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InvisiblePinback
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Snake venom hybridization method debunked (?)
    #25612171 - 11/13/18 01:53 PM (5 years, 4 months ago)

If you were on the Shroomery a few years back (ten or so), you probably know that "snake venom hybridization" was a hot topic. The idea was that one could add rattlesnake venom to agar, inoculate the agar with two species of fungi, and somehow the snake venom would allow genetic exchange to occur.

The idea was (to the best of my knowledge) first described in a paper in International Journal of Medicinal Mushrooms by John Holliday in 2004, where a "hybrid" of two Cordyceps sinensis strains was developed. I was already very doubtful when I read it the first time, for a number of reasons: low scientific quality; earlier dubious publication by the author; weak proof of hybridization; no published use of the method before or after, for example. Another problem with the method is why a proteolytic venom would weaken a chitin cell wall and thereby allow genetic exchange.

Then, the other day, for some reason I happened upon a paper with various species that have been described as anamorphs of Cordyceps sinensis (more properly called Ophiocordyceps sinensis). I quote:
Quote:

Paecilomyces hepiali Chen (strain CS-4) is probably the most popular commercial strain used to produce most of the mycelial products sold in the world marketplace as O. sinensis. It used to be called Cordyceps sinensis (strain CS-4). The strain CS-4 was isolated by the Chinese Academy of Sciences in 1972 from specimens found in Qinghai province, central China. It was tested for its medical potential, and by 1988 it had been approved in China as a medicine under the name Jin Shui Bao. At present there is a lot of reported evidence that CS-4 is not a correct anamorph of O. sinensis but belongs to a different fungus, Paecilomyces hepiali Chen.




So, what was previously called the CS-4 strain of Cordyceps sinensis is really Paecilomyces hepiali, which been found to be an endoparasite of Cordyceps sinensis.

What is really interesting here is that one of the "Cordyceps" strains that was used for hybridization in the Holliday paper was CS-4 (note: it is written as CX4 in the paper, but that is clearly a typo as it is written CS4 here).


So, I would suggest that what was isolated is not a "hybrid" of two Cordyceps sinensis strains, but rather Cordyceps sinensis parasitized by Paecilomyces hepiali.

I also want to make it clear that I have no reason to question that the paper was written in good faith, or that the authors isolated a different kind of mycelium, or that the metabolite profile of the isolated mycelium was different. In fact, many microorganisms produce more and different metabolites when stressed. However, I think it is clear that the isolated mycelium is not a hybrid, and that the snake venom method for hybridization is not valid.

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OfflineThesunbeam
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Re: Snake venom hybridization method debunked (?) [Re: Pinback]
    #25612187 - 11/13/18 02:02 PM (5 years, 4 months ago)

I remember this, from what I understand the venom was supposed to break the cell spores and allow the genes to mix or something? I would think that the venom would destroy the genetic material.

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Re: Snake venom hybridization method debunked (?) [Re: Thesunbeam]
    #25612190 - 11/13/18 02:04 PM (5 years, 4 months ago)

Wouldent this make venomous mushrooms that turn you extra super gay?

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Re: Snake venom hybridization method debunked (?) [Re: Thesunbeam]
    #25612195 - 11/13/18 02:06 PM (5 years, 4 months ago)

I had a bottle of ancient anti-venom from the Vietnam war but thats lost or been used.

Was Shroomery made in 1997. I have a vivid memory seeing the forum on an old hp.

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Re: Snake venom hybridization method debunked (?) [Re: Sofaking420]
    #25612199 - 11/13/18 02:09 PM (5 years, 4 months ago)

Didn't Roger rabbit use this method to create PE6?
I doubt he would be lying about that.

Just out of interest does anyone actually know how cube hybrids are created? Is it just monokaryotic mycelium from two different varieties mated on a Petri?

Workman the creator of APE would surely know the answer..


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Re: Snake venom hybridization method debunked (?) [Re: 2in1]
    #25612259 - 11/13/18 02:43 PM (5 years, 4 months ago)

It is not about lying. It is about a flawed method.

