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OfflineKizzle
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Re: P. cubensis Genetics Info [Re: bodhisatta]
    #21494693 - 04/02/15 10:43 PM (9 years, 1 month ago)

This has some interesting information in it. In one of the experiments the psilocybin was being enzymatically dephosphorylated into psilocin using enzymes extracted from the mushroom. When the concentration of psilocin reached a certain level the psilocin stopped increasing and the blue discoloration started to appear at the same time.

That's enough to convince me that the bruising and the psilocybin/psilocin are probably linked in some way at least.
https://circle.ubc.ca/bitstream/id/70093/UBC_1978_A6_7%20W35.pdf

Although it's hard to take it seriously when the author's name is Wei-Wei Wang :lol:


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Edited by Kizzle (04/02/15 10:50 PM)

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Offlinekmetric
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Re: P. cubensis Genetics Info [Re: bodhisatta]
    #21494768 - 04/02/15 11:09 PM (9 years, 1 month ago)

Interesting thread..have you started on the GFP shrooms yet, g3n3? I'd love to see those..

I'm picturing a walk through the forest at night with green fluorescent shrooms all over the place.

Contam resistance is interesting as well. Let's say we find a GMO vegetable that has resistance to a certain pest that mushrooms suffer from as well, how difficult would it be to give the mushrooms the same gene modification?

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Invisiblemicro
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Re: P. cubensis Genetics Info [Re: EncyclopediaBrown]
    #21495385 - 04/03/15 06:45 AM (9 years, 1 month ago)

Quote:

EncyclopediaBrown said:
Quote:

micro said:
Quote:

EncyclopediaBrown said:
I was under the impression the enzymatic process that occurs was dephosphorylation of psilocybin to psilocin. Then the psilocin oxidizes.




Enzymatic degradation doesn't happen without enzymes.

That doesn’t mean there's not random shit in there that can oxidise it.



Psilocybin can't be oxidized, the phosphate acts as a protecting group against this action. It is indeed true that enzymatic degradation doesn't happen without enzymes, but there are phosphatases in mushroom tissue that perform this.




PSILOCIN <==


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Offlineg3n3h4x0r
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Re: P. cubensis Genetics Info [Re: micro]
    #21516103 - 04/07/15 05:43 PM (9 years, 1 month ago)

It's been pretty off topic, but interesting.  Let's apply this topic experimentally since this is an experimental thread.

Question:  How do different cooking methods affect resulting potency?

Hypothesis:  We all seem to have our own.  I think we actually need to cook these things rather than sticking to the melting point data on wikipedia - think about cooking meats, the center of the cut never reaches the temperature of the cooking container.  I personally think there will be degradation, but it's not a big of a deal as we're making it out to be.

Exp. Design:  Many people here are cultivators, so there will need to be sacrifice of several grams of mushrooms.  The current best extraction method needs used as well. 

Cond. 1: Fresh Mushroom
Cond. 2: Dried, Mushroom
Cond. 3: Fresh, sliced, pan fried mushroom in light oil (saute?) (thickness of the cut should be included)
Cond. 4: Fresh, mushroom tea (optional, imo)
Cond. 5: Dried, mushroom tea
Cond. +: Maybe other fresh cooking methods - battered and deep fried?
Cond. 0: (optional) Another species mushroom without any chemicals that can be precipitated from the extraction.


Condition 1 and 2 will serve as the comparative controls.  Several replicates (I suggest 4-6) should be used.

Condition 3+ will serve as the experimental conditions.

Condition 0 should be utilized as a control for the extraction, but it's optional since nobody can get published out of this.  I would greatly suggest, but this is up to the experimenter

Method:  (Here's where suggestions/help should come in!) 

- Exactly 20g fresh or 2g of dried mushroom should be weighed out for each condition and replicate (can change).  The time after the drying process of the dried condition should be included in the report for quality purposes.

- Dried mushroom tea will utilize 2g of dried mushroom and freshly boiled water (try and do as much damage as possible).  Extraction should be done on the entire drink.  The process for this tea should be documented - I'm not sure what the current 'recipe' is.

- Pan fried is difficult.  2 tbsp of oil should be added to a pan for the 20g of fresh mushrooms.  A digital probe/grill thermometer should be used to measure the temperature of the pan and it should be kept constant across all replicates - the temperature of the mushrooms shouldn't matter, just cook them til they look delicious.  Let cool on a paper towel and extract.

- Other conditions:  Cleanliness of the method should be considered.  I think battered and deep fried could be fun, but extracting can be a sloppy mess.  Baked could be a great option, as oven temperatures are constant.

- The weight of the extracts should be measured.  To keep environmental variables at a minimum, all replicates from a condition should be extracted at once.  The replicates will provide an average breakdown of chemical content and, if 6 or more are done, should begin to provide statistical evidence that a certain cooking method degrades an important amount of content.

Results:  I think the results will be beneficial as it will provide a percentage degraded for each cooking condition - calculations can be done afterward to actually standardize doses considering protocol is followed.  eg.  If pan frying degrades 66% of the product, the content can multiplied by the reciprocal (3x) to reach the desired dosage.  This opens doors for people who can't stomach them or just want to get fancy.

Considerations:  I'm not too sure how well the extraction solvents will fare with the introduction of fats/oils - I haven't looked up any of the extractions.  Maybe a wash of sorts can be performed if needed.  Consider and include the efficiency of the extraction if it's documented.

If any of the math, etc. is daunting, I have a ridiculous mathematical background and could do 100% of it - just provide decent data!

