New & Noteworthy

Follow the CSHL Cell Biology of Yeasts Meeting on Twitter!

November 03, 2013

Wish you were going to Cold Spring Harbor for the Cell Biology of Yeasts meeting this week, November 5-9?  SGD will be live tweeting from CSHL, highlighting topics from talks and posters. Keep up with events at the meeting by following @yeastgenome on Twitter or searching #YCB2013 for all tweets!

Categories: News and Views

How Yeast May One Day Help Michael J. Fox

October 31, 2013

Folks, yeast has been on a roll lately with regard to helping to understand and finding treatments for human disease. Last week we talked about how synthetic lethal screens may find new, previously unrecognized druggable targets for cancer. And this week it is Parkinson’s disease.

Now of course yeast can’t get the traditional sort of Parkinson’s disease …it doesn’t have a brain.  But it shares enough biology with us that when it expresses a mutant version of α-synuclein (α-syn) that is known to greatly increase a person’s risk for developing Parkinson’s disease, the yeast cell shows many of the same phenotypes as a diseased neuron.  The yeast acts as a stand-in for the neuron.

In a new study out in Science, Tardiff and coworkers use this yeast model to identify a heretofore unknown target for Parkinson’s disease in a sort of reverse engineering process. They screened around 190,000 compounds and looked for those that rescued toxicity in this yeast model. They found one significant hit, an N-aryl-benzimidazole (NAB) compound. Working backwards from this hit they identified its target as Rsp5p, a Nedd4 E3 ubiquitin ligase. 

The authors then went on to confirm this finding in C. elegans and rat neuron models where this compound halted and even managed to reverse neuronal damage. And for the coup de grace, Chung and coworkers showed in a companion paper that the compound worked in human neurons too. But not just any human neurons.

The authors used two sets of neurons derived from induced pluripotent cells from a single patient.  One set of neurons had a mutation in the α-syn gene which is known to put patients at a high risk of Parkinson’s disease-induced dementia.  The other set had the mutation corrected.  The compound they identified in yeast reversed some of the effects in the neurons with the α-syn mutation without significantly affecting the corrected neurons.  Wow.

What makes this even more exciting is that many people thought you couldn’t target α-syn with a small molecule. But as the studies here show, you can target an E3 ubiquitin ligase that can overcome the effects of mutant α-syn.  It took an unbiased screen in yeast to reveal a target that would have taken much, much longer to find in human cells. 

The mutant α-syn protein ends up in inclusion bodies that disrupt endosomal traffic in the cell.  The NAB compound that the authors discovered restored endosomal transport and greatly decreased the numbers of these inclusion bodies.  Juicing up Rsp5 seemed to clear out the mutant protein.

The next steps are those usually associated with finding a lead compound—chemical modification to make it safer and more effective, testing in clinical trial and then, if everything goes well, helping patients with Parkinson’s disease.  And that may not be all.

The α-syn protein isn’t just involved in Parkinson’s disease.  The dementia associated with this protein is part of a larger group of disorders called dementia with Lewy bodies that affects around 1.3 million people in the US.  If everything goes according to plan, many of these patients may one day thank yeast for their treatment.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight, Yeast and Human Disease

Tags: alpha-synuclein, Saccharomyces cerevisiae, ubiquitin ligase, yeast model for human disease

Using Yeast to Find Better Cancer Treatments

October 24, 2013

Current cancer treatments are a lot like trying to destroy a particular red plate by letting a bull loose in a china shop.  Yes, the plate is eventually smashed, but the collateral damage is pretty severe.

Ideally we would want something a bit more discriminating than an enraged bull.  We might want an assassin that can fire a single bullet that destroys that red plate. 

One way to identify the assassin that can selectively find and destroy cancer cells is by taking advantage of the idea of synthetic lethal mutations.  “Synthetic lethal” is a genetic term that sounds a lot more complicated than it really is.  Basically the idea is that mutating certain pairs of genes kills a cell, although mutating each gene by itself has little or no effect.

