CUBE Chatshaala:- The Causerie will still be on!

drishtantmkawale · April 11, 2020, 7:18pm

So, this reference is on following today’s CUBE Webinar discussion on our Fresh-water crustaceans, Moinas!!
Gave a break to our flying beasts Fruit-flies!
Well, we started from the very basics starting with what is Moina and what research question are we actually addressing with Moinas.
How does low-oxygen induce the red colour in Moina?
We had left out jargons like Histone remodelling and stuff like that and focussed on knowing and making everyone clear about what are we actually doing!!

So I started with the culture techniques, then I told about what can actually be done using Moinas at home during the lockdown period!

Then, we came on our research question that how are we actually making use of these tiny-creatures to study the 2019-Nobel Prize-winning work on hypoxia and in detail how cells sense and adapt to low oxygen conditions?

So, we continued that at Normal dissolved oxygen condition or Normoxia, the Moinas are fed with (just giving an example) 1 drop of milk daily in 250mL Dechlorinated Water and after 7 days or say a week, we observe that still the Moinas remain colourless.

But when we treat the Moinas with 6 drops of milk (not daily, but at the start of the culture with 5-10 moinas, we feed them with 6 drops of milk on alternate days because initially their number is less, and if they are overloaded will milk, they will die, so till their number increases to 40-50, they will be fed on alternate days and henceforth, daily. Reaching 40-50 Moinas when initially starting with ~10 Moinas takes 3-4 days.), at the end of 7 days we observe that their colour doesn’t remain colourless but their they change to Red.
We get ~100 Moinas out of which ~70 are Red and rest 30 are colourless-yellow.

So their colour change occurs along a gradient.
Day 1-2: Colourless
Day 3: Pale Yellow
Day 4: Yellow
Day 5: Yellow
Day 6: Orangish-Red
Day 7: Orangish-Red to Red

How is the colour change occurring?

So, according to the Paper Hypoxia-induced Haemoglobin Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent by Thomas Gorr and Team, it says that Daphnia, a close relative of Moina, has four Haemoglobin Genes in its Genome which give rise to Haemoglobin protein which is responsible for the red colour of the Daphnia/Moina!

It also says that “Protein products of the hb3 and hb2 genes (of the four known globin genes 5′-hb4-hb3-hb1-hb2-3′) are strongly induced by hypoxia, whereas the up-regulation of the hb1 gene is only moderate”

By this, I understand that the two genes hb3 and hb2 get up-regulated strictly during hypoxia and hb1 needs moderate stimulus.

Which means that in Normoxia, only a single gene is responsible for the production of Hb protein. I think that because Hb is produced at normal levels, the Moina appears colourless to us even after 7 days.

Which means that at hypoxia, all four genes will be actively producing extra Hb and the Moina will be visible as Red.

But we cannot rule out the fact that Hb will be synthesised always!
Yes, Hb is the main oxygen carrier and it would be there even in Normoxia but normal levels although it appears to be colourless, Haemoglobin would be produced.

So, Savithri Ma’am from Delhi (I guess) questioned that Why is that human blood appears more reddish when oxygenated and brown red or less red when deoxygenated and the same is ulta in case of our Moinas i.e. the oxygen-deprived Moinas appear Red and Oxygenated Moinas appear Colourless!!

Why that??

Relating this to the 2019 Nobel Prize-winning work on Hypoxia or in simple words, “how cells sense and adapt to low oxygen levels?”

There are proteins called Hypoxia-Inducible Factors or HIFs which by their name, say that they induce hypoxia. Further, HIF consist of transcription factors.

What are transcription factors?
Transcription factors are proteins which guide the enzyme-linked with DNA transcription to the promoter site of the required DNA.

HIF consists of HIF-1-alpha and ARNT or HIF-1-beta as the transcription factors.
So these Hypoxia-inducing factors bind to the promoter sites of HREs.

What are HREs or Hypoxia-response elements?
These are DNA segments present before the (in our case) Haemoglobin genes and this place is where HIFs (protein) come and bind.

So, the three Nobel-laureates discovered that in Normoxia, when there is more oxygen in the cell, HIF protein is degraded in the cytoplasm (otherwise it will induce hypoxia na?). More oxygen or normal oxygen in the cell will lead to the activation of enzyme Prolyl Hydroxylase which will help in the Prolyl hydroxylation which involves tagging of HIFs through Ubiquitin and VHL proteins (both proteins - Von hippel Lindau protein).
This tagging leads to degradation of HIF in the proteasome.

Then, in hypoxia, the cells have less oxygen so HIFs will not get degraded.
If HIFs will not get degraded then Hypoxia will be induced!!
And that is what happens here!
Hypoxia is induced.

How is hypoxia induced by the cell?
The active Hypoxia-inducible factors go to the Hypoxia response elements (HREs) INSIDE THE NUCLEUS and bind to them.
This binding of HIFs to the promoter region leads to activation or up-regulation of Hb genes.
And so our Moina turns Red.

This can be confidently seen as our Hypothesis!!

Please correct me if I went wrong anywhere!!
@Arunan @GN @KiranKalakotiR @mayur @saida786110 @Zahra_R17 @bivasnag @Priti_Kanade @magpie @jaikishan

Arunan · April 12, 2020, 3:41pm

Why not give some reference link to the studies of others and statements you have referred to? @drishtantmkawale
Looking for comments from @Abhishek @Akshitha @abhijith…@batul

drishtantmkawale · April 12, 2020, 4:52pm

Oh yes!!

https://www.jbc.org/content/279/34/36038.full
Hypoxia-induced Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent

The above reference is for the confirmation of four Haemoglobin genes in Daphnia magna and also this tells us that two genes get up-regulated strictly in hypoxia and one needs moderate stimulus.

