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Invisibleteknix
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Wild Reishi Clone Grow. * 1
    #27002833 - 10/25/20 02:07 PM (3 years, 4 months ago)

So I found these Reishi in the Wild and decided to clone it and cultivate it.



I had dried them all at low temps around 90F, and stored them for a few months in sandwich bags until I felt like taking on the project.

For cloning I just poured a 10% bleach/water solution in one jar and plain water in another jar for rinsing. I took the softest tissue from the specimen I had dried and dunked it in the bleach water. I poked the tissue into the water, stabbing it down with the forceps to bbbreak the water tension on the surface of the tissue. I let it soak up bleach from 2 minutes to probably about 7 minutes, which was the length of the process.

After all the tissue I was going to put to agar was sitting in the bleech solution for 2 minutes I started the process by using forceps to take out a tissue sample and dry it on a paper towel, I then dunked it in the glass of water and then again dried it on a paper towel, (which I had set out in advance). I then put the tissue onto agar. I did this for the rest of the tissue until I had made a total of 8 plates.



(The plates are fully colonized now and in a fridge minus a few that got contaminated, which was acceptable considering I was open air inoculating and using a time spread for the bleach soak.)


I then moved some wedges from a couple of plates to sterilized WBS and allowed it to colonize. (fresh out of popcorn t.t)



I then proceeded to spawn the WBS to sterilized fuel pellet/sawdust blocks with about 10% verm added and a sprinkling of calcium carbonate. I used roughly 4 teaspoons of spawn per bag, so 1 quart spawned all 7 bags. There is roughly 3 quarts of substrate per bag.



Now I wait :smile:

Edited by teknix (10/25/20 03:47 PM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003151 - 10/25/20 05:08 PM (3 years, 4 months ago)

Awesome, I love wild clone grows!

Do you know what species you're working with?


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27003208 - 10/25/20 05:33 PM (3 years, 4 months ago)

Best guess is Ganoderma Sessile, considering the G. Lucidum that I have has growth characteristics that is much different. I might do some taxonomy if I get some decent results to find out for certain. It is definitely KOH positive for Ganoderic Acid tho.

I had spread a G. Lucidum Slurry around the area the previous years, but this mycelium grows quite a bit different from the G. Lucidum mycelium. I didn't particularly inoculate the tree so I'm guessing it's not the G. Lucidum.

I also have a run of G. Lucidum incubating to compare it to later.

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003217 - 10/25/20 05:38 PM (3 years, 4 months ago)

Nice, looking forward to see how it fruits.  Ganoderma is one of my favorites to watch :smile:


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003223 - 10/25/20 05:41 PM (3 years, 4 months ago)

The G. Lucidum are almost done incubating so, unfortunately, I won't be able to do a true side by side.

But the substrate and spawn ratios are very similar.


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003244 - 10/25/20 05:53 PM (3 years, 4 months ago)

Here are a few specimen I dried and kept from my first and last grows, can you tell which is which? :P



Hopefully I can get some conks to sporalate again. Since the spores contain the highest concentrations of Ganoderic Acid(?), that is most desirable to me.

Edited by teknix (10/25/20 07:11 PM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27006592 - 10/27/20 03:34 PM (3 years, 4 months ago)

Great overview of your methods - thank you for sharing!  Will come back and update here if I try it.  :smile:

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27016804 - 11/02/20 06:03 AM (3 years, 4 months ago)

Quote:

teknix said:

Hopefully I can get some conks to sporalate again. Since the spores contain the highest concentrations of Ganoderic Acid(?), that is most desirable to me.




Just remember you'd have to crack the spores to get that stuff out of them. It's not easy, but there's some threads around on methods


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27018312 - 11/02/20 09:01 PM (3 years, 4 months ago)

Haha yeah, that's why you do the alcohol extraction first right? :smile:

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27019928 - 11/03/20 05:44 PM (3 years, 4 months ago)

I'm pretty sure even alcohol doesn't get much of the stuff inside spores when they're not cracked.  Cracking them requires either germination (which reishi has a terribly low germination rate), or some pretty intense mechanical methods.


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27019995 - 11/03/20 06:38 PM (3 years, 4 months ago)

It looks like you are right about the insolubility of chitin in alcohol. I found a decent write-up that tested the solubility of chitin in various solutions.

https://www.intechopen.com/books/solubility-of-polysaccharides/solubility-of-chitin-solvents-solution-behaviors-and-their-related-mechanisms

I'm not sure if the Ganoderic Acid in a spore would become bio-available through digestion? I'll have to look for more research about that.