A synthetic lethal strategy seems tailor made for cancer treatments.  After all, a big part of what happens when a cell becomes cancerous is that it undergoes a series of mutations.  If scientists can find and target these mutated genes’ synthetically lethal partners, then the cancer cell will die but normal cells will not.

This is just what Deshpande and coworkers set out to do in a new study in the journal Cancer Research.  They first scanned a previous screen that looked at 5.4 million pairwise interactions in the yeast S. cerevisiae to find the best synthetic lethal pairs. They found 116,000 pairs that significantly affected cell growth only if both genes in the pair were mutated.

A deeper look into the data revealed that 24,000 of these pairs had human orthologs for both genes. In 500 of these pairs, at least one of the partner genes had been shown to be mutated in certain cancers. Using a strict set of criteria (such as the strength and reproducibility of the synthetic lethal effect, and the presence of clear one-to-one orthology between yeast and human), the authors narrowed these 500 down to 21 pairs that they decided to study in mammalian cell lines.

When the authors knocked down the expression of both genes in these 21 gene pairs in a mammalian cell line, they found six that significantly affected growth.  They focused the rest of the work on the strongest two pairs, SMARCB1/PMSA4 and ASPSCR1/PSMC2.  These mammalian gene pairs correspond to the yeast orthologs SNF5/PRE9 and UBX4/RPT1, respectively.

The authors identified two separate cancer cell lines that harbored mutated versions of the SMARCB1 gene.  When this gene’s synthetic lethal partner, PMSA4, was downregulated in these cancer lines, the growth of each cell line was severely compromised. The same was not true for a cell line that had a wild type version of SMARCB1—this cell line was not affected by downregulating PMSA4.  The authors used a synthetic lethal screen in yeast to identify a new cancer target which when downregulated selectively killed the cancer without killing “normal” cells.

This proof of principle set of experiments shows how the humble yeast may one day speed up the process of finding cancer treatments without all those nasty side effects (like vomiting, hair loss, anemia and so on).  Yeast screens can first be used to identify target genes and then perhaps also to find small molecules that affect the activity of those gene products.  Yeast may one day tame the raging bull in a china shop that is current cancer treatments.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, Saccharomyces cerevisiae, synthetic lethal

Memorial Gathering for Dr. Fred Sherman

October 23, 2013

A memorial gathering in memory of Fred Sherman will be held at 10:00 am on Friday, December 6, 2013. The gathering will be held in the Ryan Case Methods Room (Rm #1-9576) of the University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester NY 14642. This event will be a celebration of the life and science of Fred, comprised of reminiscences about Fred by some who knew him well, followed by an opportunity for any guest to say a few words about Fred. For more information, contact Mark Dumont (Mark_Dumont@urmc.rochester.edu), Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642 (Phone 585-275-2466).

Categories: News and Views

A GLAMorous New Role for Prefoldin

October 17, 2013

The prefoldin complex seemed like an ordinary housekeeper. It sat in the cytoplasm and folded protein after protein, just as Cinderella spent her days folding laundry for her stepsisters.

In the old story, the handsome prince searched the kingdom for a girl whose foot would fit the glass slipper. Using this crude screen, he finally found Cinderella and revealed her to be the true princess that she was.

In a new study, Millán-Zambrano and coworkers did essentially the same thing for the prefoldin complex.  They searched the genome of S. cerevisiae for new mutations that would affect transcription elongation. They found the prefoldin complex subunit PFD1 and went on to establish that in addition to its humdrum cytoplasmic role, prefoldin has a surprising and glamorous role in the nucleus facilitating transcriptional elongation.

The researchers decided to cast a wide net in their search for genes with previously undiscovered roles in transcriptional elongation. Their group had already worked out the GLAM assay (Gene Length-dependent Accumulation of mRNA), which can uncover elongation defects.

The assay uses two different reporter gene constructs that both encode Pho5p, an acid phosphatase. One generates an mRNA of average length, while the other generates an unusually long mRNA when fully transcribed. The acid phosphatase activity of Pho5p is simple to measure, and correlates well with abundance of its mRNA. If there is a problem with transcriptional elongation in a particular mutant strain, there will be much less phosphatase activity generated from the longer form than from the shorter one. So the ratio of the two gives a good indication of how well elongation is working in that mutant strain.

