229 replies to this topic

[John Schloendorn]

Caver, you'd be a LysoSENS hero forever. If you're in the US, great. If outside, first check with your post-office if mailing animal fecal samples is OK (we know soil is OK, but haven't tried fecal). If official docs from the recipient biosafety facility are any help, we can provide them. In any case make sure it's smell-proof ;-)

[caver]

I do live in the US in fact. If all goes well, I'll try and collect a guano sample within the next month (heavy schedule at present), and get it/them off to you. I'll include the GPS cooridnates of the cave entrance.

[JonesGuy]

See, this is what I love about people. Because there is no way that recording GPS coordinates of caves is EVER going to be part of my life ...

... it isn't even something I'd dream of. My life is just so different. I'm a little jealous.

[olaf.larsson]

I have the following proposition:
Go to your local sloutherhouse ask for the eyes of animals, (preferably cattle since they have big eyes). Explain you want them it for a biological experiment, and ensure that you are not a member of a bizarre cult or something.. Dig them down at various locations take the soils after one week.

[John Schloendorn]

A week would almost certainly not be enough, because decomposition of the juicy, proteinaceous material would generate a millionfold excess of the type of bugs we don't want. I did (briefly) consider spraying some pure A2E into some piece of forest, but then thought that doesn't really seem to have any advantage over taking a piece of forest to my culture dish right away. Please feel free to prove me wrong.

[ag24]

> ensure that you are not a member of a bizarre cult or something

Hang on wolfram - he IS a member of a bizarre cult

... or something

:-)

[John Schloendorn]

Heh, but if you want something badly and immediately, it most unfortunately can still help to ensure people that we are not... (sometimes). It's changing!

[ag24]

"assure" vs "ensure"

[Mind]

A slaughterhouse is not too bad of an idea, but you would want to dig in places where they dump excess or unusable parts. The stuff is probably picked up by a rendering or waste disposal company but the area of ground around the waste pick-up area or dumpster probably has years of animal waste products built up in the soil. Maybe that would be a good place to look.

[John Schloendorn]

What can I say, it's always good to see you criticise my grammar rather than my science!

[olaf.larsson]

did (briefly) consider spraying some pure A2E into some piece of forest

Yes I have also thought about this. Im not sure it is the same thing. If you spray diluted A2E in the forest any useful organism "passing by" would have the chance to expand their population. If you take forest sample you will only get the bactria present in the sample. I have a new altenative proposition:

Put diluted A2E in agarplates in various places (preferably at places where people passing by do not see the plates). If any usefull microorganism lands on the plates it will start to grow. The A2E in the solution will not be destroyed by this experiment contrary to if it would be sprayed out in the forest.

By the way I can remind you that it was in a similar way that Alexander Fleming discovered penicillin in 1928, When Penicillium notatum by accident landed on his Staphylococcus culture.

Edited by wolfram, 28 January 2006 - 10:04 PM.

[Da55id]

I never thought I'd ever admire bacteria so much :-0

[ddhewitt]

The class of compounds most relevantly similar to A2E are probably retinoids. They are found in fruit, flowers, other colored natural compouds, and are also enriched in some carnivorous vertebrates. (I found no A2E degraders in kitten feces, but maybe a tiger would do. Game anyone?)
Any place associated to the regular presence and/or decomposition of those should be a good place to check out, but please also be creative. Nature is huge and incomprehensibly complex, so seeking out any type of bizarre habitat is in principle a great idea. Retionids aren't exactly infrequent. (I'm really looking forward to giving Guy's latest deep sea ones a shot!)

Wouldn't the backyard compost heap be a good place. That is where all my left over bit of fruits and veggies end up. I would expect them to be richer sources of retinoids than meat. These fruit and veggie bits have travelled from all over the world to end up in my compost heap and probably bring all sorts of microorganisms with them creating a diverse ecology.

Duane

[jmmathieu]

Yes I have also thought about this. Im not sure it is the same thing. If you spray diluted A2E in the forest any useful organism "passing by" would have the chance to expand their population. If you take forest sample you will only get the bactria present in the sample. I have a new altenative proposition:

Put diluted A2E in agarplates in various places (preferably at places where people passing by do not see the plates). If any usefull microorganism lands on the plates it will start to grow. The A2E in the solution will not be destroyed by this experiment contrary to if it would be sprayed out in the forest.