This is a photograph taken from the 2019-Nobel Prize-winning Work by William G. Kaelin, Jr., Peter J. Ratcliffe and Gregg L. Semenza for their discoveries of how cells sense and adapt to oxygen availability.

This tells us about HIFs’ degradation in Normoxia and mechanism of binding to HREs in the nucleus and so on.
I have attached the official summary of the 2019-Nobel Prize.

press-medicine2019.pdf (1.8 MB)

saida786110 · April 12, 2020, 9:32pm

So in yesterday’s discussion we discussed about lipid molecule
Lipids are a larger class of molecules while fatty acids form a small part of it. Fatty acids are long chain hydrocarbons with a carboxylic acid group at one end. They can either be saturated (no double bonds) or unsaturated (one or more double bonds along the C-chain).

As we observe coconut oil in winter it is in solid form and in summer it is in liquid form

So does it have saturated fat or unsaturated fats?

Coconut oil contains more of saturated fat as it can also be seen in the solid form
But whether it should be solid or liquid depends on the temperature

We also discussed that how essential it is to clean the things that we bring in from outside these days
Mostly fruit and vegetables:carrot:

So people often use soap for cleaning fruits and vegetables
So is it helpful?
But as vegetables have pores in them soap might enter the vegetable and this may give as gastrointestinal problems

“Cleaning fruits and vegetables with soap can cause gastrointestinal issues that mimic the symptoms of COVID-19.”

We are not aware of how gastrointestinal issues can mimic the symptoms of covid-19 which affects the respiratory tract

So can we think of something that works similar to that of soap and can clean vegetables and fruits without affecting us??

something like Potassium permanganate can be used for cleaning vegetables and fruits as they are used to kill viruses.and which can be cheaply available on any chemist shop as well

Arunan · April 13, 2020, 4:13pm

Huffpost article is spurious sounding!

Lydia · May 6, 2020, 11:26am

4th MAY, 2020

Webinar Summary

Possibilities or the sources of contamination in the home made media.
We discussed 3 main parts which could help us in solving out the problems!
1. The components or the ingredients used for making the homemade media
2. Cooking
3. Autoclave

1.When we look at the first part i.e. components or the ingredients used for making the homemade media…

While comparing with the Standard Corn Meal Media
Dextrose and sucrose are a source of sugars,
Maize powder a source of carbohydrates,
Yeast extract a source of proteins, vitamins and minerals and
Agar as a solidifying agent.

Now when we look to the ingredients in our Tomato Rava Homemade Media,
Table sugar is a source of sugar as in cornmeal media
Rava is a source of carbohydrates (73g in 100g rava)
Tomato puree is a source of proteins, vitamins and minerals
Rava here in this media acts as a solidifying or a binding agent as well.

So when we look at the components or the ingredients used…they are just perfect when compared with each other!
We were now clear with the first part!

2. Coming to the second part i.e. COOKING

We are not cooking just for the purpose of cooking for some time or so.
There is a purpose behind cooking.

What if we don’t cook?
was the question raised by @Arunan Sir.
We have in our mind that we require a solid consistency of the media.

Why do we need a solid consistency?
was the next question.
If we get a liquid medium then that won’t support the culturing of flies in a bottle
The flies would get stuck to the media and then they die!

What is this solid consistency, like how is it’s shape?
The media after pouring in the bottle should take the shape of the bottle or the container and should have an even surface!
If not an even surface, then the flies might get stuck somewhere and then they die!
All these factors should be kept in mind!

What is this cooking? Till what time do we cook? What should we achieve by cooking?
We need a homogenised mixture or a medium.
For this we need to cook properly.
What do we mean by cook properly?
We need to cook the ingredients in water till we are sure that all the components have dissolved in the water and are mixed completely.
Here the time take for cooking is not what is concerned rather, the homogenisation of the media is what we are looking for!
This is what is done with the standard cornmeal media too!

If there is excess of water, we can evaporate it by cooking while the ingredients are being mixed in the water.
Should not overcook as added by @ankitcube
Do not cook till the media gets thick or till it gets too thick in its consistency
Otherwise it would be difficult while pouring this media in the bottles.

If this much is taken care of, then we can to very much extent prevent the growth of bacteria or fungi or whatever unwanted growth was seen!

3. The third part i.e. AUTOCLAVE
We all have a pressure cooker at home.
As suggested by @Arunan Sir, we can pressure cook it for once and then our media is ready.

This much can be tried out and we can achieve what we want!

drishtantmkawale · May 6, 2020, 11:34am

Why is pressure cooking needed?
We are already cooking it right?

KiranKalakotiR · May 7, 2020, 1:43pm

Update on CUBE Room Webinar : Saturday,11 April 2020

Cubist from different Places/Cube Centers had joined the Cube Room Webinar, Saida Sayyed, Drishtant Maruti Kawale, Lydia Mathew, Isha Pawale, Mandar Chavan, Yash Shergare, Jai Kishan, Aashutosh Mule, Rafikh Shaikh, Zahra, and Priti Kanade are from CUBE HBCSE, TIFR Mumbai.

