAC1-G661C,G771A mutant conferred a TmR phenotype when expressed in the ire1Δ strain (Fig. 1B, row 7), suggesting that the G771A mutation derepressed HAC1 mRNA translation in the absence of splicing. Consistent with this interpretation, immunoblot analysis revealed substantial Hac1 protein levels without an increase in the level of the HAC1 mRNA (Fig. 1D, lane 4) when the HAC1-G661C,G771A mutant was expressed in either hac1Δ (Fig. 1C, lane 4) or ire1Δ (Fig. 1C, lane 7) cells. These data demonstrate that a single base mutation in the intron is sufficient to eliminate translational control of the HAC1 mRNA.
Translational Control of the HAC1 mRNA Is Sensitive to Alteration of a Single Base Pair Interaction between the Intron and 5′ UTR
To gain further insights into HAC1 translational control and the importance of base-pairing contacts between the intron and the 5′ UTR of the HAC1 mRNA, mutations designed to disrupt single or double base-pairing interactions were introduced into the intron or the 5′ UTR of a human influenza hemagglutinin (HA) epitope-tagged version of HAC1. In this construct, the HA tag was inserted between residues Ser-10 and Asn-11 of the HAC1 ORF. Importantly, the HA-tagged HAC1 allele functioned like untagged WT HAC1 and complemented the TmS phenotype of a hac1Δ strain (Fig. 2A, row 2) in an IRE1-dependent manner (Fig. 2B, row 2). As shown in Fig. 2B, the single C−23G (row 3) and the double C−23G,C−24G (row 4) mutations, located in the 5′ UTR of the HA-HAC1 mRNA and designed to disrupt the base-pairing interaction with the intron (Fig. 1A), suppressed the TmS phenotype of an ire1Δ hac1Δ double-mutant strain. As no HAC1 mRNA splicing takes place in the ire1Δ hac1Δ strain, these results indicate that, like the G771A mutation in the intron (Fig. 1B, row 7), point mutations in the HAC1 5′ UTR can derepress Hac1 production. Mutations in other intron residues involved in the base-pairing interactions also led to prominent TmR phenotypes. As shown in Fig. 2B, the G767C (row 5) and G767C,G768C (row 6) mutations in HA-HAC1 conferred strong TmR phenotypes in the ire1Δ hac1Δ strain, suggesting that mutations in either the 5′ UTR or the intron allowed ribosomes to produce the Hac1 protein from the unspliced mRNA.
Immunoblot and Northern analyses were used to assess the impact of the 5′ UTR and intron mutations on Hac1 protein and mRNA levels. As the cells lack Ire1 protein and no splicing of the HAC1 mRNA will take place, extracts were prepared from cells grown in the absence of ER stress. Whereas no HA-Hac1 was detected in the extracts from cells expressing WT HA-HAC1 (Fig. 2C, upper panel, lane 2), very low or low levels of HA-Hac1 protein were observed in the extracts prepared from cells expressing the C−23G or C−23G,C−24G mutant alleles, respectively, of HA-HAC1 (Fig. 2C, upper panel, lanes 3 and 4). Thus, low level expression of HA-Hac1 protein from the HA-HAC1-C−23G allele was apparently sufficient to confer a TmR phenotype. Consistent with the more pronounced TmR phenotype, the HA-Hac1 protein was readily detected in extracts prepared from the ire1Δ hac1Δ strain expressing the G767C (Fig. 2C, lane 5) and G767C,G768C (lane 6) mutant form of HA-HAC1. Northern analyses revealed that the levels of HA-HAC1 mRNA were similar in cells expressing the WT or various mutant alleles (Fig. 2C, lower panel, lanes 3–6 versus 2), suggesting that differences in protein expression were not due to altered mRNA levels. These results provide further support for the hypothesis that mutations designed to disrupt the 5′ UTR-intron interaction derepress Hac1 production by enabling translation of the unspliced HAC1 mRNA.
The random mutational screen (Fig. 1B) revealed that the G771A mutation in the HAC1 intron derepressed Hac1 synthesis. In the unspliced HAC1 mRNA, G771 is predicted to form a base-pairing interaction with C−27 (Fig. 1A). To test the importance of this predicted base pair, three additional HAC1 mutants were generated. The single mutants HAC1-C−27G and HAC1-G771C were made to destroy the pairing and the double mutant HAC1-C−27G,G771C was constructed to restore the pairing in the opposite orientation. As shown in Fig. 2D, both HAC1-C−27G (row 3) and HAC1-G771C mutants (row 4) conferred a TmR phenotype in the ire1Δ hac1Δ strain. In contrast, the HAC1-C−27G,G771C double mutant (Fig. 2D, row 5) functioned like WT HAC1 (row 2) and conferred a TmS phenotype in the ire1Δ hac1Δ strain. As expected, the Hac1 protein was detected only in the extracts obtained from cells expressing the HAC1-C−27G mutant (Fig. 2E, Western, lane 3) and the HAC1-G771C mutant (lane 4). The mutual suppression of the HAC1-C−27G and HAC1-G771C mutants in the HAC1-C−27G,G771C double mutant is consistent with the proposed base-pairing interaction between these residues and further demonstrates that repression of HAC1 expression by the 5′ UTR-intron interaction is sensitive to loss of a single base pair interaction.
When expressed in a hac1Δ strain (IRE1 intact in the chromosome), the HAC1-C−27G, HAC1-G771C, and HAC1-C−27G,G771C mutants, like WT HAC1, conferred a TmR phenotype (Fig. 2F, rows 2–5). Based on the results of these mutational analyses, we conclude that 5′ UTR-intron interaction and, in particular, the C−27/G771 base pair plays an important role in repressing HAC1 mRNA translation.
Introduction of an In-frame AUG Start Codon Upstream of the Secondary Structure in the 5′ UTR Derepresses HAC1 Synthesis
The secondary structure in the 5′ UTR could impair Hac1 synthesis by blocking ribosomal scanning to the start codon or, as has been proposed (
), by impairing translation elongation. To differentiate between these models, AUG start codons were inserted at three sites in the 5′ UTR of the HAC1 gene (Fig. 3A). All three of the introduced AUG codons were in-frame with the HAC1 ORF and thus would be predicted to encode Hac1 proteins with N-terminal extensions. To exclusively monitor translation initiating at the inserted AUG codon, the adenine of the authentic HAC1 start codon was mutated to guanine, generating the HAC1-A1G allele, and the adenine of the AUG codon encoding Met3 was likewise altered to guanine to generate the HAC1-A7G mutation (Fig. 3A). As shown in Fig. 3B, the HAC1-A1G,A7G mutant allele failed to complement the TmS phenotype of a hac1Δ strain (row 3). As the A1G,A7G mutation did not reduce HAC1 mRNA levels (Fig. 3D, lane 3), this result is consistent with the notion that, even following splicing, scanning ribosomes fail to initiate at the HAC1 start site (a GUG codon in the A1G mutant), and no Hac1 protein was produced (Fig. 3C, lane 2).
