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Please help Lyso-SENS!


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#1 John Schloendorn

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Posted 23 November 2005 - 01:46 AM


As most of you know, Lyso-SENS is a recent effort by the Methuselah Foundation to target age-related storage diseases, which are caused by the accumulation of some pathogenic chemical substance in the body with age. Examples of age-related storage diseases are Alzheimer's disease (amyloid and tau aggregates), heart disease and stroke (cholesterol and oxidized cholesterol species in the artery wall), age-related macular degeneration (lipofuscin of the retinal pigment epithelium) and skin wrinkles (advanced glycation end products of the extracellular matrix). As such, Lyso-SENS targets three of the seven SENS pillars: Intracellular aggregates, extracellular aggregates and extracellular crosslinks.
Lyso-SENS works by using methods from environmental bioremediation to find soil microbes capable of degrading the target substance. We then hope to isolate the enzymes used by these microbes to do so and use them for therapy in a way that is similar to current enzyme replacement treatments for congenital lysosomal storage diseases. However, not all soil microbes are amenable to our screening methods, not all enzymes we may discover will work in the human physiological environment, some will have deleterious side-effects, and so on. So we need many sources of different microorganisms to begin with.

Posted Image

This is where you come in. Please send us soil samples from your area, from places that you think are likely to contain microbes capable of degrading age-related aggregates. About a handfull of soil (100-200 grams) will be plenty. The more microbial diversity we can put into these experiments, the better will be our chances of sustained success. So your participation will truly increase the chances of success of the Lyso-SENS project.

Now what kinds of places would be the right choice? We do not know for sure, but one can speculate. As a rule of thumb, age-related aggregates get degraded in nature, whereever aged vertebrates die and rot on a regular basis. There are animal graveyards, places where farms or slaughterhauses drop their excess or simply forests or lakes rich in wildlife.
Initially, we have also thought of human graveyards, and I still have not completely rejected the idea. However, please remember to be responsible when obtaining samples from anywhere, and always uphold local regulations and traditions.
Another possibility is industrial wastewaters. Chemistry plants generally have a high diversity of organic compunds in their effluent and some of them may by chance resemble age-related aggregates enough to drive the selection for enzymes we can use.
In general, highly biodiverse regions are preferrable over ones dominated only by a few species. I would be happier to have a sample from the great barrier reef, rather than one from a pond that is overgrown with only one type of algae.
There is also no need to obtain samples from recently deceased animals, since at first it is the nutricious, easily digestible stuff that gets degraded. Recalcitrant, age-related aggregates will follow only when most other parts are gone. We do not know the exact timeframes of these processes, so it is best to choose a region where decomposition takes place continously or regularly.

The highest priority targets we are pursuing right now are 7-ketocholesterol (7KC) and A2E. The chemical structures of these compounds are shown below.

7KC is a major troublemaker in the atherosclerotic lesion. It can impair the removal of cholesterol from such lesions, can trigger apoptosis in arterial smooth muscle cells and macrophaes and has a number of other pathogenic effects. Chemically, it resembles cholesterol, steroid hormones and (to a lesser degree) also bile. 7KC occurs naturally in some vertebrate arteries, but most animals do not get as bad atherosclerosis as humans do, so it should be most abundant in human remains. Though it should also be worth checking out places where animals rot that did not move a lot and had a bad diet, such as high-throughput farm dumps. I think wastewater or chemical outlets may be worth going for, too.

A2E is a pathogenic fluorophore of the lipofuscin of the retinal pigment epithelium in the eye, that has been shown to cause a whole bunch of pathological effects in age-related macular degeneration, the leading cause of blindness in the elderly. It has vitamin A derived side-chains, which also occur in plants and some synthetic chemicals. Another part of the molecule is a pyridinium ring, which also occurs in diverse molecules of life and synthetic chemistry, so therapies may in principle come from any places that are rich in such compounds. However, the combination of the two features is rare, and except for places where eyes rot I am not sure where to find A2E specifically in nature. Use your imagination.