Joined bySavithri Singh Former Principle of AND College Delhi and Vaishanavi Mirdha.

KiranKalakotiR · May 7, 2020, 2:15pm

Update on Cube Room Webinar: 06.05.2020

Picture of Yesterday’s Cube Room Webinar( Date:06.05.2020) that was conducted on Google Meet Audio- Video Conferencing app and joined by Prof. MC Arunan, Drishtant Maruti kawale, Isha Pawle, Lydia Mathew, Saida Sayyed, Zahra and Kiran Yadav from Cube HBCSE Mumbai, Dr. Sarita Kumar and Komal Kotra from Cube AND College Delhi , Shalu Kumari from Cube Patna, Hina Mudgal from Cube Kanpur, Aswathy Suresh and Lakshmi P J from Cube SN Natika College

Above given is the link through which one can join the Cube Room Webinar, Everyday in the Evening from 5:30pm to 8:00pm.

Lydia · May 8, 2020, 1:19pm

Summary of Yesterday’s Online Discussion by @yash_sheregare
07/05/2020

Will Acetic acid be a mutagen to fruit flies ?
@aswathy a CUBist from CUBE SN Nattika who for some time is very well trying and learning about fruit flies, how to culture them, designing home made media

In one of her tomato rava medium, She added to her medium, ~ 10ml of Vinegar to a 100 ml medium containing bottle
(a lockdown substitute for propionic acid which is eliminate other fungal growth at low pH)
And now she had a burning question…
whether the acetic acid added to this medium for killing fungi, whether will it have any mutagenic effect on fruit flies?

We all tried to discuss her question and come to an easy suggestion
That Vinegar is evidently used by majority of people in various foods like pickles salads
If would have been a mutagen, it wouldn’t be a choice for us to use it for pickling or adding to salads
@Drishtant through some reference then found out that Acetic acid is not a Mutagen!

Now , we have a new opening and a question that sir asked as to whether propionic acid that is added in std corn meal agar medium, is it a mutagen?

Goof ups of no Expectation and Anticipation!
@YashSheregare, from CUBE Institute of Science, I try to explain everyone about the new tomato medium that is tested if it is a good medium for culturing fruit flies, larvae and pupae for 1 or 2 generations with no contamination.
2 medium bottles in which 10 and 6 fruit flies were added and comparing the observation made previously of a fantastic tomato slice that gave rise to next generation fruit flies, I should be expecting pupae in 4 days.

However I didn’t observe my bottles properly after 2 days for larvae and didn’t expect larvaes the next day when gravid females after laying eggs on the same day!

There is a very high possibility that gravid fruit flies will lay eggs in the medium the same day they will be transferred which got ignored…

Highlights of Gooof up - The goof up that I did was not observing and anticipation of what is to be OBSERVED and EXPECTED everyday!!
The idea of Feeling for the organism should be practised by me and others who working on model organisms!

Discussions on How should we include Goof ups and the importance of Highlighting goof ups in our fruit fly ebook

Lydia · May 8, 2020, 2:04pm

Here we are trying to design a protocol that would exactly be like the standard corn mean media that we make in the lab for our standard CsBz flies i.e. Drosophila melanogaster flies.

It would be better if I explain the method of preparation of the standard corn meal media (250ml).

Dextrose - 12.5gms
Sucrose - 6.25gms
Maize powder - 20.75gms
Yeast extract - 3.75gms
Agar - 4gm
Mix all these components in water.
Add water to make up the volume of the mixture to 250ml
Mix all the components in the water well.
Then cook the media mixture on a medium flame.
Cook till all the components in the media are homogenised completely and that there are no lumps in the media.

Next comes autoclaving
We autoclave using a pressure cooker.
The reason for autoclaving is to kill the spores and microorganisms present and to sterilize everything.
We sterilize the glass bottles and the media by autoclaving in a pressure cooker.
Trying maximum to minimize the possibilities of getting any fungal or bacterial growth we autoclave

Once the media is autoclaved,
We add acids in the 250ml media
Orthophosphoric acid - 0.17ml
Propionic acid - 1ml

This is what we want to do exactly in our homemade media.
So that we can standardize our homemade media as well.

This is a site as a reference for the standard cornmeal media that we make…

drishtantmkawale · May 8, 2020, 4:41pm

Can we summarize the work each of us has been doing since the lockdown started?

I think there has been a lot of work done in the past two months.
We have done many improvisations in the Homemade Fruitfly medium, changed our traps, discussed on different media, etc, discussed the identification of flies and much more.

Every day I get to see a lot of photos and videos!
But, the point I would like to emphasise on is that do we learn or infer something from those photos, small write-ups!!!?
Writing a summary about the discussion and our work would help us in gathering points for the E-Book as well as our own knowledge and furthermore, it would make it clear that how much do we understand what we do?
Are there any conceptual gaps?
There must be many gaps which we can fill by understanding it collaboratively leaving no stone unturned!

And also, while starting something new or continuing the previous work, we should put the following things (which I have learned and have been learning in CUBE) in the discussion
Starting with the Plan of Work which will include the things which we are planning to do in the coming days or on the same day, Design of the Experiment/Set-up which will tell us that how is our experiment going to work exactly!, then will come to our Hypothesis which should say what do we anticipate/expect from this experiment and regardless of the result we should propose a hypothesis which we would be disproving or proving by our experiment along with cited references and cross-references from authentic authors, journals, websites, labs and universities will be helpful! and then the Procedure of the experiment.