We first characterized the effect of replacing nucleotides −42UAA−40, immediately upstream of the 5′ UTR-intron interaction (see Figs. 1A and 3A), with an AUG codon to generate the HAC1-AUG−42,A1G,A7G allele. As shown in Fig. 3B, the HAC1-AUG−42,A1G,A7G allele conferred a TmR phenotype when expressed in a hac1Δ strain (row 4), suggesting that a functional Hac1 protein with an extra 14 amino acids at the N terminus was expressed from the AUG−42 codon. To directly examine Hac1 production, Western analyses were performed on WCEs prepared from ER-stressed cells using an antibody prepared against recombinant Hac1 protein. Consistent with the introduction of 14 extra N-terminal residues, the Hac1 protein in the HAC1-AUG−42,A1G,A7G cells was of higher molecular weight than the WT Hac1 protein (Fig. 3C, lanes 1 and 3). It is important to note that these experiments were performed using a hac1Δ strain in which the chromosomal IRE1 gene was intact. Thus, it is likely that the HAC1-AUG−42,A1G,A7G mRNA was spliced in these cells, and we conclude that a 14-residue N-terminal extension does not impair Hac1 production or the ability of Hac1 to promote yeast cell growth on medium containing tunicamycin. However, the intent of these experiments was to determine whether the introduced AUG−42 start codon could promote translation of the unspliced HAC1 mRNA. Therefore, we expressed the HAC1-AUG−42,A1G,A7G allele in an ire1Δ strain where the mRNA will not be spliced. As shown in Fig. 3B, the HAC1-AUG−42,A1G,A7G allele failed to complement the TmS phenotype of the ire1Δ strain (Fig. 3B, row 5), and Western analyses showed that the Hac1 protein was not produced (Fig. 3D, lower panel, lane 2). These results indicate that the unspliced HAC1-AUG−42,A1G,A7G mRNA is translationally repressed, and we propose that the adjacent secondary structure from the 5′ UTR-intron interaction may preclude scanning ribosomes from accessing the AUG−42 start codon or may impede formation of a productive 80S ribosome on the AUG−42 codon.
To further test the ability of inserted AUG start codons to overcome translational repression on the unspliced HAC1 mRNA, two additional mutants were constructed. First, the nucleotides −33CCU−31 in the HAC1-A1G,A7G allele were substituted by an AUG triplet to generate the HAC1-AUG−33,A1G,A7G allele (Figs. 1A and 3A). As this mutation is predicted to disrupt two base pair interactions between the 5′ UTR and the intron (Fig. 1A) and as we previously saw that disruption of a single base pair interaction was sufficient to derepress HAC1 mRNA translation (FIGURE 1, FIGURE 2), the AUG−33 mutation was expected to likewise derepress HAC1 expression. Accordingly, as shown in Fig. 4A, row 3, and in contrast to WT HAC1 (row 1), the HAC1-AUG−33,A1G,A7G allele conferred a TmR phenotype in the ire1Δ hac1Δ strain, and Hac1 protein was detected in extracts from cells expressing the mutant but not the WT protein (Fig. 4B, lane 3 versus 1). These experiments with the HAC1-AUG−33,A1G,A7G mutant provide further support for the hypothesis that disruption of the secondary structure formed by the base-pairing interaction between the 5′ UTR and intron is sufficient to derepress translation of the unspliced HAC1 mRNA.
Because the AUG−42 insertion may have failed to derepress HAC1 mRNA translation due to the close proximity of AUG−42 to the 5′ UTR-intron secondary structure, the third mutant was constructed by inserting the AUG codon further upstream from the secondary structure. To avoid inserting the AUG codon too close to the 5′ end of the mRNA, where it may be susceptible to being skipped by scanning ribosomes (
), the HAC1-AUG−60,A1G,A7G allele was generated by inserting an AUG codon followed by 18 additional nucleotides at position −39 (Figs. 1A and 3A). The inserted nucleotides consist of two 9-nucleotide repeats of the sequence normally found between positions −15 and −7 (5′-−15ACAACAACC−7-3′) of the HAC1 mRNA. As this sequence is not part of the 5′ UTR-intron interaction and is rich in adenine and cytosine residues, it is predicted to be unstructured and thus serve as a simple spacer between the inserted AUG start codon and the base-paired element in the HAC1 5′ UTR. In this new HAC1-AUG−60,A1G,A7G allele, the inserted AUG−60 start codon is located ∼26 nucleotides from the 5′ cap (
) and ∼22 nucleotides upstream of the start of the base-paired elements.
Interestingly, when introduced into the ire1Δ hac1Δ strain, the HAC1-AUG−60,A1G,A7G allele conferred a TmR phenotype (Fig. 4A, row 5). Consistent with this growth phenotype, Western analysis of WCEs from the strain grown under non-ER stress conditions revealed a prominent Hac1 protein signal (Fig. 4B, lane 5). Importantly, the Hac1 protein in the HAC1-AUG−60,A1G,A7G strain migrated more slowly in SDS-PAGE than the Hac1 protein produced in the HAC1-AUG−33,A1G,A7G strain (Fig. 4B, lane 3), consistent with the longer N-terminal extension for the protein initiating at AUG−60. As the HAC1-AUG−60,A1G,A7G mRNA is unspliced in the ire1Δ hac1Δ strain, and thus the 5′ UTR-intron interaction is intact, the ability of AUG−60 codon insertion to derepress Hac1 production leads to two important conclusions. First, in contrast to the previously proposed model of a translation elongation block on the unspliced HAC1 mRNA (
), elongating ribosomes are able to translate through the 5′ UTR-intron interaction. Second, the data are consistent with a model in which the 5′ UTR-intron interaction impedes translation initiation by blocking ribosomes scanning to the native AUG start codon.
Disruption of the 5′ UTR-Intron Interaction Results in Greater Association of HAC1 mRNA with Polysomes
The genetic suppressor and AUG insertion mutant studies support the model that ribosomal scanning on the WT unspliced HAC1 mRNA is paused at the 5′ UTR-intron interaction site. Accordingly, the point mutations that weaken the 5′ UTR-intron interaction allow scanning ribosomes to traverse the 5′ UTR and initiate translation at the AUG start codon of HAC1. If this model is correct, few ribosomes should associate with the unspliced WT HAC1 mRNA, whereas the HAC1 mRNA containing the point mutations that disrupt the 5′ UTR-intron interaction should be readily translated and thus show a greater association with polysomes. To test this possibility, the WT chromosomal HAC1 allele (translationally repressed) in the ire1Δ strain J751 was replaced with the HAC1-G767C,G768C mutant (translationally active; see Fig. 2, B and C, lane 6) to create strain J1167. Next, WCEs from strains J751 and J1167 were subjected to velocity sedimentation in 7–47% sucrose gradients, and then the gradients were fractionated while being scanned at 254 nm (see “Experimental Procedures”) to visualize the free 40S and 60S ribosomal subunits, 80S monosomes, and polyribosomes (Fig. 5). To monitor the sedimentation of mRNAs in the gradients, RNA was isolated from the gradient fractions and analyzed by Northern blot using probes to detect the HAC1 mRNA and the ADH1 mRNA. As shown in Fig. 5A, the WT HAC1 mRNA in unstressed ire1Δ cells showed strong association with 40S subunits (lane 2) and only ∼53% of the HAC1 mRNA associated with polysomes. In contrast, the ADH1 mRNA was primarily (∼80%) associated with the polysome fractions. Disruption of the 5′ UTR-intron base-pairing interaction in the HAC1-G767C,G768C mutant resulted in more (∼70%) of the HAC1 mRNA associating with polysomes (Fig. 5B, lanes 5–8). These data provide independent support for the idea that the 5′ UTR-intron interaction blocks ribosomal scanning, resulting in a substantial fraction of the HAC1 mRNA associating with a single 40S subunit. Accordingly, disruption of the secondary structure releases the initiation block and enables more ribosomes to translate the HAC1 mRNA, resulting in more of the mRNA associating with polyribosomes.