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OfflinePeter65199
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Re: Snake venom hybridization method debunked (?) [Re: Pinback]
    #25613140 - 11/13/18 09:49 PM (5 years, 4 months ago)

I have never tried using rattlesnake venom so cannot say 100% but that first paper you quoted seems entirely feasible. Besides the proteolytic enzymes you mentioned the venom in question also contains chitinase.It has the lytic enzymes necessary to digest the cell wall and expose the required protoplasts. I am no expert on snake poison so can only speculate that like Abrin in the paper below it also contains some sort of ribosome inhibiting protein (or strong proteolytic activity ect) which facilitates somatic fusion between protoplast and DNA.  Obviously venom constituents will vary depending on size, age, diet, location ect ect of the snake and I would imagine would vary wildly. And the right concentration would have to be tweaked. As I said I have never used or seen it used, but theoretically it is sound.

The paper below uses a very similar technique on Cordyceps Militaris using the ridiculously potent toxin Abrin, they actually cite the Holliday paper in question.

An effective approach of strain improvement in Cordyceps militaris using abrin

"Is it just monokaryotic mycelium from two different varieties mated on a Petri?

Yep! But they said it would have taken them a long time and lot of hassle to do what they did, by the traditional method you described.

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Re: Snake venom hybridization method debunked (?) [Re: Peter65199]
    #25613150 - 11/13/18 10:00 PM (5 years, 4 months ago)

I have not read any of the relevant papers here but crossing two strains of the same species does not result in a hybrid.


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OfflinePeter65199
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Re: Snake venom hybridization method debunked (?) [Re: inski]
    #25613195 - 11/13/18 10:25 PM (5 years, 4 months ago)

@Pinback I meant to add above look into PEG mediated somatic fusion, could have potential?????


Hi Inski! Maybe we are using the same word for different things, the definition I am using is;

Quote:

Hybrid definition is - an offspring of two animals or plants of different races, breeds, varieties, species, or genera.
Hybrid | Definition of Hybrid by Merriam-Webster



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Re: Snake venom hybridization method debunked (?) [Re: Peter65199]
    #25613342 - 11/14/18 12:33 AM (5 years, 4 months ago)

Quote:

Peter65199 said:
I have never tried using rattlesnake venom so cannot say 100% but that first paper you quoted seems entirely feasible. Besides the proteolytic enzymes you mentioned the venom in question also contains chitinase.




I tried searching for that but could not find anything. Do you have a reference?

Not that it matters, because hardly any conclusions about "hybridization" can be drawn from the Holliday paper. Again, the CS-4 isolate they used in the hybridization experiment has been found to be a parasite of Cordyceps sinensis, and not a Cordyceps isolate that the authors seem to have believed. This is a very important point as it makes any conclusions drawn about hybridization invalid!

Whether to call it a hybrid or not -- I am using the authors' description from the paper. I would myself call their intended result a cross.

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Re: Snake venom hybridization method debunked (?) [Re: Peter65199]
    #25613736 - 11/14/18 07:42 AM (5 years, 4 months ago)

Quote:

Peter65199 said:
@Pinback I meant to add above look into PEG mediated somatic fusion, could have potential?????


Hi Inski! Maybe we are using the same word for different things, the definition I am using is;

Quote:

Hybrid definition is - an offspring of two animals or plants of different races, breeds, varieties, species, or genera.
Hybrid | Definition of Hybrid by Merriam-Webster






Mixing varieties together results in a varietal hybrid which isn't a hybrid at all. Because you mixed the same species together which is really nothing special.

Like a lab and a poodle make a labridoodle but its still a canine.
A true hybrid would be mixing a canine with a fox or wolf.


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Re: Snake venom hybridization method debunked (?) [Re: bodhisatta]
    #25614064 - 11/14/18 10:58 AM (5 years, 4 months ago)

That's right, or you could create an interspecific hybrid by crossing different species from the same genus.

If you cross two varieties of the same species you get an intraspecific hybrid or intervarietal hybrid.


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OfflinePeter65199
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Re: Snake venom hybridization method debunked (?) [Re: inski]
    #25614938 - 11/14/18 05:23 PM (5 years, 4 months ago)

But this is just semantics... Inski is correct in biology you are taught that you can have intra or inter hybrids, both are by definition hybrids.

@Pinback, I sincerely apologize I also can not find what I was looking at when I said Crotalus Atrox has chitinase in its venom. I have papers that document other snakes that have it. But I dont know why I was under that impression, I think I was confusing it with the somewhat chitinolytic vipericidin in Crotalus durissus terrificus(wrong rattlesnake)? :facepalm3: As you said it does not matter that much anyway since chitin only makes up small percentage of the fungal cell wall(similar to lignin in wood, a structural polymer). And as you said the venom is proteolytic, and could degrade/inhibit the vast majority of the cell wall anyway allowing creation of protoplasts and exchange.