Writing or posting this in the groups or discussing all this before performing the actual experiment is essential because someone would suggest any changes or we think that this should be done and this should not be done i.e. improvisations will be suggested which would take us a step ahead in doing it.

The most important thing would be posting regular updates with a breaking news! as @Arunan Says, if there won’t be any catchy headline, no one will be interested in reading which means that our breaking news is the bait!
These updates should include each and every detail which was observed by us because Science is very weird (for me), one day we would observe one thing, then the next day something other which tells us that something unexplored is waiting for us!

And reporting the Goof-ups which happened during the course time is essential as it would have a direct impact on our results if some mistakes will go unnoticed!

URGENT!

drishtantmkawale · May 9, 2020, 8:21pm

8th May 2020 Summary of the CUBE Room Webinar!

So, Nikhil Sharma from Acharya Narendra Dev College (AND College) had joined along with @GN, @KiranKalakotiR, Aswathy Suresh from Nattika, Oyindril Dutta from Asansol, Abhishek from Kolenchery, @Arunan from Mumbai and myself.

So, the discussion started with Kiran asking Nikhil about his further plans on the Drosophila Model System on which he was working. He said that he is going to set traps for trapping the flies with his further plan to study the life cycle, life span and classify the fruitflies on the basis of their characteristics!

I know all these are a lot of things at once so we started step by step by asking the design of his experiment.
We came to know that he was just planning to keep one trap but CUBE doesn’t believe in keeping all the eggs in the same basket!

So, we made him understand the importance of having replicas.

Nikhil said that he will be taking plastic bottles and in that, he will be adding banana as the trap in one bottle and banana peel in the other bottle which will act as a medium too. But I asked, why two different traps?

To which he answered that he wanted to try out, how will it work?
So, we suggested that he should try both and see along with replicates and put regular updates. But GN added, there would arise a problem with the banana peel bottle. He asked what?
So, Sir said that we should find out the answer by ourselves!
How?

We should try keeping a banana peel in an empty bottle and then close it and observe what happens over a period of time.
And GN asked, what are our expectations!
The most important thing!
To which I replied, as the bottle will be closed, there are no chances of air passing inside, so the banana peel will get dried up, it will darken, it would shrink.

Then GN asked only these many things?
I said, yes.

Then asked about something which we have been learning in our textbooks - Spontaneous Generation!
What is it?
Spontaneous Generation means something arises from nothing!
Here, the something can be fungal or any other type of invasion on the banana peel!
We can say, that earlier nothing was visible on the peel but later, there is a chance that as we are not using a sterile (free from contamination) bottle or a sterile banana peel, we can observe growth in the form of fungus and the chances of it getting inside the bottle are more from the banana peel as compared to the bottle as the banana peels are having cells from which the fungus is deriving its nutrition!

So we saw, we got a context here which was directly related to our curriculum.

Then, carrying on with our discussion, Nikhil said he wants to study the life cycle of the fruitfly for which he will need at least some flies to start with.
But the question arises, is Nikhil actually culturing fruitflies!!?
How do we confirm that the animal which we have inside the bottle is actually the ones which we are looking for!!?

Here is where Taxonomical Classification pitches in!
Classification on the basis of its characteristics!
Kingdom, Phylum, Class, Order, Family, Genus and Species, this is the hierarchy of classification.

Crow and Fruitflies fall in the same kingdom!
What makes them different?
Their size?
Yes, but Noo.
@Arunan joked, does the difference in the height of Sir himself and Amitabh Bachchan make them fall in different species!!?

No right!!

So, what is that which makes them different?
Fruitflies and freshwater-crustacean Moina fall under the same phyla because of the presence of a segmented-body, compound eyes, jointed appendages and chitinous exoskeleton!

What makes them different?
The presence and absence of wings!

This diverges them into a totally different Order, which is Diptera for Drosophila and Branchiopoda for Moina.

Dipterans (Di- two and ptera- wings) are characterised by the presence of a pair of wings.
And Branchiopods (Branchio-something related to bronchioles of the lungs/gills and poda-feet) are characterised on the basis of the presence of gills on some of their appendages!

I have been working on Moina Model System from the past 8 months but this is when I recalled the meaning of Branchiopoda!
And this is how to further both the organisms differ in their classification!

We see that at a point, two distinctly different organisms are related!
This is too, a beauty of Science in itself where to some, taxonomy seems booooooring and to CUBists, it solves questions!!
If we develop an interest in what we work, we will be able to pass in on to others very beautifully!

And in the end, we discussed the relation between Cardamine and Drosophila!
Yes!
Botany and Zoology, the offsprings of the same parent Biology, but often considered un-related, but we CUBists have an opportunity to prove that they are related!

Further, we should discuss this and also we should bring back our Nobel-Prize winning works (as much as 6 Nobel Prizes for the wandering beauty - Drosophila!) and see whether we are able to understand these works and relate them!