Overexpression of eIF4A Restores the UPR and Derepresses Hac1 Production in Yeast Expressing the HAC1-AUG−42,A1G,A7G Variant
Whereas insertion of an AUG codon at position −60 far upstream of the secondary structure in the HAC1 mRNA led to constitutive derepression of Hac1 synthesis (Fig. 4), the insertion of an AUG codon at position −42 immediately before the secondary structure failed to derepress Hac1 synthesis in the absence of splicing (Fig. 3). As our data indicate that the secondary structure formed by the base-pairing interactions between the intron and 5′ UTR in the HAC1 mRNA interferes with translation initiation, we reasoned that scanning ribosomes lack sufficient secondary structure unwinding ability to access the AUG−42 start codon. However, because the AUG−42 codon is located just upstream of the secondary structure, the scanning ribosome may only need to partially melt the secondary structure to access the AUG−42 codon. Accordingly, we reasoned that overexpression of the translation factors that function in mRNA binding andscanning might derepress Hac1 production on the HAC1-AUG−42,A1G,A7G mRNA. Consistent with this hypothesis, overexpression of eIF4A, an RNA helicase that promotes melting of RNA secondary structures (
) conferred a tunicamycin-resistant phenotype in an ire1Δ hac1Δ strain expressing the HAC1-AUG−42,A1G,A7G allele (Fig. 6B, right panels, row 2). This derepression of HAC1 synthesis was dependent on eIF4A helicase activity as overexpression of the eIF4A-D170E mutant that lacks helicase activity (
), failed to promote growth of the strain expressing the HAC1-AUG−42,A1G,A7G allele on tunicamycin medium (Fig. 6B, right panels, rows 2, 3, and 4). Consistent with these growth phenotypes, Western blot analyses demonstrated that overexpression of eIF4A, but not eIF4E, eIF4B, or non-functional eIF4A-D170E, promoted synthesis of Hac1 in from the HAC1-AUG−42,A1G,A7G mRNA (Fig. 6C). These data support the model that increasing the cellular amount of eIF4A enables the scanning ribosome to melt secondary structure on the HAC1-AUG−42,A1G,A7G mRNA and gain access to the AUG−42 start codon. In contrast to the ability of eIF4A overexpression to derepress translation of the HAC1-AUG−42,A1G,A7G mRNA, overexpression of eIF4A failed to confer a tunicamycin-resistant phenotype in cells expressing the wild-type HAC1 mRNA (Fig. 6B, left panel, row 2). Thus, we propose that overexpression of eIF4A can promote the modest level of secondary structure melting required for the scanning ribosome to access AUG−42 but is not sufficient for the ribosome to traverse through the entire base-paired region and access the wild-type HAC1 start codon.
The Hac1 transcription factor is the primary regulator of the UPR (
). Under conditions of ER stress, HAC1 expression is increased. The Hac1 protein then promotes the expression of a variety of proteins including ER chaperones that help alleviate the stress conditions. As production of Hac1 and stimulation of the UPR in the absence of ER stress would be wasteful, it is not surprising that cells have adopted a mechanism to limit Hac1 synthesis in unstressed cells. Intriguingly, in yeast the mechanism to limit HAC1 expression under non-stressed conditions is linked to the unique cytoplasmic splicing of the HAC1 mRNA (
). Translation of the unspliced HAC1 mRNA will terminate at a stop codon in the intron, whereas translation of the spliced HAC1 mRNA will generate a protein with an altered C terminus that terminates in the second exon. Previous studies (
) and the results in this report demonstrate that both forms of the Hac1 protein are functional and enable cells to grow under ER stress conditions. However, production of Hac1 protein from the unspliced mRNA is inhibited by base-pairing interactions between sequences in the intron and in the 5′ UTR of the HAC1 mRNA. Within a span of 19 nucleotides, 16 form base-pairing interactions between the 5′ UTR and intron including a stretch of 11 consecutive base pairs. Splicing of the HAC1 mRNA removes the intron and thus eliminates the base-pairing interactions and enables HAC1 mRNA translation. As previously demonstrated by Rüegsegger et al. (
), replacement of the 5′ UTR base-pairing element with the sequence from the intron or replacement of the intron element with the sequence from the 5′ UTR enabled constitutive translation of the HAC1 mRNA. Moreover, when the 5′ UTR and intron elements were swapped simultaneously to allow the base-pairing interaction to occur, but with opposite orientation compared with the original context, HAC1 mRNA translational repression was restored. Although these studies clearly demonstrated that the intron played an important role in limiting HAC1 mRNA translation, the molecular mechanism of the repression was not clarified.
To gain insights into the mechanism of the translational repression of the unspliced HAC1 mRNA, we employed two mutational strategies. First, the cleavage site between exon 1 and the intron was mutated to eliminate splicing of the HAC1 mRNA, and then a second-site suppressor mutation was identified that restored Hac1 synthesis. This suppressor mutation disrupted a single base pair in the 5′ UTR-intron interaction. Likewise, using a traditional site-directed mutagenesis approach, we showed that altering a single base pair interaction between the 5′ UTR and intron was sufficient to derepress HAC1 mRNA translation in cells lacking the Ire1 endonuclease and was thus unable to splice the HAC1 mRNA. Although Rüegsegger et al. (
) showed that eliminating all 16 base-pairing interactions between the 5′ UTR and intron eliminated the translational repression, our studies reveal that loss of a single base pair interaction within or at the end of the 11 consecutive base pairs is sufficient to allow translation of the unspliced HAC1 mRNA. Thus, it appears that the HAC1 mRNA structure is poised on the threshold of strength required for translational repression, perhaps indicating that further strengthening of this interaction with additional base pair interactions might be deleterious. Likewise, it seems possible that conditions or factors that promote melting of mRNA secondary structures might be able to derepress translation of the unspliced HAC1 mRNA.