If you choose to send us samples, please do so immediately after you obtain them. Do not store them unnecessarily. For packaging we would recommend to wrap solid samples in plastic, liquid samples in some sort of screwcap bottle and put them in a suitably sized parcel. 100-200 grams ("a handful") will be a plentiful amount. Cooling or sterile handling will not be necessary. And of course be sure to add some chocolate, packaged separately. Please also add a brief description of where and when you took the sample. We will be ready to receive samples from Nov 28, 2005.

Please send your sample(s) to:

By regular mail:

John Schloendorn
The Biodesign Institute
PO Box 875701
Tempe, AZ 85287-5701
USA

(The above address cannot receive FedEx / UPS shipments)

By FedEx / UPS:

John Schloendorn
The Biodesign Institute
1001 South McAllister Ave
Tempe, AZ 85287-5701
USA

(Apparently, the above address cannot receive regular mail -- sorry about the trouble)

I can reimburse you for the cost of shipping if required, but you can also donate shipping to Lyso-SENS. I will make sure you get appropriate credit for this somewhere.

If you are unsure whether the sample source you are having in mind is a good choice, you are most welcome to ask here. And even if you are sure, you are cordially invited to share your ideas and story.

Also, if you like to collaborate in other ways, please get in touch with me. There are opportunities to put skills in sequence bioinformatics and/or synthetic organic chemistry to good use.

[Edit: spelling, fixed address, minor details]

Edited by John Schloendorn, 07 May 2007 - 05:43 PM.


#2 John Schloendorn

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Posted 23 November 2005 - 01:46 AM

Our current target molecules, 7KC and A2E:

Attached Files



#3 John Schloendorn

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Posted 23 November 2005 - 01:49 AM

A more technical paper on the Lyso-SENS method is available here: Medical Bioremediation.

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#4 jrhall

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Posted 23 November 2005 - 04:07 PM

Hi John,

I am happy to see LysoSENS moving forward. I think this is exactly the sort of thing that needs to be done in order to generate some momentum for SENS. I shall be sending my samples next week.

I did have a question, if you took a pile of aged dead mammals and left them out, wouldn't they soon be colonized by assorted bacteria, fungus etc who would breakdown the whole organism? Could you then watch the intracellular junk and see who is degrading it?

Jeff

#5 eternaltraveler

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Posted 23 November 2005 - 06:51 PM

The largest meat packing plant in the country isn't to far from me. I'll look into where they dispose of the waste.

#6 JonesGuy

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Posted 23 November 2005 - 08:37 PM

John, what's your marginal cost on processing each sample to satisfaction? There has to be some reason why we just don't collect samples from EVERYWHERE.

#7 Mind

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Posted 23 November 2005 - 09:52 PM

I will be sending you a sample. Also, I want to thank you for your research efforts and for reaching out to the Imminst membership. This is an opportunity for us all to help advance anti-aging research.

#8 John Schloendorn

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Posted 23 November 2005 - 10:41 PM

Thank you so much, guys! You truly make me wonder how mainstream environmental biotech persons get along without such wonderful community support.

Jeff,

if you took a pile of aged dead mammals and left them out, wouldn't they soon be colonized by assorted bacteria, fungus etc who would breakdown the whole organism? Could you then watch the intracellular junk and see who is degrading it?

Well, as I sort of alluded to above, such measurements would be greatly confounded by a millionnfold or more excess of fast-growing microbes on easily digestable stuff that we are not interested in. Also there is such a huge diversity of chemicals in a rotting cadaver that there would always be some things that look very similar to the one we want to measure, which can compromise our measurement. That is why we need to isolate the specific compounds we want to target.
Also, it is possible that the relevant creatures would simply not find a dead mammal that we drag out only now. So we are interested in ecological niches that have been around for a long time so that different microbes had a chance to move in, establish themselves, compete with each other under constant selection for the best degraders, and so on.

Qjones,

John, what's your marginal cost on processing each sample to satisfaction? There has to be some reason why we just don't collect samples from EVERYWHERE.