Please contribute and start posting regular updates as well as a summary!
@Lydia @yash_sheregare @saida786110 and others!

saida786110 · May 11, 2020, 4:52pm

[5/11, 10:05 PM] Saida: in today’s discussion we talked about floral dip method in cardamine plant
floral dip method is basically diping the flower:hibiscus: in plasmid and detergent.
plasma containing gene of interest
and we are using detergent so that it can make pore in the cell membrane of the plant so that the plasmid can get incorporated in it.
[5/11, 10:05 PM] Saida: and the gene of interest that we talked about was antibiotic-resistant Gene that is kanamycin resistant gene
so why antibiotic resistance gene?
does antibiotic affect plants
certainly not antibiotic is used to kill bacterias and not eukaryotes😅
now the question is how would an antibiotic will effect a plant if yes then how🙄
[5/11, 10:05 PM] Saida: so as an example we took penicillin which affects the cell wall of the bacteria
and even plants have cell wall so can antibiotic effect plant cell wall
the cell wall of the plant is made up of cellulose basically glucose β(1→4)-glycosidic bonds.
and cell cell wall of the bacteria is made up of peptidoglycan. peptide is a compound consisting of two or more amino acids and glycans are compounds consisting of a large number of monosaccharides linked glycosidically.
so if penicillium affects the glycosidic bond of bacteria then and it can also affect plant cell wall. but Penicillin prevents peptidoglycan from cross-linking and cross linking is through peptide so penicillin will not be able to effect plant cell.
[5/11, 10:05 PM] Saida: now coming back to kanamycin. kanamycin prevents Protein synthesis and there is protein synthesis in plants so maybe it can stop
Protein synthesis in plants as well .
but kanamycin bind to the 30s subunit of bacterial ribosome. and in eukaryotes there is no 30s subunit of ribosome there is 40s and 60s so it should not affect plants but plants also have mitochondria and cytoplasm that are of bacterial origin which have 30s and 50s subunits of ribosome. so technically kanamycin can affect eukaryotes as well
[5/11, 10:05 PM] Saida: so supposingly if I take animal cells there is no cell wall as well only cell membrane and I take some detergent to interrupt the cell membrane of the cell and add some plasmid will there be a gene transfer taking place??
[5/11, 10:05 PM] Saida: we have figured out how would kanamycin will affect the plant cell and if there is gene transfer of kanamycin resistant Gene then cardamine can be grown even in kanamycin.
but how would we by detergent disrupting the cell membrane of plant incorporate our gene of interest?!

drishtantmkawale · May 19, 2020, 10:14am

18 May 2020, CUBE Room Webinar Summary

We started with @Rechel_tirkey ⁩ from CUBE Ranchi updating about her Moina cultures in her home lab.

Then, Gaurav Aggarwal⁩ studying at the Central University of Punjab joined us. We came to know that he has been working on Soil Worms or Nematodes.

We asked him about the things done by him before the lockdown.
So, as every nematode CUBist starts with, Gaurav also started collecting soil samples from the campus in a petri plate

Then, he prepared 2% Agar media and then poured it into another petri plate.

Why was 2% Agar used?

He also mentioned that 70% Ethanol was used by him as an attractant for the nematodes.
Why 70% Ethanol?
For me, it is used to make aseptic conditions and here it is being used as an attractant!
And Gaurav also said that Escherichia coli, a bacterium will be used by him to feed the nematodes.
I am sure that in their natural habitat, i.e. soil, not only E. coli, but many other bacteria would also be there on which the nematodes would feed.

So, Why specifically E. coli?
I suggested if bacteria are only needed, then the simplest source of bacteria would be milk which has nutrients in the form of carbohydrates, proteins, vitamins, minerals and fats as well as bacteria!
Instead of giving a Rajshaahi E. coli, milk bacteria can be used.

Can we start isolating the nematodes in our home labs?
What would be the procedure or design?

Originally asked by @Arunan
@Anshu_kadam_2813 ⁩ @bivasnag⁩ @⁨Kunal Kadam⁩ @⁨Omkar Badnale⁩ @Shivamkumar_Sriwas - How will you guys start isolating the soil worms with the minimal required things available at our homes!!?

All this would be further explained by Gaurav.
This was done as his immediate objective was to isolate the nematode and identify it.

But @Lydia and many others questioned, how do we identify the nematode or the worm which we have got.
Gaurav⁩ said about DNA sequencing!
It is not a thing which we can do at home! Is it?

@PChitralekha added that sequencing can be done for identifying the animal or plant at the species level. She gave the example of Chlorella algae which at the species level, would need DNA Sequencing.

Arunan Sir⁩, @Lydia, Mayur Gaikwad Sir⁩, Aashutosh Mule Sir⁩, @yash_sheregare ⁩said that can’t we proceed in identifying the nematode taxonomically?
Caenorhaditis elegans is the soil nematode which has Nobel-Prize on its name!
So, we can start identifying the nematode and take the characteristics of C. elegans as the Control or Standard, to match with, along with pictures and references.

First of all, how do we know that the animal which we have is an animal?
If we start with addressing such basic questions, we will we able to get a context which we can relate to the school/college curriculum!

Taxonomy is anyways thought of as a boring thing to teach as well as to learn.
So why not make it interesting by discussing with the help of a whole library in our hand; our mobile phone.
Along with gathering some pictures and examples at each taxonomic hierarchy.

Answering questions like how can a soil nematode be differentiated from an Earthworm!? would give us a good start.
If we show a microscopic image of a nematode, a child would say, "Arey, this is a small Earthworm!"

If I show a layman, my Moina culture bottle, he would say, “Aye, ye toh keeda hai”! (Oii, this is an insect!)!
For them, it may be a "keeda* or an insect, but for us, we know that it is not. So, how do we convince people about our organism!!?

