Moina as an Epigenetic model organism: CUBE Elphinstone

Moina as an Epigenetic model organism: CUBE Elphinstone
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Hey Everyone, This is Drishtant Maruti Kawale [F.Y. B.Sc. (H) Biotech] from CUBE Elphinstone College, Mumbai.

I am working on the model organism Moina. Moinas can be studied as a model organism because first of all, it is easy to culture, gives more offsprings in less time (parthenogenetically) /short life cycle due to which gene expression can be studied in less time, it is sensitive to changes in water chemistry.
So, the future scope is to study epigenetics of Moina in detail, first understand the process on the observational basis rather than the experimental one. This is an interesting field of study and can be studied on cancer and other knowledgeable fields.

Introduction:

Classification:

Kingdom: Animalia

Phylum: Arthropoda

Subphylum: Crustacea

Class: Branchiopoda

Order: Cladocera

Family: Moinidae

Genus: Moina

Objectives:
Immediate: Culturing of moinas and to maintain a red Moina culture.
Short term: To check for colour change in Moinas after a week or so.
Long term: To study epigenetics (the study of inheritable phenotype without altering the DNA sequence) and specifically DNA acetylation in Moinas using HDAC inhibitor Valproic acid (VPA) and to study the effect of unfavourable conditions like hypoxia in Moina and the probable reasons for the responses (changing to red colour) shown by Moinas in such conditions.

So, today 3 culture bottles L1, L2, L3 were prepared at CUBE HBCSE, Mumbai. The Moinas in these bottles were taken from the existing cultures at HBCSE.

L1 & L2: 750ml DC Water + 5 colourless Moinas + 3 drops of milk (1 drop - in 250ml DC so 3 drops in 750ml)
L3: 250ml DC Water+ 5 colourless Moinas + 4 drops of milk (Hypoxia)

Tomorrow’s Expectations: 5-7 Moinas in all bottles

We are performing the VPA set up so we will need more culture bottles, (in fact more red moinas because we will check the effect of the HDAC inhibitor on Red Moinas which would take more days to become colourless at various concentrations of VPA) which will be prepared in the coming days.
I know that more culture bottles could have been prepared today itself!

We’ll keep posting literature regarding our set up along with the references.

What is VPA?
These are medications primarily used to treat epilepsy and bipolar disorder and to prevent migraine headaches.

Why are we using it on Moinas or as an HDAC inhibitor?
Well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic centre of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic (stem cells which give rise to other blood cells) progenitor (parent) cells and leukemic blasts from acute myeloid leukaemia patients. Moreover, tumour growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy.

Reference for VPA as an HDAC inhibitor: Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells.

So, the question may arise that why to study Haemoglobin of Moina?
Haemoglobin in Moina:

Haemoglobin is a protein which carries oxygen (1 Hb molecule carries 4 Oxygen atoms) to all parts of the body in humans as well in Freshwater Moina s. It is present throughout the bloodstream in the body in humans and it’s level increases or decreases as per the requirement of the organism. But, in moinas, Haemoglobin expression can be seen in a span of few days by altering their environmental conditions like Normoxia (Normal Oxygen concentration in their environment i.e. water) and Hypoxia (Less Oxygen concentration in their environment). As a result, the moinas turn pale yellow to red in colour after keeping them in hypoxic condition because, as there is less oxygen already, more Hb would be required by the organism to fulfil its bodily functions as it is the carrier of oxygen. It restores back to colourless or white colour after keeping it back in the normoxic condition.

Literature:

In normoxic conditions i.e. When there is normal dissolved oxygen concentration, the moinas are colourless, but a change in colour is observed after some days when they are kept under hypoxic (low dissolved oxygen) conditions, their colour changes from colourless to pale yellow and then to red. They tend to develop more or extra haemoglobin to increase the uptake of oxygen from the water. The Hb uptake varies in response to environmental changes. The form of Hb protein which contains oxygen is Oxy-Haemoglobin and is responsible for the red colour of moinas in hypoxic condition, the quantity of whose depends upon the dissolved oxygen gas concentration in the water. We have observed that the amount of dissolved oxygen is inversely proportional to the amount of Hb produced.

We hypothesise that change in the colour of moinas is due to the change in their gene expression. Of the four Hb genes: dmHb1, dmHb2, dmHb3, dmHb4 (all located on a single chromosome), in the *Moinas *, any two (which two exactly are not known to us) are active i.e. Produce Hb in normoxia and all four are active in hypoxia producing extra Hb, as, for that period of time it is irreversible change and not a mutation, we thought it as to be an epigenetic (phenotypic inheritable change in morphological/observable characteristics without change in DNA sequence) change induced due to change in environmental conditions.