By the way I can remind you that it was in a similar way that Alexander Fleming discovered penicillin in 1928, When Penicillium notatum by accident landed on his Staphylococcus culture.

This does pose an interesting question for John: are you investigating the degradative capabilities of fungi as well? Personally I throw away all cultures that have been contaminated with fungi. In the beginning I was only interested in obtaining bacteria that could degrade 7KC and really wasn't concerned with aseptic techniques until I had growth and needed to obtain pure cultures, so I had some fungal growth. 7KC has proven to be a relatively easy compound to degrade, so I never considered using fungi, but if you are having problems finding bacteria to do the job for A2E would you consider fungi?

[John Schloendorn]

Hmm Wolfram, I see your argument does have some force... The experiment would have to be modified in a couple of ways. First there should be no agar, but silica plates, since I found that agar-degraders are abundant even on the mainland (Interestingly, some of my 7KC degraders are also agar-degraders). Second, A2E is light-sensitive, so one would need a mechanism to let microbes in but keep almost any non-red light out. Third, I would need to persuade my collaborator that this plan is worth to have her make the rather copious amounts of A2E it requires. Fourth, I'd need a forest, or some other biodiverse habitat. This is Arizona, all I have is sun-baked desert and golf-courses!
While these problems all do not seem impossible to overcome in one way or another, their combination does render your plan less than optimally efficient. So yes, depending on the outcome of the latest series of screens, I'll consider it. Thanks for your input!

Duane, sounds good to me. We have had one compost-heap sample, which didn't work, but that may have been bad luck, or it might have been too old when I started the A2E experiments (it was one of the first submissions).

Jacques, I sure as hell would prefer bacteria by a great deal, but if fungi is all I get, then fungi is what I'm gonna learn to deal with... I have only one fungal 7KC degrader and he isn't even very good at it.

[JonesGuy]

Yeah, my compost heap contains a lot of diverse fruits. Bananas, avocados, oranges, etc. from all over the world. So, it may not have been the melting pot I was hoping for. But it was worth a try.

I haven't yet be able to get permission to take a sample from the buffalo slaughter farm.

[eternaltraveler]

As I've spoken with John about, I'm attempting to put together a lysosens project for my masters thesis in microbiology.

I'll probably try to find degraders for several aggregates. I'd like to find one for hyperphosphorylated tau or better yet a dephosphorylator. Though dealing with microgram quantities will make it rather tough.

As I said though, I will try to do several aggregates so hopefully I get some good data about at least one.

Hmm Wolfram, I see your argument does have some force... The experiment would have to be modified in a couple of ways. First there should be no agar, but silica plates, since I found that agar-degraders are abundant even on the mainland (Interestingly, some of my 7KC degraders are also agar-degraders). Second, A2E is light-sensitive, so one would need a mechanism to let microbes in but keep almost any non-red light out. Third, I would need to persuade my collaborator that this plan is worth to have her make the rather copious amounts of A2E it requires. Fourth, I'd need a forest, or some other biodiverse habitat. This is Arizona, all I have is sun-baked desert and golf-courses!
While these problems all do not seem impossible to overcome in one way or another, their combination does render your plan less than optimally efficient. So yes, depending on the outcome of the latest series of screens, I'll consider it. Thanks for your input!

if I can get any A2E I could put these silica plates in a tropical rain forest. As far as protecting them from light, what about burying them under an inch or so of soil with some kind of highly porous covering?

[JonesGuy]

Hey, do you still need dirt samples, or has that part shown itself to be over?

[John Schloendorn]

Good point, here's the plan:

I am for the moment busy with data analysis and characterizing the bugs I've got, and am also out of A2E. I will try to schedule another A2E delivery in some 3-6 weeks, and this is also when I expect to receive some glucosepane, to add a third target compound. This is the quantitatively most important collagen advanced-glycation end-product (AGE) in aging and diabetes.

The optimal time to receive further samples will be when these target compounds arrive.