Millán-Zambrano and coworkers used this assay to screen the genome-wide collection of viable deletion mutants. They came up with mutations in lots of genes that were already known to affect transcriptional elongation, confirming that the assay was working. They also found some genes that hadn’t been shown to be involved in elongation before.  One of these was PFD1, a gene encoding a subunit of the prefoldin complex. As this deletion had one of the most significant effects on elongation, they decided to investigate it further.

Prefoldin is a non-essential complex made of six subunits that helps to fold proteins in the cytoplasm as they are translated. The authors tested mutants lacking the other subunits and found that most of them also had transcriptional elongation defects in the GLAM assay, although none quite as strong as the pfd1 mutant.

Since prefoldin is important in folding microtubules and actin filaments, the researchers wondered whether the GLAM assay result was the indirect effect of cytoskeletal defects. They were able to rule this out by showing that drugs that destabilize the cytoskeleton didn’t affect the GLAM ratio in wild-type cells, and that mutations in prefoldin subunits didn’t confer strong sensitivity to those drugs.

If prefoldin has a role in transcription, it would obviously need to get inside the nucleus. It had previously been seen in the cytoplasm, but when the authors took another look, they found it in the nucleus as well. Furthermore, Pfd1p was bound to the chromatin of actively transcribed genes! And besides its effect on transcription elongation, the pfd1 mutant has lower levels of RNA polymerase II occupancy and abnormal patterns of histone binding on transcribed genes.

There’s still a lot of work to be done to figure out exactly what prefoldin is doing during transcriptional elongation. Right now, the evidence points to its involvement in evicting histones from genes in order to expose them for transcription. But even before all the details of this story are worked out, this is a good reminder never to assume that an everyday housekeeper is only that.

With the right screen we can find new and exciting things about the most humdrum of characters. A glass slipper screen revealed the princess under that apron and chimney soot. And a GLAM assay revealed the sexy, exciting transcription elongation factor that is prefoldin.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: prefoldin, RNA polymerase II, Saccharomyces cerevisiae, transcription elongation

Yeast Winnows Down GWAS Hits in Autism

October 10, 2013

Cheap and easy genome sequencing has been both a blessing and a curse. We are able to find an incredible wealth of variation, but for the most part we have no easy way to tell whether a difference might contribute to a disease or not.

The poster child for this problem is autism. Lots of genome wide association studies (GWAS) have been done and lots of rare variants in lots of different genes have been found – unfortunately, way too many to pick out the ones that really matter.

Luckily our friend yeast can help. Various researchers have identified a number of variants in the human cation/proton antiporter gene NHE9 that associate with autism. In a new study, Kondapalli and coworkers used the NHE9 ortholog NHX1 from S. cerevisiae as an initial screen to identify which variants impact the activity of the NHE9 protein. They found that two of the three mutations they looked at compromised the activity of yeast Nhx1p.

They then set out to confirm these results in mammalian cells.  When they looked at protein activity in glial cells, they found that all three mutations compromised the activity of NHE9.  This is obviously different from what they found in yeast.

Now this doesn’t mean that yeast is useless for this approach (God forbid!).  No, instead it means that it is probably only useful for a subset of autism mutations.  Kondapalli and coworkers had suspected this, but apparently the subset is smaller than they initially thought.

The first thing they did was to generate a rough three dimensional map of the NHE9 protein in order to see which parts the two proteins shared.  The idea is that they could then do a quick screen in yeast with mutations that affect the shared structure.

While the structure of NHE9 has not been solved, we do have the structure of its distant bacterial relative, NhaA.  Kondapalli and coworkers aligned the two along with the yeast ortholog Nhx1p and identified conserved regions.

Three of the NHE9 mutations associated with autism—V176I, L236S, and S438P—were all predicted to be in shared, membrane-spanning parts of the protein.  The researchers introduced the equivalent mutations into NHX1—V167I, I222S, and A438P. 