So, when epigenetic changes take place changes in morphology, physiology also takes place but no change in DNA content takes place.

In case of our freshwater crustacean, this occurs during the hypoxia condition where with low oxygen concentration, the Hb concentration increases up to 10 times the normal concentration due to the upregulation of Hb. Upregulation in simple terms means on the action of external stimulus, the cell increases the number of cellular components mainly RNA or protein. Here, Hb is upregulated which is why moinas appear red in colour.

The hypoxic condition stimulates the changes in the body of Moina which leads to histone modifications.

The gene is a part of DNA. In moinas during the hypoxic condition, some enzymes like HAT (histone acetyltransferases) become active which leads to acetylation of histone protein. During the acetylation, the acetyl group CH3COO- removes the positive charge from the histone protein which was a positively charged protein due to the presence of amino acids Lysine and Arginine which are basic in nature due to the presence of –NH2 group in them. Earlier, there was an octamer between histone and DNA which were bound by the nucleosome which is the fundamental unit of DNA packaging but now, as a result of acetylation, there will be no electrostatic force of attraction between DNA and Histone protein due to which DNA becomes loose and hence RNA polymerase with the help of transcription factor, HIF-1-alpha or hypoxia-induced factor 1 alpha reads the DNA strand and transcribes it to RNA and then translates it into Haemoglobin protein which is needed by the moinas. The promoter regions of the globin genes each contain hypoxia response elements (HREs) as potential binding sites for HIFs.

So, after the addition of Valproic acid, the reverse of the above process happens. The HDAC (histone deacetylases) are not able to deacetylate the histone as a result of which the Hb protein will be produced until the concentration of VPA decreases and hence the Moinas will remain red.

Proposed Experimental Setup

Objective: To study the effect of HDAC inhibitor Valproic Acid (VPA) on Red Moinas at different concentrations.

Requirements: Valproic Acid tablet 200mg, test tubes, DC water and milk.

We are going to use 200mg Valproic acid tablets as the HDAC inhibitors. We are going to take different concentrations of the VPA solution – 5ug, 10ug, 50ug, 100ug, 500ug (ug is microgram) and going to check the number of days taken by the HDAC inhibitor to inhibit the deacetylation in the Moinas at the above-mentioned concentrations.

Preparation of Valproic Acid solution as well as its different concentrations:

Take 200mg VPA tablet and dissolve it in 200ml DC water. The solution now is of 1ug/ml.

Now, to prepare 5ug concentration, take 5ml of the above 1ug/ml solution and add it into 995ml of DC water.

Repeat the above step to prepare 10ug, 50ug, 100ug, 500ug concentrations.

Take 12 test tubes and add 10ml of DC water in each of them. To that add 5 red moinas (except in the colourless control one – where 5 colourless moinas would be added) which were obtained by inducing hypoxia.

Now, add the prepared concentrations dropwise in 10 of those test tubes (5 main and 5 duplicate) and the remaining 2 test tubes would be Control tubes – one red control and the other colourless control.

After that the moinas need to be fed with milk in terms of dilutions.

As per the protocol, we add 1 drop of milk in 250ml of DC water, so for 25ml of DC water, we will add 1:10 dilution of milk i.e. 1 drop of milk and 10 drops of water and now for 10ml of DC water, 1:25 dilution.

We hypothesise that as the moinas are kept in different concentrations, they will get colourless in varying time. For example, the 5ug moinas will turn back into colourless in less time as compared to the 50ug ones due to the lesser concentration of VPA. The red moinas which are to be kept in the water containing VPA solution at different concentrations will take more to revert back to colourless at different concentrations because, in the presence of VPA which is an HDAC inhibitor, the DNA deacetylation will take time. This will only take place when gene regulation will happen by DNA acetylation.

This assay would be performed only and only when there are a considerable number of red moinas with us!