If the paper was flawed you would hope that the peer reviewed journal in which appeared would have picked it up (but it happens sometimes), but why then did the researchers in the paper above even attempt this, quote from paper;
Quote:

The present study is based on the study conducted by
Holliday et al. (2004) using Snake venom for somatic fusion of C. sinensis and the developed strains
were reproducible strains and showed improved quantitative bioactive compounds. A similar effective
somatic fusion was observed in the present study using abrin, a plant poison.




And why did it succeed?
They used Novozyme 234 to create the protoplasts and it is not that hard to get.....
Quote:

Abrin was extracted by soaking 10 g pulverized seeds of Abrus precatorius in PBS (pH 7.7)
with 5 % NaCl for overnight at 4°C. The homogenate was filtered and centrifuged at 10,000 rpm for 30
min at 4°C.The crude abrin was dialyzed against distilled water against 10 mM Tris-HC1 pH 7.7 for 24
h each. The dialyzed solution was centrifuged at 15, 000 for 20 min, and the partially purified abrin was
used for hybridization.




Easier to get and keep than fresh rattlesnake venom, but insanely toxic. It could be tested. Also I read that link about Holliday and it definitely did not help his cred, but the paper/method still seems solid. I was reading an old thread with you and RR (roger rabbit) and he said he had used it before and did not doubt the paper either???

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Re: Snake venom hybridization method debunked (?) [Re: Peter65199]
    #25615016 - 11/14/18 06:02 PM (5 years, 4 months ago)

IIRC RR never has used venom only repeated the Holliday story. I csn investigate later


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OfflinePeter65199
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Re: Snake venom hybridization method debunked (?) [Re: bodhisatta]
    #25615110 - 11/14/18 06:46 PM (5 years, 4 months ago)

Snake Venom Into Hybrids <- Link to post.
This is an image he posted;

Quote:

I first learned of rattlesnake venom from attending a medicinal mushroom conference in 2005. John Holliday did a presentation on it. Here's a link to a paper he wrote. John Holliday is director of research at alahomedicinals.com and there's lots more info on his website.
RR
http://www.nwbotanicals.org/nwb/lexicon/hybridcordyceps.htm




Here is hes quote to what he used it for;
Quote:

Search this forum for the Redboy thread(s). I had some 20 year old redboy spores that simply would not germinate, no matter what I tried. After placing the non-viable spores on venomated agar for a couple of weeks, I released monokaryotic PR mycelium to crawl over the dish. Low and behold, when the monokaryotic mycelium reached the area where the spores were near the edge, two separate rhizomorphic strains emerged. Somehow the snake venom allowed the PR mycelium to access the nucleus of the spores for mating. I had tried this without the venom to no avail. The very first time venom was used, the result was dikaryotic mycelium, and the redboy strain was back as a 50/50 cross with PR. The experiment was repeated with the offspring, resulting in 75% Redboy(perhaps? I'm not a geneticist), which was then released to the community.
RR




And when Pinback said it he doubted the hybridization would work, and Holiday was not a real mycologist RR replied;
Quote:

John Holliday IS a real mycologist.  Write him to ask for more info if you wish.  He owns www.alohamedicinals.com





@Pinback; I hope you dont take this an attack on you, its far from it! I actually respect your style, it would serve you very well when researching. I like people who dont just take things at face value, and dig deeper. Anyway, I hope we can work together on anything one of us may be having trouble with in the future.... :thumbup:

Edited by Peter65199 (11/14/18 06:48 PM)

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Re: Snake venom hybridization method debunked (?) [Re: Peter65199]
    #25615669 - 11/15/18 04:33 AM (5 years, 4 months ago)

John holliday is fake news.

The International journal of medicinal mushrooms or whatever is or was started by him.  Probably to push his own products.

Hes definetly about as much of a doctor as I am, it's an honorary degree from a chinese uni.

He's also the kind of guy to double down rather than admit fault, when confronted with a mistake.

I know people that had the misfortune of hiring him on mycology related projects that ultimately failed, costing investors hundreds of thousands of $$$.  Since the mushroom farming biz is so small, there's a lot less fools for him to prey on now...


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Re: Snake venom hybridization method debunked (?) [Re: drake89]
    #25616319 - 11/15/18 11:52 AM (5 years, 4 months ago)

Thanks for the comments.