 A yeast deleted for NHX1 grows poorly in high salt and low pH and also has increased sensitivity to hygromycin B, as compared to a yeast with a functioning NHX1.  Two of the mutant genes, carrying A438P or I222S, failed to rescue these growth defects.  The other mutant gene, with the V167I change, worked as well as wild type NHX1 at rescuing the yeast.  So at least in yeast, two of the three mutations appear to impact protein activity.

The next step was to see if the same was true in mammals.  Easier said than done!  Ideally they would want to investigate whether these mutations affected the protein in the cells where NHE9 is usually active.  Too bad no one knows this protein’s natural habitat.  This is why the researchers starting slicing mouse brains to figure out when and where the protein is expressed.

While we don’t have time or space to go into all the details here, Kondapalli and coworkers found that when and where in the brain NHE9 was expressed made sense as far as a possible contribution to autism.  They also found that glial cells had about 1.2 fold more NHE9 transcripts than did neuronal cells.  They therefore did their assays of protein activity in a type of glial cells called astrocytes.

While they couldn’t completely knock out NHE9 in mouse astrocytes, they were able to knock down its expression by over 80%.  When they added back the mutant NHE9 genes, they found that all three failed to mimic the effect of adding back wild type NHE9 to these cells.  This is different than what they found in yeast, where only two of the mutations impacted protein activity. 

When they went back to their 3D model, they saw that the mutation that differed, V167I, affected a less defined part of the structure.  This points to the fact that for the quick yeast screen to work, they need to be looking at parts of the protein where the structure is shared between the yeast and the human version.  In a perfect world they would have had crystal structures of each to work off of instead of having to kludge together a model.

In any event, this is the first step towards validating yeast as a quick screen for identifying mutations that can impact protein activity and so are good candidates for being involved in disease.  Yeast may help scientists separate the wheat from the chaff of GWAS and so help figure out how diseases happen and maybe help find treatments or even cures.  Well done yeast.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight, Yeast and Human Disease

Tags: autism, Saccharomyces cerevisiae, yeast model for human disease

Randy Schekman and the Awesome Power of Yeast!

October 07, 2013

Congratulations to Randy Schekman, James Rothman, and Thomas Südhof, who have been awarded the 2013 Nobel Prize in Physiology or Medicine for their work in understanding how the cell organizes its transport system. Randy Schekman used the awesome power of yeast to identify and characterize genes required for vesicle traffic. James Rothman characterized these and other proteins in mammalian cells, and Thomas Südhof showed the critical role of vesicle trafficking in nerve cells. You can read summaries of their Nobel winning work at Nature, The Scientist, and The New York Times, or search SGD to see how each of these researchers has used our model organism in their research: Randy Schekman, James Rothman, Thomas Südhof.

Categories: News and Views

Yeast Muad’Dib

October 02, 2013

Remember in Dune when Paul Muad’Dib took a sip of the “Water of Life” and needed weeks in a coma to turn it into something that let him survive and emerge even more powerful than before?  Turns out yeast sometimes have to do something similar.

Now of course the yeast aren’t consciously moving molecules around to deal with a poison like Paul did.  No, instead they sometimes need to transcribe low levels of a mutated gene over a long period of time to survive in a new environment.  

This process is called retromutagenesis.  The idea is that a cell gets a mutation that would allow it to survive and prosper in a new environment if only it could replicate its DNA.  Unfortunately the new environment is so unforgiving that the cell can’t replicate. 

The cell escapes this catch-22 by transcribing the gene with the mutation so that the mutant protein can get made.  Once enough of this protein is made, the cell manages to get up enough steam to power through a cell cycle.  Now the mutation is established and the yeast can make lots of mutant protein and happily chug along.

In a new study in the latest issue of GENETICS, Shockley and coworkers hypothesize that something like this is happening in their experiments.  They were studying oxidative damage to DNA and found that some of their mutants required many days before they could grow in the absence of tryptophan (trp).  They argue that these late arising revertants were due to the cells having to wait until retromutagenesis allowed enough functional Trp5p to be made so the cell could replicate.

The authors have created strains of yeast with various mutations in the TRP5 gene that cause the yeast to be unable to grow in the absence of trp.  What makes these strains so useful is that they are set up in such a way that six different, specific point reversions can result in a functional TRP5 gene.  They can then analyze any Trp+ revertants to see what types of damage lead to which type of mutations.