References

  1. NFC: Moina – Russian Red Daphnia, Tuesday 14 September 1999 – Hemsath Gay
  2. Moina (Crustacea: Anomopoda, Moinidae) in the Czech Republic, November 4, 2002,– Adam Petrusek Department of Hydrobiology, Charles University, Prague, Czech Republic
  3. The Haemoglobin of Daphnia, 5 August 1947 - H. Munro Fox F.R.S., Belford College, University of London
  4. HIF at a glance, Journal of Cell Science, 2009 – M. Christiane Brahimi-Horn, Jacques Poussegur
  5. Hypoxia-induced synthesis of haemoglobin in the crustacean Daphnia magna is hypoxia-inducible factor-dependent, Journal of Biological Chemistry, May 28, 2008, Thomas Gorr Division of Haematology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, Yamagata H, Bunn HF, Cahn JD.
  6. Transcriptional and Translational Relationship in Environmental Stress: Frontiers in Physiology, 02 July 2018 – Jingyi Jia, Lu Li, Xiangjiang Liu, Chengqiang Lei, Ying Dong, Guoqiang Wu and Guangfu Hu – College of Fisheries, Huazhong Agricultural University, Wuhan, China
  7. Sequencing of Hb Gene 4 (dmhb4) and Southern Blot Analysis provide Evidence of More than Four Members of the Daphnia magna Globin family: Bioscience, Biotechnology and Biochemistry, 22 May 2014 Marc Wolf, Frank Nunes, Ralph Pirow, Alexandra Wendler, Desiree Spiering, Rudifer J. Paul.
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Can you also hyperlink the references please?

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Yeah why not!

http://www.fao.org/3/W3732E/w3732e0x.htm

http://www.nslc.wustl.edu/elgin/genomics/srf/Daphnia6-8.pdf

https://www.tandfonline.com/doi/pdf/10.1271/bbb.69.1193

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What a pity , there is no reference to the current Nobel Prize related work! :sleepy:

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Can you try out an experiment where you have bottles with different oxygen concentration and see if the inverse curve holds true, i.e. lower the oxygen concentration, higher the percentage of red-coloured Moinas being produced?

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Yes, why not?

We will surely keep this is mind and make a design of the experiment!

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What did the nobel laureates say…?


A rough analysis done by me at CUBE HBCSE, Mumbai.

Still, there are some questions to ponder upon.

Reference: press-medicine2019.pdf (1.8 MB)

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Can you summarize the Nobel Prize of this year in a short write up @drishtantmkawale

I am interested more in knowledge representation of structure and process. In the ongoing discussion, there was a discussion of the normoxic and hypoxic conditions of moina with reference to HIF-1alpha

Here is a representation. can anyone (students) explain the above process in words by looking at the representation diagram…

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So yesterday, at CUBE HBCSE, Mumbai I, @Arunan sir ishanikishan sir @Harshad sir and @Lydia discussed about the issues the Moina group in CUBE Elphinstone is facing.
We figured out that our design of the experiment was not proper (We said that our cultures were not surviving) , there are many conceptual gaps like we do not know what is normoxia… we say it is the normal oxygen/dissolved oxygen in the DC water but how much of dissolved oxygen? has anyone counted… No!
So if we take 250ml of DC water in one bottle, and another 250ml in another bottle, there is a possibility that both may have different dissolved oxygen!! Isn’t it?
Another problem was we know that we add milk to the bacteria, but we don’t know why is it added, what happens after it’s addition! A C6H12O6-Glucose compound gets converted into CO2 + H2O + Energy (heat+ATP) just like that?
No, there are cycles like Glycolysis, Krebs cycle, Electron Transport Chain (ETC) which help or I must say initiate the process of energy formation which helps the bacteria in reproduction and in other processes. And that bacteria in turn are eaten by moinas.
So if we add more milk, more bacteria will be there (and they will reproduce obviously), which will consume the so called dissolved oxygen so no or very less oxygen would be remaining for the moinas to survive that is why @Zahra_R17 was saying that our moinas are dying!!
And we also figured out that there is a need to standardise our culturing protocol!

Other gap is that we are not able to relate things!
Arunan sir said :100:% right that this year the Nobel prize had gone to Hypoxia and HIF but we don’t know how the mechanism happens at molecular level!
HIFs, HREs… What are these??
God particles?
No!! Proteins and DNA sequences respectively!

God doesn’t do these molecular processes!
Arunan sir said that God must be sleeping while these processes happen!:joy::joy:

So yes, these conceptual gaps need to be filled through discussion…

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How do you plan on filling this gaps?
Are you going to measure oxygen levels?
If yes then how are you planning to do it.?

Winkler Method Reference :

See the above link. I have been searching for ways to monitor oxygen level since a month and this one is the least complicated and cheapest method I found. What do you think about it? Do you have any better alternative? If yes please share.

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Please give the update on the success of your culture of Moina. Please provide photographic and other evidence

Please give updates @drishtantmkawale and discuss goof-ups, if any!

Reference please @drishtantmkawale. Are you sure 2 hb genes are expressed in normoxic conditions?

Please give a design of the Experiment.

So now what did you do to get successful cultures? How many moina are you having now m? Wid pics as evidence.