Peter: not at all, I appreciate your comment. That abrin paper is also flawed, for a number of reasons.
1. No mention of any negative control experiment. Very significant flaw.
2. No analysis or test to ensure that a cross had formed, other than visual examination. Very significant flaw.
3. No mention of a test to ensure that the strains used were indeed dikaryotic (monokaryotization could have occured before the experiment. Perhaps unlikely, but would have been nice to see it tested)
4. No evidence for somatic fusion. It could in theory be that monokaryotization and subsequent mating happen, which would give the same effect here, i.e., that a cross is formed. But again, no cross has been demonstrated in my opinion.
5. No replicates for biomass and metabolite production. It is unclear what the natural, inherent variation is.

These are not small, technical points. They in principle makes it very difficult to draw any conclusion from that paper (regarding abrin at least).

drake89: I have not heard that it was started by him, and think I would like a reference for that too :smirk: I did see that he is on the editorial board for the journal, but I do not know if he was that at the time of the paper publication. After all, it was some time ago.

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Re: Snake venom hybridization method debunked (?) [Re: Pinback]
    #25617226 - 11/15/18 07:19 PM (5 years, 4 months ago)

I cannot dispute those points, they do seem to be missing?

As for Mr. Holiday personally I do not know him at all, but apparently his reputation precedes him.

This is why I took the position I did.

There are fusigenic agents that are present both in Abrin and C.Atrox venom. They both contain lectins which have been widely documented to agglutinate protoplasts, in the paper below remember Ricin from the castor bean is a close relative of the Abrin producing Rosary Pea(they are both Ribosomal Inactivating Proteins RIPs, which are
lectins).

Plant Protoplast Agglutination by Lectins

In fact C.Atrox venom contains some other interesting proteins like its disintergrins;

Quote:

Potato lectin: a modular protein sharing sequence similarities with the extensin family, the hevein lectin family, and snake venom disintegrins(platelet aggregation inhibitors).

"Finally, all plant chitin-binding domains examined bore a remarkable sequence similarity, particularly in the spacing of Cys residues, to the disintegrins (platelet aggregation inhibitors) which occur in crotalid and viperid snake venoms."

https://www.ncbi.nlm.nih.gov/pubmed/8054990




Crotalus Atrox venom profile;


Lysing the cell wall;

C.Atrox venom is has a high proteolytic activity compared with other snakes in general, these degrade and inhibit proteins. Since the chitin content of a fungal cell wall(in our case) is from 10% to 20%, which means 80% to 90% of the cell wall containing proteins and glucans are vulnerable. The snake venom disintergrins above bind chitin and act like some plant lectins which have some agglutination properties (agglutination is not fusion, but in a lot of cases once the polarity issues are overcome fusion can then proceed). There are also host of other enzymes I could get into like the metalloproteinase(but alas my time has run out).

Edited by Peter65199 (11/15/18 11:41 PM)

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Re: Snake venom hybridization method debunked (?) [Re: Peter65199]
    #25623109 - 11/18/18 12:01 PM (5 years, 4 months ago)

Quote:

Peter65199 said:
And when Pinback said it he doubted the hybridization would work, and Holiday was not a real mycologist RR replied;
Quote:

John Holliday IS a real mycologist.  Write him to ask for more info if you wish.  He owns www.alohamedicinals.com









I didn't realize it until now, but the Discover article about his Dictyophora paper has a quote expressing the same thing I did in 2008:
Quote:

“I might add, he is not a mycologist. His doctorate is from some unnamed Chinese university. Normally if you get a honorary doctorate you actually mention where it’s from. Honorary doctorates are only valid on the university grounds where you got them. It doesn’t make you a Ph.D. in something. It’s a token.” --Debbie Viess




Debbie Viess also wrote an article about that paper which can be accessed here.

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Re: Snake venom hybridization method debunked (?) [Re: Pinback]
    #25623307 - 11/18/18 01:54 PM (5 years, 4 months ago)

I have not read the actual paper and am not going too waste my time reading it :smirk:, so I dont know if he emphasizes that it was legend as he said he did when he was questioned about it. But I did a google with its title and huff post articles ect came up discrediting it. Of course they were going to latch onto a attention grabbing, sensational story about spontaneous orgasms that turned out to be bogus. Which would make me ask if he was doing serious research why on earth would he have done that? I can understand telling tourists and young people as a sort of local joke, but writing that paper? Was it a misguided attempt at promoting himself and his business? Anyway.....

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