We can start by relating it to some other organisms which anyone could be familiar with!
If we see, Nematodes, Earthworms, Frutiflies, Moinas… at some point in their classification, they are grouped together say in Kingdom: Animalia, then as we go further, we come to know that there are some characteristics, which make them different from each other due to which some end up in the soil, some fly in the air, and some swim in the water!

The only wait is for getting a good context to start with, rest, the CUBists will handle!

We also discussed the taxonomical classification of the Standard Drosophila melanogaster CsBz fly and Moina macrocopa, both of which are Model-organisms in CUBE.

Later, in the end, @saswathy679 from CUBE SN College Nattika, Kerala shared her Fruitfly Homelab Medium story and updated us about the cultures and current happenings at her place.

yash_sheregare · May 22, 2020, 3:52pm

Online Discussions
17/05/2020 CUBE Room Webinar

We had discussions today on varied different
Topics starting from
Our special lockdown stimulated Home Made Medium for fruit flies - Tomato Rava SugarVineger Medium (TRS-V Medium)
To Moina Culturing, Circadian Rhythm studies in fruit flies, topics like Genetic drift in brief and many more!

Most highlighted Take home from Today’s Discussion

As @mayur sir added while discussing Moina Culture for Sweta CUBE Ranchi

Don’t put all your in one basket

In our CUBE , the context goes for every model system
Be it, Moina cultures (keeping of replicates)
Fruit fly cultures
Hydra cultures
Cardamine studies
And so on!
We need to have replicates for each time we try to do anything
Be it

culturing or maintaining Moina cultures, or making a new setup for Subculturing!
Maintaining fruit fly cultures and even testing home made medium for Culturing fruit flies!
Culturing Hydra, Having replicates!

@⁨Ashwathy CUBE Nattika⁩ , a Nattika CUBist has very well tried and made a working home made TRS V medium which worked well to give rise to larvae (in three days) pupae (in another w days) and next generation fly (after another 3 days, which means next generation fruit flies in 8 days! ) in her single medium bottle! ( This result we see evidently also in our Std Corn Meal agar medium)

Now, to confirm and say, it’s a medium that functions well
Or lets say,
To make this our standard Medium, we all together need to Design and make medium with Replicates and control

Which means?
When we make and test the medium and say the medium is working well for getting us larvae, pupae and next generation flies, it shouldn’t be giving us the result in just one medium bottle but atleast 3 replicates and even better for more bottles tried and tested by all other CUBE centers working on fruit fly and culturing them during Lockdown Collaboratively!

For that we all together must discuss it in length with details
The design of medium
The protocol of medium
Modifications in the medium if any
Number of replicates to be made
Number of fruit flies added in each bottle (keeping in mind and also adding and counting number of gravid females in each bottle)
Types of Control setup needed?

Please add in your inputs!

drishtantmkawale · May 23, 2020, 12:42pm

22 May 2020 CUBE Room Webinar

Yesterday’s discussion started with CUBist Abhijeet Singh, a Class XII student from Mumbai, sharing his experience in CUBE; he told us about the model organisms he worked on, the basic understanding of it. Abhijeet has worked on the Ventral Nerve Cord Regeneration in Earthoworms, on the Fruitflies as well and has also studied on Mosquitoes.
About Mosquitoes he told us about keeping Ovitraps. Abhijeet and @Hinaiqbal_Mudgal from Kanpur were discussing about their own experiences with mosquitoes, their current status and further plan.

Then, we came to the Drosophila-Fruitflies where @saswathy679 from Thrissur has been working a lot on the Fruitflies, be it trapping, medium preparation, preparing single line cultures. So, Abhijeet was new to this so we heard the summary of @saswathy679’s work so far and her further plan.

Then from South, we went to East-India in Asansol, West Bengal where @OYINDRIL is also working on fruitflies. As of now, the Circadian Rhythm Studies are being done by him.
We would like to know about your work @OYINDRIL DUTTA…!

@OYINDRIL is also planning to prepare media to maintain his fruitflies. So @saswathy679 is also working on fruitflies. As of now, the Circadian Rhythm Studies are being done by @OYINDRIL DUTTA…
We would like to know about your work @OYINDRIL DUTTA…!

He is also planning to prepare media to maintain his fruitflies. So Aswathy, who is now confident of sharing the Procedure (secret recipe🤭) of her media, we should look forward to get updates.
So her we saw that CUBE is acting as a connecting link between Thrissur, Kerala and Asansol, West Bengal where collaboration is taking place at every step and problems are being solved!

As @Arunan said, there is lack of anticipation/expectation from our work!
We should expect something in order to get something. If we aren’t expecting only, then what are we going to look for?
And also when proposing a hypothesis, design of an experiment, we should not do it vaguely but proceed with confidence so that the fellow viewers would be eager to know what is happening.
The way of presenting our work with proper headline/breaking news, plan, expectations matters the most!

Not to forget, Evidence based Research is what we should focus on, otherwise it would be like doing a ritual of which no one cares of!
Evidences, archives in the form of Research Papers (from recognized labs, universities, journals, authors/scientists along with cross references), Photos and Videos with a proper hypothesis/strong statement are the things which we should look for, further which would convince our audience!

For example, if we are preparing the Fruitfly medium at home, then we should start mentioning the quantity of media, the requirements of it, the texture, the procedure, cooking time…
If we say that 100mL medium was prepared just like that, then some fellow CUBist trying to reproduce the same medium won’t be able to do it.