Evidently we can break down the chitin cell walls through digestion, but increasing the bioavailability would definitely be better.

https://www.researchgate.net/publication/6249199_Human_Gastric_Juice_Contains_Chitinase_That_Can_Degrade_Chitin

Looks like trichoderma has the chitinase enzyme as well:

https://www.mybiosource.com/enzyme/chitinase-trichoderma-viride/653950

So techinically we could use trichoderma to increase the bioavailability of ganoderic acid in Reishi spores? However I am currently at a loss on the procedure we would use to do so. Chitinase with the spores could cause a slow-release of ganoderic acid, maybe making more of it useful in the long run. You get your instant GAE from the extraction and save some in the spores to be released later with the enzymes.

Thanks for bringing this up Forrester, I appreciate it.

Edited by teknix (11/03/20 07:10 PM)

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27020428 - 11/03/20 10:19 PM (3 years, 4 months ago)

Also chitinase in soybean seed coats.

https://academic.oup.com/jxb/article/52/365/2283/543833

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27021991 - 11/04/20 05:45 PM (3 years, 4 months ago)

No, I wasn't referring to chitin being insoluble.  We can still get the good stuff out of mushrooms via alcohol despite that.

I was talking about what's inside spores themselves, it's an entirely different thing.


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27022090 - 11/04/20 06:43 PM (3 years, 4 months ago)

Interesting, so the higher concentration of G. Acid are largely bio-unavailable in spores:

Zhou et al. (2012) observed that dietary SGL (2 g/kg) had no effect on oxidative stress and mitochondrial dysfunction in the hippocampus of rats. The activities of spores are closely related to the status of the sporoderm, and sporoderm-broken spores of G. lucidum (SSGL) are more effective in modulating the immune responses in rats than sporoderm-unbroken spores (Yue et al., 2008).

The spores were dried at 55 Β°C for 24 h and then were broken by a supercritical fluid extraction device according to Fu et al. (2009) and Li et al. (2011). Briefly, approximately 150 g of the spores was loaded into a steel cylinder equipped with mesh filters on both ends. Liquefied CO2 was pumped into the vessel, and the pressure was raised to 35 MPa. The temperature was controlled at 25 Β°C during the processing. The pressure was released within 1 min at the end of the 4-h process. During the process of rapid depressurization, the resistance of the sporoderm created a pressure difference inside and outside the sporoderm, and the spores were broken when CO2 burst out of the sporoderm. Sporoderm-broken spores of G. lucidum were collected and stored at βˆ’20 Β°C before adding into the diets.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082644/

I saw this guy (NileRed) on youtube build a supercritical CO2 chamber to make aerogel. But it cost about $2k to build.



Here is another one, just using dry ice.


Edited by teknix (11/05/20 01:49 AM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27022212 - 11/04/20 07:40 PM (3 years, 4 months ago)

So much easier to just grow more reishi and make more tincure than do anything to the spores. But it is interesting nonetheless.

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: deadmandave]
    #27022274 - 11/04/20 08:13 PM (3 years, 4 months ago)

Deinitely, there are also ways to increase ganoderic acid production, that are much simpler like in this paper where they use Aspirin in an LC (submerged culture):

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053616

But they degrade the cell walls prior to extraction with enzymes:

Fungal cells of G. lucidum were treated with aspirin for 16 hr, fixed with 4% paraformadehyde for 1 hr, washed with PBS, and then digested with cell wall degrading enzymes (0.5 U mLβˆ’1 of driselase, 1050 U mLβˆ’1 of Ξ²-glucaronidase, 81.25 U mLβˆ’1 of lyticase, 5 mg mLβˆ’1 of lysing enzyme, and 0.015 U mLβˆ’1 of chitinase) for 30 min.

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Re: Wild Reishi Clone Grow. [Re: deadmandave]
    #27026078 - 11/07/20 04:07 AM (3 years, 4 months ago)

Quote:

deadmandave said:
So much easier to just grow more reishi and make more tincure than do anything to the spores. But it is interesting nonetheless.




^^ this.


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27026839 - 11/07/20 01:14 PM (3 years, 4 months ago)

Update 13 days since spawn:

Growth seems pretty slow and a weaker mycelial mat in comparison to the G. Lucidum. I didn't mix the spawn around at all, I just left the blocks as tightly compact as they were when they came out of the pc and put the spawn on top of it.



Here is the G. Lucidum under lights now:


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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27027393 - 11/07/20 06:35 PM (3 years, 4 months ago)

cool!  ya prob loose all the antioxidant effects getting micro dosages of paraformaldehyde and whatever extras are in the solution form manufacture... 

if get any amount of spores. maybe just ferment and all teh microbes would break down cell walls eventually. or put in disitlled water and do a couple freeze thaw cycles. maybe even with dried ice?? i dunno space and spores as they say but water and ice have a good mechanical action and knock over mountains..

one question what time frame for getting reishi to fruit?  in my mind it was 6 months + to get decent fruits but likely not the case. thanks!