I will specify a more precise time-point soon. Though I'd like your input on where to get glucosepane-degrading microbes. For example, I'm thinking of places where aged human skin particles get deposited and degraded. Behind grandpa's sofa? A piece of carpet from a cheap nursing-home that don't clean well? Also, AGEs occur in some sugary fruit. I'll keep posting more as I find out.

[JonesGuy]

Would it make sense to put some skin scrapings in some soil now, then? (By scrapings, I mean that dead skin on the elbows or heels ...

As well, what about people with dandruff? There might be something there.

[John Schloendorn]

Hm, I still have doubts as for the utility of burying stuff, but I could be wrong. Also, for using a human skin donor, I would need to obtain informed consent and ethics approval, which might not be worth the hassle... We can discuss this idea further by personal communication.

[maestro949]

I have to admit that I'm fairly ignorant to how Lyso-SENS is trying to accomplish the breaking down of the bad proteins that have accumulated in the lysosomes but I think it goes something like this...

Feed the "bad" proteins that the lysosome can't break down to many bacteria that biodegrade organisms and then see if they can also degrade the bad stuff too. Once you find one, study the enzymes that performed the proteolysis on the protein and voila, you have a candidate for a pharmaceutical drug.

If that's fairly accurate, wouldn't it also be possible to just feed entire dead cells to the same bacteria and look for the target molecules in the bacterial byproducts? Or better yet, get big chunks of the target compounds and toss them in a few peoples' gardens (burying skin cells would be tough to catch the critters that ate the lysosomal junk). If something starts eating away at them, bingo, you have a suspect. Feed them to different species too besides bacteria. Birds, dogs, cats, etc. and see if any digestive tracks can tear 'em up.

OK, i'll shut up and get back to reading

[John Schloendorn]

Maestro, yes, you got the rough idea.

"just feed entire dead cells to the same bacteria and look for the target molecules in the bacterial byproducts?

This seems to me not superior to our strategy of feeding the pure target molecule and looking for it in the "byproducts". We have discussed your other ideas above.

[maestro949]

This seems to me not superior to our strategy of feeding the pure target molecule and looking for it in the "byproducts".

I was thinking that by feeding the whole cell you could test for all of the non-edible-by-the-lysosome compounds that have accumulated could be tested for in a single shot.

We have discussed your other ideas above.

Oops. You did! Sorry, thought I read the whole thread but just realized I missed a whole chunk of it.

[John Schloendorn]

Ah, now I get your idea, not bad.

First, you would not want to do this on a community of different microbes for selection purposes, because feeding whole cells would greatly bias the culture towards fast-gowing sugar, protein and lipid feeders, which might then poison or otherwise kill a minority of slower degraders of rare stuff.

However, if you start out with a pure culture that you know degrades one of your target compounds, and this compound is in humans part of a heterogenous mixture of similar compounds in a single cell type, such as lipofuscin or oxysterols, then a method based on your idea might tell you what other relevant targets they can eat. For example, I offered both cholesterol and 7kc to my isolates in order to assess which substrate they would prefer and if the breakdown mechanism was similar. At first glance, I think offering whole foam cell extracts might be a difficult, but exciting experiment to extend these findings. Thanks for the suggestion!

Edited by John Schloendorn, 06 March 2006 - 09:11 PM.

[Da55id]

this would be like the pilot fish that clean shark's teeth :-)

[caver]

*bump*
Mr. Schloendorn et al., I'm finally going to be able to retrieve a guano sample the weekend of May 5th. I see this thread has been idle for some time; if you guys are still searching for these microbes/enzymes, I'll get one or more samples off to you. Let me know something.

[John Schloendorn]

We're still out of target compounds, and busy with the 7kc degraders, which turned out quite capable at degrading even some other compounds (esters) which also make trouble in heart disease. So we still don't have an immediate need for more samples.

However, two other target compounds are being made, and as soon as we get them there will be a major campaign calling for soils, probably only weeks in the future. Don't worry, it will be impossible to overhear ;-)

[treonsverdery]

geocities.com/treonbarleyverdery/index.html

Edited by treonsverdery, 01 November 2006 - 01:39 AM.