Secondary structural elements in the 5′ UTR of mRNAs have previously been shown to inhibit translation (
). As these structured mRNAs failed to associate with ribosomes, it was concluded that the 5′ UTR structures blocked 40S ribosome loading and/or ribosomal scanning to the start codon on the mRNA. Accordingly, the 5′ UTR-intron interaction would be predicted to impair translation initiation on the HAC1 mRNA. At odds with this hypothesis, Rüegsegger et al. (
) reported that unspliced HAC1 mRNA co-sedimented with polysomes on sucrose gradients, leading to a proposal that the 5′ UTR-intron interaction impedes translation elongation. We sought to directly test whether the 5′ UTR-intron interaction could block an elongating ribosome. Whereas inserting an AUG start codon immediately upstream of the interaction site failed to derepress translation of the unspliced HAC1 mRNA (AUG−42 in FIGURE 3, FIGURE 4), insertion of an AUG codon further upstream (AUG−60) enabled Hac1 synthesis in the absence of splicing (Fig. 4). In this latter construct, the inserted AUG−60 codon was positioned ∼26 residues from the cap and ∼22 residues upstream of the secondary structure. These gaps between the cap, AUG codon, and secondary structure provide sufficient room for a 43S PIC to bind at the cap, scan to AUG−60 start codon, and assemble a translationally competent 80S ribosome. Notably, toe-printing assays have shown that the leading edge of the ribosome is typically around 16 nucleotides downstream of the P site (
), so the 22-nucleotide gap between AUG−60 and the 5′ UTR secondary structure provides the space needed for an 80S ribosome. The ability of ribosomes initiating at AUG−60 to elongate through the 5′ UTR-intron secondary structure is consistent with previous reports demonstrating that elongating ribosomes can melt secondary structures (
). Moreover, these results demonstrate that the 5′ UTR-intron interaction does not block translation elongation, and we therefore conclude that translation of the unspliced HAC1 mRNA is blocked at translation initiation.
Polysome profiles provide an additional means to characterize translation defects. When translation initiation is impaired, fewer ribosomes associate with an mRNA, and the mRNA sediments as a free messenger ribonucleoprotein particle (mRNP) or perhaps associated with 40S subunits and smaller polysomes. On the other hand, if translation elongation is impaired, the mRNA will readily bind to ribosomes and sediment with large polysomes in sucrose gradient analyses. Rüegsegger et al. (
) found that the WT unspliced HAC1 mRNA co-sedimented with free 40S subunits as well as with polysomes, and mutation of the 5′ UTR-intron interaction resulted in loss of the association with 40S subunits and a shift of the majority of the mRNA to the polysome fractions. Likewise, Mori et al. (
) reported that the unspliced HAC1 mRNA in unstressed cells co-sedimented with ribosomal subunits, whereas the spliced HAC1 mRNA in tunicamycin-treated cells sedimented with large polysomes. In our studies we found that the association of the HAC1 mRNA with polysomes was influenced by the amount of HAC1 mRNA in the cell. Overexpression of HAC1 mRNA resulted in a relative shift in the sedimentation of the mRNA such that a greater fraction co-sedimented with polysomes.
As this result did not correlate with increased synthesis of Hac1 protein, we reasoned that the overexpressed mRNA might not be translated but, rather, associated with different complexes that co-sedimented with polysomes. To avoid this possible complication, we generated isogenic strains expressing WT HAC1 or a translationally derepressed HAC1 intron mutant. Whereas the unspliced HAC1 mRNA was roughly equally distributed between a free state, possibly a messenger ribonucleoprotein or associated with 40S particles, and a form that co-sedimented with polysomes (Fig. 5A), disruption of the 5′ UTR-intron interaction resulted in a relative shift of the HAC1 mRNA to co-sediment with polysomes (Fig. 5B). The limited association of unspliced HAC1 mRNA with polysomes is consistent with the results of ribosomal profiling studies in which the HAC1 mRNA in unstressed cells was not found to be associated with many ribosomes (
). Taken together, the results of the polysome profile analyses and site-directed mutagenesis studies are consistent with the notion that the 5′ UTR-intron interaction represses translation of the unspliced HAC1 mRNA by blocking translation initiation and that splicing relieves this block to promote HAC1 mRNA translation.
As 5′ UTR secondary structure can block scanning of the 43S PIC after binding to the mRNA (
), we reasoned that overexpression of translation factors that promote scanning might stimulate HAC1 mRNA translation. Accordingly, we found that overexpression of the RNA helicase eIF4A could derepress HAC1-AUG−42,A1G,A7G mRNA translation in the absence of splicing (Fig. 6). As overexpression of eIF4A failed to confer a tunicamycin-resistant phenotype in cells expressing wild-type HAC1 mRNA (Fig. 6), we propose that overexpression of eIF4A modestly increases the ability of the scanning ribosome to melt secondary structures. This boost in melting power is not sufficient to enable a ribosome to scan through the entire base-paired region formed between the HAC1 intron and 5′ UTR but may help melt the beginning of the base-paired region to enable the scanning ribosome to access a start codon at position −42. The finding that overexpression of a translation initiation factor can promote Hac1 synthesis provides further support for the notion that the base-pairing between the intron and 5′ UTR of the HAC1 mRNA interferes with translation initiation. Moreover, these studies suggest that HAC1 translational control can be used as a tool to gain insights into translation initiation. Much like GCN4 translational control has provided novel insights into the factors that promote Met-tRNAiMet binding to the 40S ribosome (

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JURISDICTION: United Kingdom CHANGE: NEW! Equinox IP Portal Members Directory Have you checked out our…
|Industry Trademark Application Strategy Manual for Designated Goods and Services Available!
“TIPO published the Industry Trademark Application Strategy Manual for Designated Goods and Services and Comparison Tables for Standard Industrial Classification and Goods/Services Classification, hoping to explain and offer suggestions pertaining to the selection of designated goods or services when filing applications so as to get trademark protection needed within a specific industry. They were published on the TIPO website on September 27 for all to use.
The aforementioned manual can be downloaded from the Traditional Chinese TIPO website under “Trademark” – “Information” – “Trademark Application Information for Industries” – “Industry Trademark Application Strategy Manual for Designated Goods and Services.”
“Comparison Tables for Standard Industrial classification and Goods/Services Classification Search” is a brand new function within the trademark search system, currently available in Chinese. A link to the manual is displayed on the Industry Trademark Application Strategy Manual for Designated Goods and Services webpage, offering users quick access to information, such as the classification of goods/services corresponding to a specific industry. Moreover, it provides a platform to cross-reference between Standard Industrial classification and Nice classification.”
|27 Sep 2021
|Sept. 2021 Revision of the Article-by-Article Interpretation of the Trademark Act
“To promote public awareness of the Trademark Act, TIPO published the Article-by-Article Interpretation of the Trademark Act in 2004 and has continued to revise it in response to amendments to relevant laws. After the previous revision (January 2017), the attached list to Article 19 of the Enforcement Rules of the Trademark Act and the infringement identifying process in Article 7 and 9 of the Regulations Governing Customs Measures in Protecting the Rights and Interests of Trademark have been amended. Additionally, a number of examination guidelines have also been established or amended, including the Principles for Examining Trademarks Which Include the “Cross Device”, Operational Directions for Third-Party Observations on Trademark Registration Applications, and Examination Guidelines on Procedural Examination of Applications for Trademark Registration. Trademark-related laws and judicial practices have changed considerably, hence the revision this year.
Relevant key points include “Non-traditional Trademark Types,” “Trademark Distinctiveness Assessment,” “Factors Determining Likelihood of Confusion,” “Proprietor of the Earlier Used Trademark,” “The Exhaustion Doctrine,” and related court decisions are compiled into the Article-by-Article Interpretation of the Trademark Act. Available on the TIPO website.”
|01 Nov 2021
|CIRCUIT COURT RULES (INTELLECTUAL PROPERTY) 2021
“1. (1) These Rules, which may be cited as the Circuit Court Rules (Intellectual Property) 2021, shall come into operation on the 12th day of October 2021.