One of the first things the authors discovered was that oxidative damage caused all six different reversions.  While this was interesting, the specific mutation they wanted to focus on was a G to T transversion which occurs when G is converted to 8-oxoguanine.  This is why they focused on the trp5-A149C strain.

The main way that yeast cells deal with 8-oxoguanine is by removing it with the Ogg1 protein, a DNA glycosylase.  When Shockley and coworkers deleted this gene in their strain, the number of revertants increased by 20-fold.  From this they concluded that most of the revertants were the result of the misreplication of an 8-oxoguanine. 

This is where the yeast run into a problem.  In the absence of trp, the trp5 mutants do not replicate at all…they do not go through even one cell cycle.  But to revert to a functional TRP5 gene, this strain needs to go through a cell cycle.  This is why the authors think that the first step towards reversion is a mutation in the TRP5 transcript.

Consistent with this idea is the fact that the mutated G in this strain is on the transcribed strand and that this is important for high revertant frequencies.  It also helps to explain why revertants took so long to appear.  Basically there had to be a buildup of enough functional Trp5p to allow a single cell cycle to happen.  Then the G could be converted to a T and the yeast could happily grow.  In this specialized case, it looks like reversion is dependent on retromutagenesis. 

But retromutagenesis, also called transcriptional mutagenesis, doesn’t happen only in yeast cells.  It’s being studied as a possible way that all kinds of quiescent cells, such those in the process of becoming tumor cells, or bacteria whose growth has been stopped by an antibiotic, can mutate and escape the conditions that are restricting them. Our little friend may not save the human race from destruction like Paul did, but once again yeast is proving pretty darn useful in getting results that make a difference for human health.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight, Yeast and Human Disease

Getting Into Yeast’s Genes

September 26, 2013

Yeast has been responsible for a lot of hook ups in its day (think beer goggles and margaritas on the beach).  Now it is payback time.  In a new study, Giraldo-Perez and Goddard have figured out how to make yeast more promiscuous.

No, they don’t get the yeast drunk.  Instead, they found that strains containing VDE, a homing endonuclease gene (HEG), entered meiosis more often than genetically identical strains that lacked VDE.  The yeast that contained this “selfish” gene (well, actually intein) were ready to go haploid more often than those that didn’t.

VDE and its ilk are said to be selfish because they end up getting passed down to more offspring than a certain Austrian monk might have predicted.  When a diploid is heterozygous for an HEG, the homing endonuclease cuts the sister chromosome at the equivalent spot. Then, when the diploid undergoes meiosis, the sister is repaired through recombination causing both chromosomes to contain the VDE gene.  Now instead of two spores containing VDE, all four will.

Giraldo-Perez and Goddard monitored the percentage of sporulating cells over a 30 day period and found that after five days, a higher percentage of diploids homozygous for VDE sporulated compared to diploids heterozygous for or lacking VDE.  The authors contend that under the right conditions, this increased sporulation would allow VDE to spread through a population 20 times faster than it might otherwise.  And the authors found that VDE needs something like this or it might disappear.

Like alcohol, VDE isn’t all lowered inhibitions and good times.  For example, yeast homozygous for VDE grow significantly more slowly than do yeast lacking VDE in YPD, grape juice, vineyard soil, vine bark (heterozygotes fall in between).  This obviously puts yeast carrying VDE at a disadvantage, meaning that if it didn’t have another trick up its sleeve, it would dwindle away to nothing.  That trick is speeding up sporulation. 

The authors weren’t able to determine why this little bit of DNA can have such a profound effect on the growth rate of yeast.  It is almost certainly too little DNA to affect the time it takes the yeast to copy its DNA.  And the endonuclease itself is probably not randomly nicking the chromosomal DNA in the mitotic state, since it is kept out of the nucleus by host encoded karyopherins.