The main problem is the cost of the isolated age-related aggregats that we grow them on. For example reasonably pure 7KC costs $160 a gram. It is hard to say how much per sample, because there is no set protocol. To some degree, one always has to play around with the culture conditions and see what happens before one can know what is best to do next. A2E is even more difficult, because one cannot buy it at all, so it has to be synthesized by our collaborator and shipped to us.
The processing itself is mainly limited by the manpower needed to measure the growth of microorganisms plus the degradation of the target compound, which can become quite elaborate, depending on how thoroughly one wants to do it... So without an IBG we cannot scale this up indefinitely just yet :)

I'm curious, where do you get your samples from, any ideas yet?

Also, those of you who already copied the mailing address somewhere, please note that I updated it, since matters are not always as simple as they seem here at biodesign ;-)

#9 John Schloendorn

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Posted 24 November 2005 - 09:12 PM

collect samples from EVERYWHERE

Though let me emphasize that we have the capacity to process at least several dozens of samples from different areas in the very near future and more later, which is why I am posting this! Fellow ImmInsters, please help us to run these experiments at full throttle!

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#10 JonesGuy

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Posted 24 November 2005 - 11:25 PM

Too bad you can't send us samples of dishes with 'consumables' on them. That's way too expensive. Is it possible to ship mice that are bred to suffer the conditions you're looking for? One could breed the mice and create a little graveyard for them. (Not really feasible by next week).

I'll ship some dirt tomorrow. Genetically diverse, eh? Hmmn, I'll work on that.

#11 mitkat

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Posted 25 November 2005 - 04:17 AM

I'll be more than happy to oblige! This actually involves something I study! And by that, I mean soil.

#12 John Schloendorn

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Posted 25 November 2005 - 05:23 AM

Not really feasible by next week

I agree ;-) Normal rabbits are pretty good at getting atherosclerosis with human-like oxysterol levels, but only when fed a saturated lipid rich diet.

Great Mitkat! What in particular are you studying in soil? Is it in any way similar to this project?

#13 tontaube

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Posted 25 November 2005 - 08:57 AM

Hi,

I'll send two samples from graveyards...only...have to wait till full moon to make it more "realistic" :-) Just kidding.

As they are quite well-known graveyards, the probe might be contamined. Any suggestion to prevent this (apart from not taking it near where people walk)?

#14 tontaube

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Posted 25 November 2005 - 08:58 AM

Hi,

I'll send two samples from graveyards...only...have to wait till full moon to make it more "realistic" :-) Just kidding.

As they are quite well-known graveyards, the probe might be contamined. Any suggestion to prevent this (apart from not taking it near where people walk)?

#15 Da55id

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Posted 25 November 2005 - 02:23 PM

Thanks everyone for their help! I know that you will keep in mind that the sanctity of graveyards must be carefully respected. John please confirm that this is the case with all soil samples so that we are not guilty of being Frankensteinian.

#16 Mind

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Posted 25 November 2005 - 02:29 PM

No graveyards. That is a good policy.

Also, there is an opportunity for media exposure on my end. I work at a TV station and collecting soil samples to aid anti-aging research may be something they would want to cover. There is also a local newspaper that might find the soil collection interesting and do a feature story about it. Should I go for this media exposure, or would you rather just keep it under wraps for now?

#17 JonesGuy

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Posted 25 November 2005 - 03:33 PM

I think that the soil in graveyards would be useless, in that it's all just topsoil - the bodies (widely spaced) are many feet apart, and deep underground.

#18 Da55id

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Posted 25 November 2005 - 04:51 PM

Let's keep it quiet until the first peer reviewed paper is written on this. Should happen w'in 6 months or so.

dg

#19 olaf.larsson

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Posted 25 November 2005 - 05:26 PM

I admire your industriality, finaly someone who makes something in reality!!!!!

My intuition (which ofcourse maybee be wrong) says me the following about the project:

*Organisms that degrade the compounds in some way will be easy to find.

*It will not be very easy to find the gene responsible for this. Bacteria for example do not use vitamin A as I know it. So the enzyme responsible for A2E katabolism will not be associated to vitamin A metabolism. As I have been informed the identification of the gene should be by degenerate PCR. But I dont understand how one could make degenerate primers for an unknown enzyme, this requires guessing which enzyme it is.