(2) These Rules shall be construed together with the Circuit Court Rules 2001 to 2021.
(3) The Circuit Court Rules 2001 to 2021 as amended by these Rules may be cited as the Circuit Court Rules 2001 to 2021.
2. The Circuit Court Rules 2001 to 2021 are hereby amended by the insertion, immediately following Order 5C, as Order 5D, of the Order set out in the Schedule.
Intellectual Property Claims
1. In this Order, unless the context or subject matter otherwise requires—
the “1992 Act” means the Patents Act 1992;
the “1996 Act” means the Trade Marks Act 1996;
the “2000 Act” means the Copyright and Related Rights Act 2000;
the “2001 Act” means the Industrial Designs Act 2001;
the “2019 Act” means the Copyright and Other Intellectual Property Law Provisions Act 2019;
“intellectual property claim” has the same meaning as in section 2 of the 2000 Act.
Additional particulars required in civil bill in intellectual property claim
2.(1) An intellectual property claim shall be commenced by the issue and service of an ordinary civil bill, in the Form 2A in the Schedule of Forms, with the modifications necessitated by this rule, which shall:
(i) state the registered number of any registered intellectual property right to which the intellectual property claim relates;
(ii) set out full particulars of the ownership of any unregistered intellectual property right concerned in the proceedings, or of the right of the plaintiff to claim infringement;
(iii) include particulars of the nature and extent of any intellectual property right relied upon by the plaintiff (including the capacity in which the plaintiff sues if not the registered proprietor or proprietor of the intellectual property right concerned);
(iv) include particulars of the nature and extent of the alleged infringement of any intellectual property right relied on by the plaintiff;
(v) set out the material facts relied on in support of the intellectual property claim;
(vi) state specifically the relief sought against each defendant and every provision or rule of law relied on in support of each such relief and, where relief under the 1992 Act, 1996 Act, 2000 Act and/or 2001 Act is sought, the particular provisions under which relief is sought and the grounds for each such relief;
(vii) state such facts as shall show jurisdiction in accordance with reference number 30 in the Third Schedule to the Courts (Supplemental Provisions) Act 1961.
(2) The requirements of this Order are additional to the requirements of Order 5 as regards civil bills.
Civil application for delivery up
3.(1) An application to the Court in or by way of a civil proceeding for the delivery up of:
(i) infringing goods, material or articles under section 20 of the 1996 Act; or
(ii) an infringing article, copy, or device under section 131 of the 2000 Act; or
(iii) an illicit recording, article or device under section 255 of the 2000 Act; or
(iv) an infringing product or article under section 61 of the 2001 Act
may be made ex parte to any sitting of the Court in the Circuit wherein it is believed that such infringing articles, copies or devices are in the possession, custody of control of any person.
(2) The application shall be grounded upon an affidavit sworn by or on behalf of the proprietor of the registered trade mark, the copyright owner or a person having rights conferred by Part III of the 2000 Act, or his or its authorised representative.
(3) Where an application has been made, and remains undetermined, in a criminal proceeding, for the delivery up of some or all of the same article(s), copies, devices or products, an application under this rule shall not be determined until the determination of the application in the criminal proceeding.
(4) The Court may adjourn an application under this rule and direct that a person against whom relief is sought be served with notice of the application, in such manner as the Court directs.
Civil application for erasure etc of offending sign or for forfeiture or disposal
4.(1) Subject to sub-rule (3), an application to the Court in or by way of a civil proceeding for:
(i) an order to cause an offending sign to be erased, removed or obliterated from any infringing goods, material or articles, or to secure the destruction of infringing goods, materials or articles under section 19 of the 1996 Act, or
(ii) an order for the destruction or forfeiture of infringing goods, material or articles under section 23 of the 1996 Act, or
(iii) an order for the forfeiture or disposal of an infringing copy, article or device under section 145 of the 2000 Act, or
(iv) an order for the forfeiture or disposal of an illicit recording, article or device under section 264 of the 2000 Act, or
(v) an order for the forfeiture or disposal of an infringing product or article under section 72 of the 2001 Act,
shall be by originating notice of motion grounded upon an affidavit, and shall be served by the applicant, by personal service or by prepaid registered post, at least seven days before the date of the sitting of the Court to which it is returnable:
(a) on the respondent thereto,
(b) on any other person appearing to have an interest in the goods, materials, articles, copies, devices or products concerned, and
(c) on any other person directed by the Court to be served.
(2) Where an application has been made, and remains undetermined, in a criminal proceeding, for the delivery up of some or all of the same article(s), copies, recordings, devices or products, an application under this rule shall not be determined until the determination of the application in the criminal proceeding.
(3) Notwithstanding sub-rules (1) and (2), the Court may, where it has determined an intellectual property claim, determine an application under section 19 of the 1996 Act made orally to the Court concerning infringing goods, material or articles which were the subject of that intellectual property claim, without the necessity for the issue of a further originating notice of motion, provided that the Court is satisfied that all persons having an interest in the infringing goods, material or articles concerned are before the Court and have had an opportunity to be heard.”
(This note is not part of the Instrument and does not purport to be a legal interpretation.)
These rules amend the Circuit Court Rules by the insertion of a new Order 5D to prescribe the procedure in intellectual property claims.
|Irish Circuit Court
|24 Sep 2021
|Deputy Chairperson of Sakpatenti Participated in the 8th Session of the Georgia-Azerbaijan Intergovernmental Economic Commission
“Within the 8th Session of the Joint Intergovernmental Commission on Economic Cooperation between Georgia and the Republic of Azerbaijan, the issue of signing an Agreement on Mutual Recognition of Geographical Indications between Georgia and the Republic of Azerbaijan was considered.
The session was co-chaired by Mr. Irakli Garibashvili, Prime Minister of Georgia and Mr. Ali Asadov, Prime Minister of the Republic of Azerbaijan. The meeting was attended by the members of the Joint Intergovernmental Commission on Economic Cooperation between Georgia and the Republic of Azerbaijan, representatives of relevant authorities of the Republic of Azerbaijan and Georgia, including Ms. Nino Chikovani, Deputy Chairperson of the National Intellectual Property Centre of Georgia Sakpatenti.
In the framework of the Intergovernmental Agreement “On Mutual Recognition of Geographical Indications between Georgia and the Republic of Azerbaijan”, Georgia and the Republic of Azerbaijan will recognize and protect the geographical indications and appellations of origin of the two countries.
At the session of the Commission it was noted that in near future the procedures related to the Agreement “On Mutual Recognition and Protection of Geographical Indications between Georgia and the Republic of Azerbaijan” will be formalized and the official document will be signed.
During the 8th Session of the Joint Intergovernmental Commission on Economic Cooperation between Georgia and the Republic of Azerbaijan, the parties discussed the cooperation issues related to the economy, trade, energy, culture and humanitarian areas. In addition, Memorandums for cooperation were signed between relevant offices of the two countries. “
|01 Nov 2021
|Publication of Ministerial Agreements related to Patents and Industrial Designs, Trademarks and Other Distinctive Signs
The following update has been machine-translated from Spanish.