Why?
Because we lacked in details!
This is why Arunan Sir always emphasises on giving the details because The DEVIL and the DEVINE is in the Detail!

But, if we go by saying that 100mL medium was measured using a cup or any other utensil, then it would be somewhat relatable otherwise people won’t do it saying that "we don’t have the sophisticated equipments for measuring and all… so we won’t proceed!"
@mayur added that simply relating the amount which we want by imagining/visualising the ~measurements which we use in our labs, will do the trick! (not necessary that the amount should be accurate everytime!)

Looking forward to get updates from everyone!

A short-summary after the Webinar will make sure that the discussion starts in the group!

Lydia · May 27, 2020, 9:27pm

26th May & 27th May 2020
CUBE Room Webinar (A short summary)

While discussing in the webinar for the past 2 days,
@saswathy679 and many others were believing or claiming that temperature might be a factor that gave a delayed life cycle.

So, the story goes like this…
@saswathy679 as a very good or a very thriving culture which started from 6th May.
Within 8 days, i.e. on the 8th day (14/05/2020), new generation flies ecloded from the pupae.
After 4 days i.e. on 18th of May, another set of media was made and the newly emerged as well as the parent flies from the 6th May bottle were transferred into the newly prepared 18th May media bottle.

In this 18th May TRSV media bottle, Aswathy could not locate any larvae for almost 7 days i.e. from 19th May to 25th May…but on 26th May, small and big larvae could be seen by @saswathy679 as a very good or a very thriving culture which started from 6th May.
Within 8 days, i.e. on the 8th day (14/05/2020), new generation flies ecloded from the pupae.
After 4 days i.e. on 18th of May, another set of media was made and the newly emerged as well as the parent flies from the 6th May bottle were transferred into the newly prepared 18th May media bottle.

In this 18th May TRSV media bottle, Aswathy could not locate any larvae for almost 7 days i.e. from 19th May to 25th May…but on 26th May, small and big larvae could be seen by @saswathy679 i.e. on the 8th day!

During the webinar,
Aswathy claimed that temperature might be the factor responsible for this delay.
When asked what was the temperature, it was told that it’s 27°C.

For me, and @yash_sheregare ,
We had 2 reasons for ruling out the factor of temperature!
27°C is normal (lab temperature) where we used to culture our fruit flies, the standard as well as the native single lines. And within 8 days we used to get the next generation flies.
Aswathy herself got a thriving culture in the same place at the same location using the same media (without making any changes) few days back…not even a month old story…
And I looked for the temperature of those days.

There isn’t much drastic difference in the temperature that would affect the life cycle of fruit flies giving a delayed cycle.

If you ask me what was the temperature during those days.
I’m giving a reference along with the data of the temperature on all those days from 6th May to 16th May (the bottle which had a thriving culture) and from 17th May to 25th May (the newly made same type of media which was claimed to show a delayed life cycle of almost 7 days!!)

Reference: https://www.accuweather.com/en/in/thrissur/188812/may-weather/188812

May
6th - 34°/24° C
7th - 34°/22° C
8th - 32°/26° C
9th - 33°/25° C
10th - 33°/25° C
11th - 33°/25° C
12th - 33°/26° C
13th - 33°/26° C
14th - 34°/25° C
15th - 33°/26° C
16th - 32°/25° C

17th - 33°/26° C
18th - 29°/25° C
19th - 31°/25° C
20th - 33°/24° C
21st - 31°/28° C
22nd - 32°/25° C
23rd - 33°/29° C
24th - 33°/27° C
25th - 33°/25° C

@saswathy679 prepared media on 17th May too…
But, I emphasized more on 18th media as that was a replica of the media made on 6th May.
On 18th May, the 6th May media was replicated and flies from the 6th May bottle were transferred into these 18th May media bottles.
So we have the temperature range from 17th May to 24th May as well.

Do we really see a temperature difference or drastic deviation in these day??
I dont see any!!
Is it really a temperature bhagwan at fault or is it some other factors???

Today,
when we were again discussing about the same thing,
still we were stuck in the temperature thing!
Surprisingly, diapause & humidity factors also came up…okay…agreed…

So…What is Diapause?
A period of suspended development in an insect, other invertebrate, or mammal embryo, especially during unfavourable environmental conditions.
Unfavourable conditions!!!???
What unfavourable conditions did Aswathy or her flies faced??
Humidity was an answer…okay

So…What is Humidity?
Its the quantitative amount of water vapour present in atmosphere.
How can this affect the flies which are cultured inside a bottle which is kept closed all the time and is opened only when we have to transfer flies…??

[size=4]Now, I would like to tell what could be the reasons along with the explanations for the so claimed delayed life cycle of Aswathy’s flies.[/size]

According to @saswathy679’s observations,
She saw larvae on 26th May (both small and big)
And then, today i.e. on 27th May, she got pupae!!
Can a larva turn into a pupa within a day?
Its impossible according to my experience and observaions with the standard and the native fly cultures.
It takes minimum 2-3 days for a larva to turn into a pupa.

What can we infer from this?
Observation is lacking because strong expectation is missing
LOGICAL EXPECTATION…that has a great role in science, especially in DESIGN

I’ll explain it more…
If Aswathy saw a larva on 26th May and it turned to a pupa on 27th May…
That means there were larvae in the media 3 days before.

So…
On 23rd May, the flies had laid eggs,
On 24th May, the first stage of larva (1mm size)
25th May, second stage larva (2mm size)
26th May, third stage larva (3mm size)
On 27th May, i.e. today, we got to see pupae!!