*The enzymes that degrade the compounds will degrade many other things as well so it will not be easy to find enzymes that will not do any harm when inserted to a mamal.

Once more Im happy and greatfull that you have lauched this project hopefully it could be a model for future similar projects. ...lets make an intracellular junk database, to get an overview of which intracellular junk there is to get rid of.

Edited by wolfram, 25 November 2005 - 11:15 PM.


#20 Da55id

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Posted 25 November 2005 - 10:51 PM

wolfram - This research was started very quietly earlier this year. John is the third investigator in this relay race to get rid of intracellural junk. I too am glad to see real action and success so far.

#21 olaf.larsson

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Posted 26 November 2005 - 07:01 AM

Methusalea is pointing out something important. Graveyard diging could give this negative publicity. A corpse will not only decompose on graveyards but pretty much anywhere where microorganisms can grow so I propose that you dont dig in graveyards.

Edited by wolfram, 26 November 2005 - 07:18 AM.


#22 jnagyjr

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Posted 26 November 2005 - 02:49 PM

There's a place in my backyard where I had to bury my dogs earlier this year (they were murdered), would a soil sample from that area be useful? If not there is a small stand of trees near a local church that I could grab a sample from.

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#23 JonesGuy

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Posted 26 November 2005 - 08:07 PM

Jnagyjr, it would be better than a lot of places.

I've read the paper (it was pretty good), and I have a greater insight into what John is doing. He's using the penicillin "throw everything against the wall and see what sticks" approach, I'm guessing. The advantage is that he shouldn't have much marginal cost, because either the microorganism will root on the petri dish, or it won't - so they can be reused.

I don't think that slaughter houses would be the best location for brain-type aggregates, because it sounds like you're looking for microorganisms that target age-related compounds - and cattle are slaughtered at a young age. The athersclerosis-type compounds might be there, since cattle lead a pretty obese life.

I still think that breeding 'high-risk' mice and burying them is a good idea, but too slow. Something to keep in mind though, since the opportunity cost is so low.

I wish you guys luck!

#24 JonesGuy

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Posted 26 November 2005 - 11:45 PM

Hey.

Has anybody investigated the law regarding shipping soil? I've recently heard (from a reliable source) that something like this can piss off US Customs. It might even be illegal.

#25 jnagyjr

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Posted 27 November 2005 - 06:00 AM

US Customs be damned I say. Its my soil, I'll do what I want with it, even if it means putting it in a ziploc baggie and sending it off to Arizona.

#26 Da55id

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Posted 27 November 2005 - 05:26 PM

The research is taking place in the US. Therefore US law must be obeyed. To do otherwise would destroy the research effort.

#27 JonesGuy

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Posted 27 November 2005 - 05:43 PM

US Customs be damned I say. Its my soil, I'll do what I want with it, even if it means putting it in a ziploc baggie and sending it off to Arizona.


This is not always a good policy. The laws are there for a reason, mainly to prevent biological contamination. 99 times out of a 100, we wouldn't have to worry, but sometimes you can introduce a disease that has a either a) devastating effect on the ecology or b) a devastating effect on your tax dollars as officials scramble to inefficiently do something because they're afraid of something.

Look at the huge economic effect of BSE. Billions of dollars, with $ per threat ratio being way out of proportion. Imagine the additional investigations that would have occurred (at huge cost) if people shipped cattle anyway, and didn't keep good records.

In addition, I think MM has a point, in that publishing the data would be rather important to us, so that more potential money can be poured into the research (in the competitive way scientists do).

#28 John Schloendorn

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Posted 27 November 2005 - 05:50 PM

Many thanks to everyone for the continued feedback and encouragement!

the probe might be contamined

Contaminations are in gernal not a big deal. A contamination that cannot grow on our compounds will do no harm (because it remains small) and a contamination that can grow is certainly most welcome (We will want its genes). The only problem can be contaminations with very high nutrient cofntents that might serve as an alternative food source, other than our age-related aggregates (which is why I asked to wrap the chocholate separately). But we can wash those away routinely, if they are soluble.