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: the man]
    #27027829 - 11/08/20 12:53 AM (3 years, 4 months ago)

It depends largely on the strain you are working with and if you are shaking/mixing the spawn/sub halfway through, as well as the amount of spawn that you use and even the supplementation. I think the G. Lucidum is a total of about 3 months for me from inoculation of grains to fruit. It's a rough estimate, but about 2-4 weeks to colonize grains, 2-4 weeks to colonize the substrate and 3+ weeks to fruit.

I'm using little amounts of spawn and not shaking/mixing, and the wild clone is slow to colonize so it will take longer in my case.

The ones in the humidity tent are pinning, I've been spraying them with a J.Crows Lugol's Solution dilute and it isn't looking too great.


Edited by teknix (11/08/20 01:38 AM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27028570 - 11/08/20 01:01 PM (3 years, 4 months ago)

hmm ya might not be the best idea. if glycerin or sugars or alcohol would affect growth negatively or too much iodine of course kills myc ie use in surgery and brewing to kill/clean.  does someone reccomend adding iodine?  i suspect just one spray would be enough for supplementation if that was the idea?

cheers

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: the man]
    #27028676 - 11/08/20 01:59 PM (3 years, 4 months ago)

I haven't seen anyone recommend it personally, I'm just trying it mostly as a contamination barrier. I've used it in a cloning process (alongside bleach) before and it worked, so I'm seeing if it is a viable option. Similar to bleach, Iodine/iodide dilute doesn't kill Mushroom mycelium. Maybe it will act as a stressor like the aspirin does in the above article and make them produce more Ganoderic Acid.

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27029373 - 11/08/20 11:08 PM (3 years, 4 months ago)

mushrooms create wonderful natural barriers some say the best stuff for protection. :wink: happy myc doesnt need extra stuff :smile: of course clean area doesnt hurt but obv causing more issues then nything. plus u dont want to od on iodine and u dont want to eat too much bleachy salts

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: the man]
    #27029520 - 11/09/20 03:56 AM (3 years, 4 months ago)

I drink J.Crows in my water fairly often, and more than what I put in the water sprayer. My thyroid is working just fine, I just had it checked at my last doctors visit. Thanks for your concern :smile: I'm not sure what you are getting at with the reference to bleach, could you elaborate your concerns better? (are you suggesting the amount that was used it cloning and rinsed off is going to make it to the fruits?)

Did you know Lugol's Solution is commonly used to purify (Drinking) water?

:themoreyouknow:

Edited by teknix (11/09/20 05:23 AM)

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: the man]
    #27029531 - 11/09/20 04:11 AM (3 years, 4 months ago)

Also, thanks for bringing it up; here is a link to some risks and benefits that can be associated to Lugol's solution. It should go without saying, but don't drink bleach, straight iodine or peroxide! :lol: J. Crows (2%), which I am referring, is a Lugol's formulation, which isn't the same as Tincture Iodine. (Tincture Iodine is labeled as "External Use Only")

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693970/


Table 1
Examples of usage of Lugol’s solution and other iodine preparations

Graves’ disease
Disinfectant
Sterilisation
Foot ulcers
Dental procedures
Histopathology
SchillerΒ΄s test
Water purification
Radiation emergency
Iodine deficiency/ cretinism
Protection from goiter

And this article also elaborates on the confusion of using measurements in drops versus ml and the dangers associated with it.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693970/

You can also use bleach for water purification, but I think it would taste nasty, and I kinda like the way iodine tastes:

Purifying by adding liquid chlorine bleach

Treat water by adding liquid household bleach, such as Clorox or Purex.

Household bleach is typically between 5.25 percent and 8.25 percent chlorine. Read the label.

Avoid using bleaches that contain perfumes, dyes and other additives. Be sure to read the label.

Cloudy water should be filtered before adding bleach.

Place the water in a clean container. Add the amount of bleach according to the table below.

Mix thoroughly and let stand for at least 60 minutes before drinking.

(Mixing table in link below)

https://www.doh.wa.gov/Emergencies/BePreparedBeSafe/SevereWeatherandNaturalDisasters/WaterPurification

Edited by teknix (11/09/20 05:51 AM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27031331 - 11/10/20 09:55 AM (3 years, 4 months ago)

Hey. I hope your grow is doing well.
I just found some Ganoderma Lucidum in two places today but some where dried and moldy. Still I did find a peace of an antler to be free of mold but it's dry.
Could you share a link or some info about how I should go about cloning that one?