I’m zahra from cube Elphinstone working with @drishtantmkawale, we currently have some 600/800 moina in total from which 50/100 moina are red and our next objective is to do dissolved oxygen assay

What is this assay about ? Why are doing this ? What’s your objective?

CUBE Elphinstone
Moina Group
@Zahra_R17 @saida786110

Objectives:
Immediate: Culturing of moinas and to maintain a red Moina culture.
Short term: To check for colour change in Moinas after a week or so.
Long term: To study epigenetics (the study of inheritable phenotype without altering the DNA sequence) and specifically DNA acetylation in Moinas using HDAC inhibitor Valproic acid (VPA) and to study the effect of unfavourable conditions like hypoxia in Moina and the probable reasons for the responses (changing to red colour) shown by Moinas in such conditions.

Total Bottles: 13 - 2 with 500ml Capacity (P1 and X2), 6 with 1L capacity (B16, S2, B2, B3, Z3 and B1), 1 with 1.5L capacity (L1), 4 with 2.25L capacity (F1, F6, M1, M3)

P1 MD-15 Oct 19 250ml of DC water + 6 drops of milk
X2 MD - 9 Oct 19 250ml of DC water + 1 drop of milk
B16 MD - 10 Oct 19 250ml of DC water + 6 drops of milk
S2 MD - 9 Oct 19 250ml of DC water + 1 drop of milk
B2 MD - 10 Oct 19 250ml of DC water + 6 drops of milk
B3 MD - 10 Oct 19 250ml of DC water + 6 drops of milk
Z3 MD - 9 Oct 19 250ml of DC water + 1 drop of milk
B1 MD - 24 Sep 19 250 ml of DC water + 2 drops of milk (their count increased till the end of Sep but due to non-feeding, moinas died but as some were alive, their count is now stable)
L1 MD - 11 Oct 19 750 ml of DC water + 3 drops of milk
F1 MD - 14 Oct 19 1.5L of DC water + 6 drops of milk
M3 MD - 15 Oct 19 1.5L of DC water + 36 drops of milk
M1 MD - 15 Oct 19 1.5L of DC water + 6 drops of milk
F6 MD - 14 Oct 19 1.5L of DC water + 36 drops of milk

Moina Count:
P1: ~50 colourless moinas Tomorrow’s expectations: ~70 colourless moinas
X2: ~25 colourless moinas TE: ~40 colourless moinas
B16: ~150 moinas out of which 50-60 are Pale yellow coloured moinas TE: ~200 moinas, ~20-30 red moinas
S2: ~20 colourless moinas TE: ~30 colourless moinas
B2: ~30 colourless moinas TE: ~40-50 colourless moinas
B3: ~150 moinas out of which ~70 are pale yellow coloured moinas TE: ~200 moinas, ~40 red moinas
Z3: ~40 colourless moinas TE: ~50-60 colourless moinas
M1: ~30 colourless moinas TE: ~40-50 colourless moinas
B1: ~50 colourless moinas TE: ~60-70 colourless moinas
L1: Forgot to take the reading
F1: ~100 moinas of which ~50 moinas are pale yellow to red in colour TE: ~120-130 moinas, ~30-40 red moinas
M3: ~30 colourless moinas TE: ~40-50 colourless moinas
F6: ~100 colourless moinas TE: ~150 moinas
Tank Culture: ~250 moinas TE: ~300 moinas
In total ~900 moinas

So, we have around 100 moinas with Pale yellow to red colour.

After we get red moinas, we will plan to do the VPA assay.
And in the coming week, we plan to estimate the dissolved oxygen in the water through Winkler’s Method.

Also, we are going to make new culture bottles with different gradients, i.e. 1 drop, 2 drops, 3 drops, 4 drops, 5 drops and 6 drops cultures along with their replica and control. This will help us to estimate the time taken by the Moinas to change their colour at various milk concentrations.


These are our Moina Cultures at CUBE Elphinstone.

Talking about the goof ups, I didn’t follow the protocol of taking a transparent bottle as my culture bottle and took a green coloured bottle!!
I felt totally absurd when I realised that how will I observe the colour of the Moinas if they are kept in green even though the bottle is at my home.

I suggest new CUBISTs to follow the standard protocol while culturing Moinas or any other Model Organism.

  1. A 1 Litre transparent plastic bottle. A 1L one because anyone can buy it from the store or sometimes it is available at our homes itself.
  2. Colourless Moinas
  3. Since I started culturing Moinas, I follow 1drop of milk in 250ml of DC Water protocol as suggested by my seniors.
  4. Always try to make replica bottles.
  5. Discard the milk after use as it turns into curd the next day.