“Dear Users, it is reported that on Monday, September 27, 2021, the following agreements on industrial property have been published in the Diario de Centro América (link for their disposal):
Patents and Industrial Designs Department:
Ministerial Agreement No. 396-2021, Apply the International Patent Classification (CIP), established in the Strasbourg Agreement, in its current edition for the year two thousand twenty-one.
Ministerial Agreement No. 398-2021, Apply the International Classification of Industrial Designs, thirteenth edition of the Locamo Agreement.
Trademarks and Other Distinctive Signs Department:
Ministerial Agreement No. 397-2021, Apply the International Classification of Products and Services for the Registration of Trademarks (Nice Classification) eleventh edition.
|27 Sep 2021
|Draft Law amending Law 17/2001, of December 7, on Trademarks, Law 20/2003, of July 7, on the Legal Protection of Industrial Design and Law 24/2015, of July 24 , Patent
The following update has been machine-translated from Spanish.
“Range of the standard : Draft law. Subject : Industrial property. Agency : Spanish Patent and Trademark Office, OA
Summary: Through this draft law, Law 17/2001, of December 7, on Trademarks, Law 20/2003, of July 7, on the Legal Protection of Industrial Design and Law 24/2015, are partially modified, of July 24, Patents, in order to address a joint reform of the three main industrial property laws through a single legal text. Thus, it is intended to carry out an update of certain aspects related to industrial property rights, in such a way that the people who own them and the rest of the actors involved enjoy greater legal certainty, while proceeding to the clarification of certain aspects that the practice carried out since the last reforms has shown to be confusing.
In particular, the reasons that justify the need to reform Law 17/2001, of December 7, on Trademarks again, are due to the opportunity to make certain adjustments to said regulations in order to provide it with greater coherence and precision. To this end, substantive and procedural norms are introduced that are recommended by the experience achieved, as well as by the practices followed in neighboring countries. Likewise, these changes are intended to respond to different situations that have arisen in recent times in our society in order to provide greater protection to applicants and holders of distinctive signs.
In the current regulatory context, the reform of Law 20/2003, of July 7, on the Legal Protection of Industrial Design, responds to the adaptation of the regulations to the international and European context, the clarification of some of its precepts and the increase of legal security in economic traffic, as well as the need to adapt its spirit to the regulatory changes that have occurred. In particular, the proposal for legislative modification of designs responds to the current needs of a dynamic and increasingly virtual market that requires, in turn, a procedure that reflects these particularities through the greater speed and simplicity of its procedures.
More than four years after the entry into force of Law 24/2015, of July 24, on Patents, both formal and substantive reasons justify the need to address a reform of said Law. On the one hand, the experience of These years have shown the need to provide greater clarity and coherence to different provisions in order to provide greater legal certainty for users of the system. Likewise, deficiencies have been detected that are intended to be corrected with the proposed modifications. Another objective that is intended to be achieved with the legislative amendment is to adapt Spanish patent regulations to the normative and interpretative changes operated in the field of patents at the international level. Thus, for example, it has given rise to intense debates, both within the European Patent Office, and the European Union, the clarification of those inventions excluded from protection because they are essentially biological processes, as well as the products obtained exclusively from these processes. Finally, those legal figures that have led to an improvement in the patent system in neighboring countries have been introduced into our legislation, such as derived utility models.
Term of submission : Term to present allegations from October 21 to November 19, 2021 .
Presentation of allegations :
The allegations may be sent to the email address: CoordinacionJuridica@oepm.es
Only observations in which the sender is identified will be considered.
In general, the answers will be considered non-confidential and, consequently, freely disseminated. The parts of the information that are to be considered confidential must be specifically indicated and delimited in the text itself, giving reasons for such qualification.
|19 Nov 2021
|Improvements to the IPONZ online case management facility
Improvements to the IPONZ online case management facility as of 29 October 2021.
We’ve made a number of improvements to our website and online case management facility as of 29 October 2021.
Changes to hearings scheduling
Whenever a hearing is scheduled, its participants will now receive an email with a hearing date and a link to confirm their availability. Our Hearings Office will also provide further information to participants when scheduling their hearing.
This change is a first step of some significant technological enhancements to our case management facility, which will help ensure that hearings can be more efficiently scheduled.
Updated trade mark search and preliminary advice report
Much like our trade mark compliance report updates in December, our trade mark search and preliminary advice reports have been updated to provide:
Our trade mark subsequent compliance report has also been updated.
Update to data extracts for international trade marks
The World Intellectual Property Organization (WIPO) handles fees and renewals for international trade marks that designate New Zealand, instead of IPONZ. Because of this, whenever international trade marks are included in a data extract from our case management system, we will no longer list renewal due dates for those records.
|29 Oct 2021


Whitesnake - Ready An' Willing review
|Album:||Ready An' Willing|
|Release date:||May 1980|
01. Fool For Your Loving
02. Sweet Talker
03. Ready An' Willing
04. Carry Your Load
06. Ain't Gonna Cry No More
07. Love Man
08. Black And Blue
09. She's A Woman
10. Love For Sale [Previously unreleased] [2006 remastered bonus]
11. Ain't No Love In The Heart Of The City [Live At the Reading Festival 1979] [2006 remastered bonus]
12. Mistreated [Live At The Reading Festival 1979] [2006 remastered bonus]
13. Love Hunter [Live At The Reading Festival 1979] [2006 remastered bonus]
14. Breakdown [Live At The Reading Festival 1979] [2006 remastered bonus]
The Rolling Stones once famously sang "I can't get no satisfaction", but for Whitesnake there is only joyous "sweet satisfaction" to be had with their 1980 song and album of the same name: Ready An' Willing.
Released in 1980, this was the third studio album for British band Whitesnake - a band formed out of the ashes of Deep Purple by Mark III singer David Coverdale. By this stage of their career Whitesnake were pretty big in their homeland, the UK. They even headlined Saturday night at the big Reading Festival that same year. Two singles from Ready an' Willing charted in the UK - "Fool for Your Loving" reached #13 in the mainstream charts and the title track made #43. It was only later in the decade that they re-invented their look, to appeal to glam metal fans, and went on to conquer the huge US market, even re-recording earlier songs.
For Ready An' Willing, Coverdale recruited two previous members of Deep Purple, in Jon Lord on keyboards and Ian Paice on drums. They were joined by Mick Moody and Bernie Marsden on guitars and Neil Murray on bass. With this line-up in place, they created a blues-based rock album which showcases solid song writing and very competent musicianship. David Coverdale has a strong voice, Ian Paice is great on the drums (as always) and the guitar work is very well done. Whilst this is blues-based rock, the lyrical content is classic "cock-rock" - what else would you expect from a band that calls itself Whitesnake? The majority of songs centre on the theme of "loving", which is the traditional rock and roll euphemism for sexual intercourse. Songs like "Fool for Your Loving", "Sweet Talker" and the title track fall into this category. The real highlights, however, are the memorable ballads "Blindman" and "Ain't Gonna Cry No More". The album fades after this, with the closing tracks a tad unoriginal and uninspiring.