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Re: Wild Reishi Clone Grow. [Re: nosf3r4tu]
    #27031495 - 11/10/20 11:47 AM (3 years, 4 months ago)

It's going ok, just a bit slow in comparison to the established strains I generally work with.

You want to get the soft white tissue to work with (from inside the antler), or if there isn't any soft tissue just get the whitest tissue.

If you find the method in the OP doesn't work and you are getting contams from the sample you can also add 2 drops of lugols iodine to 250 ml of water and then move it from the bleach dunk (5 min) to the paper towel Then rinse it, and then move it into the iodine dunk (1 min) then to the paper towel and then to the water to rinse it and then dry it on the paper towel and put it on the dish.

You could probably use hydrogen peroxide as well, but I'm not sure on what would be an effective dilute and time. You could probably put it in pure 3% peroxide for a minute but I would probably start with a 50% dilute of 3% peroxide for 1 minute if I was going to try it. (If I was using bleach first, if I wasn't using bleach first and just H2O2 dilute I would do 5 minutes)

Oh, and if you are trying to do it open air like I did, it's kinda tricky, you have to open the dish by just lifting up the lid straight up with just enough room to put the specimen on the agar, holding the lid directly above the dish and put the specimen in the dish with forceps/tweezers fairly quickly and then put the top on the dish right away. ( I personally just open a part of the dish farthest away from me at an angle, but it's harder to see what you are doing that way) The if you want to put another specimen in the same dish just rotate it a bit and do the same thing. It's really only tricky at first though. And make sure you are wearing gloves and a mask at the least. The gloves I was using are dish gloves that go up to my forearm. But I had also scrubbed the air with an air purifier prior to doing it.

Edited by teknix (11/10/20 12:25 PM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27032776 - 11/11/20 04:56 AM (3 years, 4 months ago)

Thx.
I already cloned them and I followed the same procedure as for the previous ones I did. I think they'll do fine. The inside of the antlers weren't completely dry. Just a bit dehydrated.

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Re: Wild Reishi Clone Grow. [Re: nosf3r4tu]
    #27033190 - 11/11/20 10:43 AM (3 years, 4 months ago)

I thought u said u were spraying your fruited bags with iodine solution and previously sprayed them on the regular with bleach solution...

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27035150 - 11/12/20 12:13 PM (3 years, 4 months ago)

Quote:

teknix said:






The blue in the mutant block was from dunking it in bleach when it got contaminated, but I haven't personally ever used bleach in my water sprayer. I never processed those after dunking them in bleach, I just left them to their own accord. That block is completely dry and like 3-4 years old now. I just kept it around because it was weird, hehe.

Edited by teknix (11/12/20 12:19 PM)

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Posts: 11,953
Re: Wild Reishi Clone Grow. [Re: teknix]
    #27142449 - 01/11/21 05:31 PM (3 years, 2 months ago)

The clone bags stalled out and it looks like it was due to a hyphomycetes contam, and it is because I failed to obtain a pure culture. Using the bleach method. The resilience to bleach extends to lower fungus, so it didn't kill them either. t.t



On a brighter note, the Lion's Mane is doing pretty well in vitro on the unsupplemented fuel pellets!



I got a decent conk and lots of spores from the one of the ones in the humidity tent.



and antlers from the other:



And all the reishi growing in vitro in bags look pretty sad :frown: I plan on letting the first flush grow in vitro, then I want to try dunking them in water with iodine and instant coffee, lol :smile:



The shiitake tubs are browning:




I also made some bigger reishi block I supplemented with instant coffee this time:



And that's about it.

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Invisibleteknix
π“‚€βŸπ“…’π“π“…ƒπ“Š°π“‰‘ 𓁼𓆗⨻
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Registered: 09/16/08
Posts: 11,953
Re: Wild Reishi Clone Grow. [Re: teknix] * 1
    #27252155 - 03/14/21 07:17 AM (3 years, 15 days ago)

My best conk yet! (2 antlers from 2 different bags are making this 1 conk, lol.) This is from a couple bags I supplemented with Instant Coffee and water with J.Crows dilute:



:laugh:

Massive Shiitake caps:





Ended up with 2 quarts dried reishi and 1 quart dried Lion's mane from the invitro Reishi and Lion's mane:



Edited by teknix (03/14/21 10:31 AM)

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InvisibleGenericHero
crap dangit this sucks!


Registered: 07/07/20
Posts: 2,100
Re: Wild Reishi Clone Grow. [Re: teknix]
    #27254856 - 03/15/21 10:27 PM (3 years, 13 days ago)

Good reading. Thanks y'all for making my evening less boring.


--------------------
halfass mycology

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