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Offlineg3n3h4x0r
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Re: P. cubensis Genetics Info [Re: g3n3h4x0r]
    #21516157 - 04/07/15 05:57 PM (9 years, 1 month ago)

Quote:

Interesting thread..have you started on the GFP shrooms yet, g3n3? I'd love to see those..

I'm picturing a walk through the forest at night with green fluorescent shrooms all over the place.

Contam resistance is interesting as well. Let's say we find a GMO vegetable that has resistance to a certain pest that mushrooms suffer from as well, how difficult would it be to give the mushrooms the same gene modification?




Not quite, I've been more on a bioinformatics pursuit right now so I can make something more useful - been trying to get some sets of data.  The GFP will mainly show that the transformation works.  There shouldn't be much interest in it as it, unfortunately, takes a special UV light to illuminate them (I will always been disappointed by this fact :frown:)

I could do something with luciferase and have a permanently glowing mushroom, though!  I'm not too sure how well it would function, however.  Most pictures are extended exposures and I haven't worked with it outside of small assays.

Resistance to contamination is also something that is usually bred in species.  Many of the GMOs with the media's attention are crops that allow for advanced (safer, better) pesticide, fungicide, etc. use due to genetically introduced resistance to the chemicals.  It would be hard to incorporate a defense system as they're usually complex, and if something is secreted to kill off a contaminant, can we still eat it without a stomach ache?  =)

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OfflineMessy_Lion
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Re: P. cubensis Genetics Info [Re: g3n3h4x0r]
    #21517025 - 04/07/15 09:27 PM (9 years, 1 month ago)

I have been having very similar thoughts to your project here, and some searches into fungal transformation yielded interesting procedures. I don't have my own equipment like you, and using my university's equipment is obviously off limits. I would focus on luciferase, and finding a way to get it into the genome, and be expressed (hopefully without affecting alkaloid production :mushroom2:). If you need any help with the bioinformatics side, I could probably lend you a hand or some code, depending on exactly what you are doing. P. cubensis hasn't been sequenced very well/often, so any bioinformatic analysis on the species will probably be very limited, for the time being.

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Invisiblemicro
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Re: P. cubensis Genetics Info [Re: g3n3h4x0r]
    #21517883 - 04/08/15 01:06 AM (9 years, 1 month ago)

Quote:

g3n3h4x0r said:
Results:  I think the results will be beneficial as it will provide a percentage degraded for each cooking condition - calculations can be done afterward to actually standardize doses considering protocol is followed.  eg.  If pan frying degrades 66% of the product, the content can multiplied by the reciprocal (3x) to reach the desired dosage.  This opens doors for people who can't stomach them or just want to get fancy.




That's not really how it works.

It doesn't evaporate.

Quote:

Considerations:  I'm not too sure how well the extraction solvents will fare with the introduction of fats/oils - I haven't looked up any of the extractions.  Maybe a wash of sorts can be performed if needed.  Consider and include the efficiency of the extraction if it's documented.




You aren't going to get quantitative analysis unless you purify it. You could possibly use Grignard reagent but that's... eh, it just doesn't seem like a good quantitative analysis.

Best way to purify is take fresh mushroom and pulerize, quicly sonicate at somewhere above 106 and do a methanol extration. Then evaporate the CH3-OH with vacuum and do an acid/base extraction and try to crystalize at the end (i.e. pour in like 10X insoluble alcohol and freeze and seed if you have it handy).

That's just my best guess. I know I saw someone here do it.


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Invisiblemicro
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Re: P. cubensis Genetics Info [Re: Kizzle]
    #21517898 - 04/08/15 01:14 AM (9 years, 1 month ago)

Quote:

Kizzle said:
This has some interesting information in it. In one of the experiments the psilocybin was being enzymatically dephosphorylated into psilocin using enzymes extracted from the mushroom. When the concentration of psilocin reached a certain level the psilocin stopped increasing and the blue discoloration started to appear at the same time.

That's enough to convince me that the bruising and the psilocybin/psilocin are probably linked in some way at least.
https://circle.ubc.ca/bitstream/id/70093/UBC_1978_A6_7%20W35.pdf

Although it's hard to take it seriously when the author's name is Wei-Wei Wang :lol:




It wasn't dephosphorylated; it was never polyphosphated.

Unless I'm thinking of the wrong paper.

If you add tryptamine to the substrate you get a high percentage of psilocin, suggesting a downregulation mechanism/


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OfflineKizzle
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Re: P. cubensis Genetics Info [Re: micro]
    #21518671 - 04/08/15 09:27 AM (9 years, 1 month ago)

Page 39

The dephosphorylation of psilocybin was rapid for both enzyme extracts... The liberation of psilocin reached a maximum just before the mixture turned blue.

I'm assuming the liberation of psilocin being referred to is that resulting from the desphorylation of the psilocybin i.e. the liberation of psilocin from the phosphate group.


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Offlineg3n3h4x0r
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Re: P. cubensis Genetics Info [Re: Kizzle]
    #21520153 - 04/08/15 04:39 PM (9 years, 1 month ago)

Quote:

That's not really how it works.

It doesn't evaporate.




This was one thing I was going to add to the considerations, but couldn't remember it once I got to the end.  Sat for a long while trying to figure out what it was. 

I don't really know what the chemical would breakdown into with gradual heating without some reading.  More importantly, I don't know if that product would come out of the extraction.  I made some assumptions with the experiment, but if this question could be answered and experiment shaped around it, it could work well.  Let's all discuss this - otherwise all of this talk is just a bunch of unsupported guesses.

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