So VDE is a truly a parasitic selfish gene.  It is parasitic because it sucks a little of the life out of a yeast cell.  And it is selfish because way more daughters end up with it than might be predicted.  Sounds like a nice description for many drunk people…

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: homing endonuclease, intein, Saccharomyces cerevisiae, selfish gene, VDE

Have Your Fuel and Eat It Too

September 19, 2013

Back in 2008 and 2011 there were huge spikes in the cost of food that caused riots in various parts of the world.  These things were pretty bad and one of our favorite beast’s best products, ethanol, may have been at least partly to blame.  In an attempt to deal with global warming, governments had created incentives that made it more lucrative to turn food into ethanol to power cars rather than keeping it as food to feed people.  The law of unintended consequences reared its ugly head and caused food prices to rise high enough to be unaffordable by the very poor.   

This situation arose because right now, pretty much the only commercially viable way to make ethanol is to use sugars like those found in sugar cane or starches like those found in corn.  Ultimately this won’t be a problem once scientists learn to coax yeast or other microorganisms to make ethanol out of agricultural waste.  Until then, though, one way to lessen the impact of ethanol production on food supplies might be to engineer a yeast strain that can more efficiently turn sugars into ethanol. 

One of the most inefficient parts of yeast fermentation is that the silly thing converts anywhere from 4-10% of the sugars it gets into glycerol instead of ethanol.   In a new study, Guadalupe-Medina and coworkers have engineered a strain of yeast that produces 60% less glycerol and 8% more ethanol than other commercial strains.  If they can scale this up, it might help us feed both the world’s population and our cars.

It has been known for some time that yeast end up making glycerol during fermentation because of redox-cofactor balancing issues.  In essence, the excess NADH that is made in fermentation reactions is reoxidized by converting part of the sugar into glycerol.  One obvious way to get less glycerol would be to give the yeast some other way to reoxidize its NADH. 

Guadalupe-Medina and coworkers decided to persuade yeast to use carbon dioxide instead of sugars.  Not only would this make sugar use more efficient, but their particular plan would also convert that carbon dioxide into a precursor that could be shunted into the ethanol producing pathway.  Theoretically the yeast should now increase its ethanol production both by wasting less sugar on glycerol and by turning carbon dioxide into ethanol.  And it turns out that this idea actually worked in practice.

The first step was to introduce the Rubisco enzyme into the yeast.  Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) is really one of the key enzymes in life…it provides the foundation for almost all life on the planet by fixing carbon dioxide from the air into ribulose-1,5 phosphate.  But that isn’t the important point here.  No, the key point for this work is that in the process of doing this, the enzyme oxidizes NADH.  By putting Rubisco in yeast, the yeast should now be able to reoxidize its NADH without making useless glycerol.

Of course this is easier said than done!  Rubisco is multi-subunit in most beasts and persnickety to boot.  But with a bit of work, they managed to get Saccharomyces cerevisiae to express a working copy of Rubisco.

So they would only have to introduce a single gene, the authors used the single subunit enzyme from T. dentrificans. As expected, this gene alone was not enough.  They knew from previous work that Rubisco would not work in yeast without the help of a couple of E. coli chaperones, groEL and groES.  When they expressed all three genes at the same time, they got Rubisco to fix carbon dioxide in Saccharomyces cerevisiae.  

The next step was to introduce the enzyme phosphoribulokinase (PRK) so that the ribulose-1,5 phosphate could be converted into 3-phosphoglycerate, a precursor in the ethanol pathway.  Luckily this was much easier than Rubisco and worked on the first try.  They had now engineered a Frankenyeast that should be able to make more ethanol and less glycerol.

When they tested the new strain, Guadalupe-Medina and coworkers found they had indeed engineered a more efficient yeast.  In anaerobic chemostat conditions, this yeast made 68% less glycerol and 11% more ethanol than the usual commercial strain.  They obtained similar results, 60% less glycerol and 8% more ethanol, in batch fermentations.  They had succeeded in improving an already awesome beast.

If this strain works on an industrial scale and if commercial producers all used this strain instead of the ones they currently use, the authors calculate we could get an extra 5 billion liters of ethanol added to the 110 billion we are already making.  That might just be enough to tide us over until scientists come up with a way to make ethanol commercially from non-food sources.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: biofuel, Saccharomyces cerevisiae

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