So what happened??
Expectations were missing
Keen observation with interest was missing
Its ok, she might have missed out…this happens with many of us…but directly taking temperature as the sole reason is not right…
Human errors are to be first analysed.

Its not that temperature cannot affect.
But we need to have the proper data giving evidences that there was a drastic change in the temperature.
As well as we need to be sure that there wasnt any missing out of observations or expectations that happened from our own side.

There could be one more reason,
A normal female fly can be mistaken for a gravid fly.
May be that those female flies weren’t gravid or fertile

This is my comment on the summary of the webinar that we had.
Waiting for comments from everyone.

drishtantmkawale · May 29, 2020, 10:35am

Summary- CUBE Room Webinar
Thursday, May 28th 2020

Moina-A brief discussion

The discussion started with the freshwater-crustacean Moina. I updated everyone about the Moina cultures at my home lab.
Then, I shared the breaking news of observing yellowish-green colour on the base and walls of the bottles.
I suspect that it is Algae which is taking over in the Yeast bottles. I added that once I see more greenish growth, I will add ~10 Moinas in both the bottles.
@KiranKalakotiR and Abhijeet Singh asked that how will the Moinas survive in those bottles? If we look at their natural environment which is ponds and lakes, the Moinas feed on Algae, Bacteria and Fungi (Yeast and others) present in the fresh-water bodies there.*

It will be interesting to know about their feeding mechanism on Algae and Fungi.

Abhijeet Singh asked about my Research Question and how am I working on that.
Research Question: How do the Moinas undergo colour change (colourless to coloured and vice versa) in less-oxygen condition or hypoxia and is it due to Histone modifications?

I said that it is very simple to induce hypoxia and observe the colour change of the Moinas. Everyone (if they have Moinas at their place) can do it in their home labs and the first possible colour change would be noticed by them in 72 hours!

And once the Moinas become coloured ones, we can turn them back into colourless Moina.
How!!?
By again keeping them back in Normal oxygen condition or Normoxia.
Here too, after a day i.e. ~24 hours or less than that, the 3rd day Pale Yellow or coloured Moinas will turn into colourless Moinas.

So we see that such simple experiments which can be done at home are the base of understanding the complex mechanisms like Histone acetylation/deacetylation, Remodeling and others.

Yesterday, a major goof-up happened by me that I burdened everyone present in the Webinar by explaining these complex mechanisms (without the help of any diagrams as such) instead of starting with the basics and explaining it simply!

My Apologies to everyone

I learnt that evidence based research is essential in explaining a complex system.
I have data, so I am able to explain.

Later, many others questions were asked.
Let us discuss them here. CUBists please repost the questions here.

Then, there came an issue of misunderstanding Milk as a Mutagen!!
@Vaibhavib99 and later @Shalu added that many people assume that the colour change of the Moinas from colourless to coloured ones and vice versa is due to mutations and reverse mutations respectively!

How is that even possible?
Does that mean many of us are consuming a mutagen?
No way!

Mutations take place when there is a change in base-pair sequence of the DNA.
They take time to get expressed.

And as I mentioned earlier, only the configuration of DNA and Histone is changed and rest all remains the same.
THE DNA SEQUENCE DOES NOT CHANGE!

So milk in any form, is not a mutagen which will cause mutations in Moinas!!
This is where we go wrong in understanding the TEXTBOOKISH things!

Instead of ending it here, we took it somewhere else.

Fruitflies-the flies on demand

We discussed about the current status of the fruitfly cultures maintained by @saswathy679 at her home lab in Thrissur, Kerala and @Hinaiqbal_Mudgal too, at her home lab in Kanpur, Uttar Pradesh.

As they are having many bottles with as many flies in them, all of us suggested that they should start with the identification of the dead frutiflies in their bottles.
Even if they do not belong to a single line culture of flies, any random fly can be used just as a warm-up for identification.

Starting with the family identification and moving further on.
Yash, Aswathy , @Lydia and others please add to this and let us start with the identification procedure and its design!

Shedding some light on the slow-mo guys: Snails
As we were wrapping up, we came upon the Snails which are maintained by @Anjani from CUBE Elphinstone, Mumbai, in his home lab.
His collaborators Manasi Prasad, Nishmita Amin, Nikita Dhuri updated that one of his snails died.
How do we know that a Snail has died? It may be that the snail must be inactive or sleepy!

They said that a stench was coming from that snail.
Was that a result of any bacterial, fungal infection?

Zahra Risalawala said that she had read a paper (please share that!) where it was saying that the excess of salt can harm the Snails.
Is it?
What will happen if we take a mouthful of salt?
What happens when our fellow Earthworm is exposed to high salinity.
I have seen that the earthworm tries to move away from it.
It may shrink too.

Why?
Does dehydration take place?

Nishmita Amin said that it is osmosis taking place.
What is osmosis? Do we really understand it?

We can simply start saying that because there is high salt concentration outside and low/normal salt concentration inside the body cells of the Earthworm, the salt will try to diffuse inside and in the process, it will dehydrate the Earthworm as more salt would be present inside which is harmful.

Why is it harmful?
Is it not a CONTEXT TO CURRICULUM question?

Let us solve it!

Arunan · May 29, 2020, 11:11am

Which curricular questions are being addressed here? @drishtantmkawale Please like at them with an one line description.