John please confirm that this is the case with all soil samples so that we are not guilty of being Frankensteinian.

Of course, desecrating graves cannot be in anyones interest, and I will not process samples where I have reason to believe that this happened or that come without a declaration of where they were obtained. If you wish to collect graveyard samples, I strongly recommend to obtain permission of the respective authority first (just like with samples from any place that is owned by somebody who is not you). I will be happy to confirm the intended research use of the samples in writing if required.

Should I go for this media exposure

I agree with Dave. It is not a good idea to go public with research without being able to point to your results in the form of a peer-reviewed article. Later, especially if the results should turn out positive, I think it would be a great idea.

I think that the soil in graveyards would be useless, in that it's all just topsoil - the bodies (widely spaced) are many feet apart, and deep underground.

Which is why the bacteria need to crawl around in the soil, to get to the target compounds in the first place. They have little sense for what is going on in the area, so they will be crawling randomly most of the time. That means you should be able to catch them close to the surface and away from the immediate grave area. Remember, in theory we need only a single replication-competent bacterium to grow an indefinite amount of them. For example, I think there is a perfectly good chance to find them just outside the graveyard perimeter fence.

The enzymes that degrade the compounds will degrade many other things

Possible, but it is also possible that some are specific enough. This is one of the reasons we are so interested in diversity.

Organisms that degrade the compounds in some way will be easy to find

Those of you who have been to SENS2 will remember that Mark and Jacques, who were the first LysoSENS workers already found creatures that seem to be growing on 7-Ketocholesterol (there was no work on A2E though). But easy to find heh, Mark why don't you tell him what you went through to get the samples ;-)

But I dont understand how one could make degenerate primers for an unknown enzyme

Because of the conservation of functional domains. For example, an A2E-degrading enzyme might have a domain that shares a certain percentage of its sequence vitamin-A binding domains of known vitamin A hydroxylases. If you can pinpoint just which bases are conserved in a couple of such known enzymes, and which are variable, then you can design primers with constant bases against the conserved regions and open bases against the variable ones. I think I sent you some papers that visualize the concept nicely. The second primer might for example target the extracellular secretion tag, which is also highly conserved, so that you can amplify the sequence in between.

There's a place in my backyard where I had to bury my dogs earlier this year (they were murdered), would a soil sample from that area be useful?

Murdered? Oh my god, is this the humanity we are trying to save? I think the idea of your dogs helping the fight against aging in such a way is quite remarkable. So sure, let's give them a chance.

either the microorganism will root on the petri dish, or it won't - so they can be reused

True, the limiting factor is probably the accumulation of soil nutrients and non-growing organisms in the medium that will eventually serve as an alternate food source and divert selective pressure away from the target compounds.

Hey. Has anybody investigated the law regarding shipping soil?

When you ship an international parcel, you will generally be asked to declare the contents. Please by all means do so accurately. In particular, you should indicate that the soil is a native and unmodified sample that will be used by a suitably equipped laboratory for research purposes alone. If any problems arise, we will by all means need to go the official way to solve them. Eventual questions by the post office can be directed to my email address or by phone: USA - 480 727 0731. I cannot process samples if the parcel gives me any reason to suspect that it was not accurately declared.

#29 olaf.larsson

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Posted 27 November 2005 - 06:02 PM

But easy to find heh, Mark why don't you tell him what you went through to get the samples


If a single or a few persons are able to find a microorganisms that do this by hard work in a relatively short time,
I would consider it relatively "easy". I consider "difficult" things to be things that many people work with for 20 years without any
obvius results, they are not uncommon in our area. Yes please tell me about the previous experiment..
Have you identified the organism?

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#30 JonesGuy

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Posted 27 November 2005 - 07:42 PM

Hrrm,

I've been surfing for regulations regarding the shipping of soil samples, and I have to say that government websites always frustrate me in their lack of usability.

I think I'll just ship the sample, and let the post office know what I'm doing. They can handle it.




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