In the context of the time of its release, other British bands were exploring new ground that isn't evident here. The New Wave of British Heavy Metal bands (often abbreviated as NWOBHM) were pioneering a heavier and faster form of rock music, with Iron Maiden, Judas Priest and Diamond Head (to name just a few) releasing significant albums at this time. Ready An' Willing was more a continuation of an older style of blues-based rock music. Overall, this is well worth a listen, especially if you want some good-time classic rock to lighten your mood.
Hits total: 3032 | This month: 11


text: The hypothalamus is crucially involved in the circadian timing of the sleep-wake rhythm, yet also accommodates the most important thermoregulatory neuronal network. We have shown before that adults with pituitary insufficiency and history of chiasm compression due to a tumor with suprasellar extension fall asleep later and sleep shorter than those without such history and presumed hypothalamic involvement. To solidify the hypothesized link between vigilance and thermoregulation by the hypothalamus, we aimed to test the hypothesis that the presumed hypothalamic impairment in these patients also affects skin temperature and its association with sleep onset. In a case-control study of 50 patients (54.7±14.5 yrs of age, 30 males) with pituitary insufficiency, 33 of whom had a history of chiasm compression, ambulatory distal and proximal skin temperatures were assessed continuously for 24h. Sleep parameters were assessed via questionnaire. Group differences in mean skin temperature, calculated over the wake and sleep periods separately, and group differences in the strength of association between pre-sleep skin temperature and sleep onset latency were compared. Results showed that patients with a medical history of chiasm compression had lower proximal skin temperature during the day (34.1°C±.7°C vs. 34.6°C±.7°C, p.045). Additionally, the typical association between sleep onset latency and pre-sleep distal-to-proximal skin temperature gradient was absent in these patients (r-.01, p.96), whereas it was unimpaired in those without chiasm compression (r-.61, p.02). Thus, patients with history of chiasm compression show impaired skin temperature regulation in association with disturbed sleep. The findings support the hypothesis that a medical history of chiasm compression affects hypothalamic regulation of both vigilance and temperature, possibly by chronically affecting relevant nuclei, including the ventrolateral preoptic area and anterior hypothalamic preoptic area. (Corresponding Author: email@example.com) © Informa Healthcare USA, Inc.


text:
Whether you’re looking for a life mate or just to have fun, dating is an intricate and complex endeavor. It https://mail-order-bride.com/blog/how-to-deal-with-an-emotionally-unavailable-woman calls for opened interaction, self-awareness, and compassion. Dating customs in America vary depending on the region, ethnic lineage, and faith. A powerful dating landscape that calls for more quality and knowing is created by these variations, along with societal changes and changing gender dynamics.
In general, Americans are more receptive than ever to dating people with diverse backgrounds and life views. The vast majority of songs claim that they would be open to dating somebody who practices a diverse church or is of another race or ethnicity than they are. Additionally, the majority of Americans are willing to date someone who is disabled or who earns significantly more money than they do.
But, there is still much work to be done to eliminate prejudice and bias in the dating world. The majority of American grownups claim that when looking for a deadline, they have encountered some sort of discrimination or harassment. These experience have in some scenarios been severe and traumatic. Even worse, some people have been attacked while out on a time. In other instances, the abuser was a part of their own family.
Despite this, dating in the United States is still more of a personal preference than it is in other nations. In actuality, men are more likely to question ladies out on times than the other way around. American are also more likely to be open to dating a guy who practices another religion or who is disabled. Numerous changes in dating customs have resulted from the# Metoo movement. While some of these adjustments are good, others are not. For instance, some people have claimed that in the age of physical abuse and misconduct, it is more difficult for them to know how to act on deadlines.
How people choose where to go on a first time and who should paid is another significant shift in how citizens date in America. If you are fresh to the nation and its dating tradition, these queries can be nerve-wracking.
In the us, plates, coffee shops, and franchises are the most well-liked locations to meet anyone for a second time. Additionally, some people feel more at ease meet at job or a friend’s house. Making eye contact with someone while you are speaking is crucial. This demonstrates your interest in them and your attention to detail.
Having a wingman or woman you get beneficial for those who are not familiar with American dating customs. This friend keeps the discussion going, helps you meet new people, and ensures that you look your best. This name may become familiar to you if you enjoy the television series How I Met Your Mummy.

domain: sociology


As a member owned orchestra, the Seattle Phil offers musicians a rare opportunity for participation within the organization. Being a musician for the Philharmonic is more than rehearsing and playing music, it is an entry into one of the longest running orchestra communities in the Puget Sound Region.
Formal Auditions are held each fall with Music Director Adam Stern, personnel director Dick Griffith, and selected section principals. The orchestra will hold immediate ad-hoc auditions for sections with more pressing needs.
The Seattle Philharmonic is a volunteer orchestra and positions in the orchestra are un-paid.
To request an audition, or for more information, please visit our contact form.
A program of music by composers who were also major and influential conductors. Music by Leonard Bernstein opens the program: the overture to his landmark musical West Side Story. Felix Mendelssohn, lauded in his day as a superb conductor, is represented by his concert overture The Hebrides. SPO Principal Clarinet William Blayney will perform Mozart's Clarinet Concerto, still the greatest for the instrument. The concert concludes with the Northwest premiere of the Symphony No. 1 by the great Hungarian-born composer-conductor Antal Doráti, a favorite piece of Music Director Adam Stern's for decades and his first performance of it.
Benaroya Hall houses two performance halls in a complex that is thoroughly integrated into downtown Seattle. Occupying an entire city block at the very core of the city, the development celebrates the vital role of performance events while maintaining the continuity of commercial life along one avenue and providing a much-needed public space, in the form of a terraced garden, along another.
West Side Story: Overture
The Hebrides: Overture
Concerto for Clarinet and Orchestra, K. 622
William Blayney, soloist
Symphony No. 1


text:
BPH (benign prostatic hyperplasia) is the term used to describe the enlargement of the prostate gland. The prostate gland is found at the base of a man's penis. It is shaped about like a doughnut and the urethra passes through the "hole" of the doughnut. As a man ages, the prostate grows. Sometimes this growth can accelerate causing abnormal enlargement and almost a "pinching off" of the urethra.
You can imagine this is quite painful for the guy that has to endure the inability to pass urine! He could suffer such symptoms as:
-constant "urge" to urinate
-sleep disturbance from waking multiple times each night
-blood in the urine
As a natural health consultant, I usually suggested that gentlemen experiencing these types of symptoms use things like pumpkin seed, saw palmetto, zinc and more. Those guys would come back with testimonies of great results. However, those great results would take a while to "kick in." I have been scouring the information for something natural that would produce quicker results with no side effects.
I HAVE FOUND IT!
How does it work?
Equol is a natural substance that binds to an enzyme in the body (5-alpha reductase enzyme). This enzyme converts testosterone into a substance called DHT. It is this DHT that fits into receptors on the prostate gland and causes it to grow. By binding to this enzyme, testosterone does not convert. No conversion means there isn't as much DHT to even try to attach to the prostate gland.
There are medications that do this very thing. HOWEVER, medications carry side effects. These side effects can include:
-Decreased sex drive
-Increased ejaculatory dysfunction (such as a smaller amount of semen ejaculated or ejaculation of semen into the bladder instead of out through the penis)
-Difficulty getting an erection
-Breast tenderness or enlargement
-Weakness or fatigue
-Lightheadedness, dizziness, or fainting

domain: biology


text:
Dr. Herrera performed a delicate extraction under a bridge with excellent results. He was very caring and made the procedure as easy as possible. The recovery was simple. The whole staff was very helpful and made the visit go smoothly. The view from his operating area was calming and relaxing.
- Kathleen G

domain: medical


Ridgeview have completed the unthinkable in the history of English wine. For the third time in seven years Ridgeview have won the trophy for “Best International Sparkling Wine”.
The family company situated on the South Downs of England are proving that English Sparkling Wine is a serious contender on the world stage. Ridgeview Grosvenor (Blanc de Blancs) 2007 was awarded the trophy for the “Best Bottle Fermented Sparkling Wine” in the 2011 International Wine and Spirit Competition (IWSC) one of the most respected wine competitions in the world. Over 85 countries enter the competition annually and all wines are blind tasted by an esteemed judging panel.
Ridgeview previously won the same accolade in 2005 and a similar trophy last year in the Decanter World Wine awards for overall Best Sparkling Wine; the first time this was awarded to a wine other than Champagne. Mike Roberts Director and winemaker commented; “We are thrilled with winning this much sought after trophy for the second time. It is a great reward for all the family and team efforts that is put into creating our wine. What we are most pleased about is that we are proving it is not a one off and English sparkling wine is now a serious condender in the International wine market.”
Ridgeview was founded by the Roberts family in 1994, focusing on traditional Champagne grape varieties and methods on the South Downs of Sussex. Since the release of their wines a decade ago, Ridgeview has now amassed over 150 trophies and medals, putting English Sparkling Wine on an International stage. The late burst of sunshine in 2011 perfectly coincided with the recent Ridgeview October harvest, which will ensure the quality of this year’s grapes are excellent for the current vintage which will be available in 2013.
This latest accolade tops off an amazing year for Ridgeview after their Fitzrovia (Rosé) sparkling wine was served by her Majesty the Queen at Buckingham Palace in honour of President Obama’s visit in May, and founder Mike Roberts was awarded an MBE for his services to the English Wine Industry in June.


text:
Vietnamese Gov’t pushes for a safe and transparent capital market
The Ministry of Public Security has swiftly acted to clamp down on false information over Vingroup Chairman Pham Nhat Vuong, saying it has caused negative impacts on business operations and the stock market.
The Vietnamese Government remains steadfast in ensuring a safe, effective and transparent capital market, seen as a key factor to stabilize the macro-economic conditions and a sustainable capital mobilization channel for the economy.
The move was revealed in the Government’s Resolution No.86 on the vision for capital market development.
Under the plan, the Government would focus on finalizing regulations to support market operation and restructure the stock and monetary markets.
“The stock market is expected to become the main capital mobilization channel for the economy in mid-and long-term,” noted the resolution, expecting to ensure even development among monetary, and securities markets.
The Government bond market should become a standard in the finance market and help meet the Government's capital demand for socio-economic development.
By 2025, the Government expects the size of the stock market to reach 100% of the GDP, with outstanding loans of the bond market representing at least 47% of the GDP, of which that of the corporate bonds at 20%.
Meanwhile, the resolution calls for greater IT application in supervising the operation of the stock market.
The banking sector is tasked with restructuring credit institutions and resolving bad debts under the Government’s plan for the banking sector in the 2021-2025 period, targeting a bad debt ratio among banks below 3%.
Among the key tasks, the Government would tighten the supervision of corporate bond transactions and companies’ use of capital in the post-issuance period to ensure transparency and lawful rights of investors.
The stock market authorities are responsible for upgrading the infrastructure system for market healthy development and improving the quality of information disclosure, and financial statements of issuers.
The Government stays focused on addressing any violations in the market to protect investors and build the market’s confidence, especially on cracking down market manipulation.
The long-term goal would be for Vietnam’s stock market to be upgraded from the current frontier market status to an emerging one, which would allow more investment capital, including foreign investors.
The Ministry of Finance, the State Bank of Vietnam, the Ministry of Planning and Investment, and the Ministry of Security, are expected to follow closely the market situation to timely intervene, if needed, to stabilize the market.
“Government agencies are required to maneuver both monetary and fiscal policies in flexibly and effectively to promote sustainable development of the market while promoting the involvement of new participants,” it added.
Clamping down false information over Vingroup’s owner
The Ministry of Public Security on July 11 said it is dealing with nine individuals from seven provinces/cities for allegedly fabricating news over Chairman of Vingroup Pham Nhat Vuong, saying the tycoon is being banned from exiting the country.
According to the Chief of the Ministry of Public Security’s Office, Major General To An Xo, the Hanoi Department of Information and Communications will impose a fine against To Vi Hoan (Nam Tu Liem District), 38, on charges of “disseminating harmful information" related to Vingroup.
Such an act has undermined the credibility, and lawful rights of businesses, and caused negative impacts on the stock market, said Xo.
Hoan was fined VND7.5 million (US$320) by the municipal Department of Information and Communications and was asked to remove the false information.


text:
There are approximately 20 different disorders that are classified as “vasculitis”. “Angiitis” and “Arteritis” are both synonyms for vasculitis, literally meaning “inflammation within blood vessels” or “inflammation in arteries.” Because there are so many types of vasculitis, the group is sometimes referred to in the plural: vasculitides (pronounced “vas que lit’ i deez”).
There are many different types of diseases that belong to this category. Although the diseases are similar in some ways, they often differ with respect to which organs are affected, which medications are used to treat them, and other characteristics.
Characterized by the triad of mouth ulcers, genital ulcers, and eye inflammation. However, other organ systems may also be affected.
Buerger’s Disease (Thromboangiitis Obliterans)
Mainly affects smokers, leading to decreased blood flow to the hands and feet.
Eosinophilic granulomatosis with polyangiitis (EGPA, formerly known as Churg Strauss)
Associated with asthma, nasal polyps, sinusitis, elevated eosinophil counts, and vasculitis. EGPA has a tendency to involve lungs, peripheral nerves, skin, kidneys, and heart.
Characterized by recurrent purpura (red dots) on the lower extremities and may be associated with hepatitis C virus infections or paraproteinemias.
Giant Cell Arteritis
This disease was previously named “temporal arteritis”, and is the most common type of vasculitis in adults in North America. It is a large vessel vasculitis that affects people over the age of 50 (although most individuals affected are 70-80 years of age). It can be characterized by fever, headache, and jaw/scalp pain.
This disorder is often followed after an upper respiratory tract infections and is often, but not necessarily, self-limited.
A systemic vasculitis affecting small and medium–sized blood vessels associated with the autoantibody, ANCA.
The prototype of systemic vasculitis, involving many different organ systems and focussed on medium–sized arteries.
A vasculitis that sometimes complicates the course of rheumatoid arthritis (RA). Usually occurs in patients with a history of severe RA.
A large vessel vasculitis that affects the aorta, its major branches to the extremities, and sometimes internal organs. Usually occurs in young women (< age 50 years).
Granulomatosis with Polyangiitis (GPA, formerly known as Wegener’s)
A systemic disease that involves the lungs, kidneys, upper respiratory tract, and other organs and associated with the autoantibody, ANCA.