In the first post of this series, @sco shared his suffering with us, and now it’s my turn.

This post is mainly devoted to Ph.D. students, a very vulnerable group of people, often trapped on bad projects.

Funny and nerdy!

My Story

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I started my career in 2010, with a relatively straightforward task:
• isolate plasma membranes of corn
• do EPR spin-trapping
• and play a bit with the conditions

Great! What could possibly go wrong, they are doing it for 30 years, I guess it will run smoothly.
I will probably do my thesis in less than 3 years…

But all the people I interacted looked at me with compassion.

Oh, no, you are doing those membranes :(

I didn’t realize what’s the catch, but I noticed something unusual.
All the people from that research group needed 7-10 years to do their Ph.D. Thesis.

The requirements were high but reasonable, 2 related papers, signed as the first or second author. In biology, it’s really reasonable to have several papers per year.

About a month later, I realized what was wrong…

Isolation of membranes

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I came with the high expectations of our top research institute for maze/corn.

But… Everything was really, how to say it gently, amateurism and improvisation.

Corn was never the same; sometimes it was very large with the dark roots, sometimes fragile with light roots…
And it’s strange because that corn was grown from the same seeds, in laminar *(a large box with controlled temperature, light…).

Something like this:

It's not me. And it was not my laminar source

Isolation was a continuous labor of 8 hours. I started with the mortar, to crush the roots and get membranes.

After that, the slurry was purified several times using the centrifuge.

And finally, there was the purification with the (sucrose?, I forgot) gradient.

You can find some Slides about this.

For those unfamiliar with the biochemistry, the idea is to do partition.
And there was also the “two-phase system”, similar to this:

Not the membranes, but the principle is the same, link

The idea is to have two solvents with the different properties that don’t mix, like oil and water. After the injection of the substance that you want to purify, some components go to one phase while the others go to another - simple.

After several days I got my membranes:

And I could see, using my eye-photometer, also known as “naked eye” that instead of homogeneous membranes, I got stripes and strata. In other words, inherent error No.1

EPR spectroscopy, spin-trapping

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The method itself is good, actually, it’s great and the only possible choice.
EPR spectrometer is the machine with the principle of work similar to NMR/MRI.

The sample is placed between the strong magnets and irradiated by the microwaves.
Stable paramagnetic species (some environmental radicals, some oxidation states of metals) can be detected directly, but the unstable radicals that we find in biological systems need to be trapped by the probe molecule that becomes stable radical itself.

The trap of choice was DEPMPO, capable to distinguish different types of radicals, remains stable for 60 hours easy, in contrast to DMPO, *OH and *OOH adducts (trap + short-lived radical) remain stable.

You can buy some from Enzo Lifesciences, only 2.250 Eur per mg!!!

But…
Serbia is not a member of the EU, why – don’t ask me.
And we have border control. In practice it means that every packet must be checked – that is ok.
What is not ok is the fact that they are not in any hurry, no, no… Because every day your order stays on the border control is charged to – you.

When we get DEPMPO, it’s contaminated

So we need to purify it. It’s a simple procedure, but...

it includes vacuum concentration and you simply can’t have the same trap again

Now it is getting worse…

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Every time you freeze the membranes to store them and melt them to use them – membranes change

How? Simple, they burst and spontaneously form vesicles that can be “inside-out”. Or… Not form anything. Or form the vesicles with the right orientation. And it’s different, every single time.

And worse…

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Our spectrometer was old, older than me, and not very sensitive for this purpose (later we tried with the latest Bruker – better, but still pointless) and the

signal to noise ratio was 2:1 or 3:1 in best case

And the effect was negligible.

But the worst thing…

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My boss had the vision about the malic acid, a common molecule as the most important thing in Universe.

Because? Reasons…! No proofs, no supporting data, nothing. And before me, 4 people failed to find any effect.

Summary:

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• I had different plasma membranes, every time
• Membranes that change their structure, every time
• Detected by the probe that is different, every time
• Signal to noise ratio is 2:1, spectra are complex and
• the effect is not even visible, BUT PROVE IT!

So, how it ended… Well, simple, and that is the most important thing to learn from this, if you are Ph.D. student.

I kept doing my things’

I learned Matlab, chemometrics, statistics. I learned everything I could. And I was building the connections and got more papers than my own boss.

When the moment for the defense came, my ex-boss wanted to block me.
I refused, sent him to hell, and done my own and even got fired!
*(and found the job that same afternoon, because - networking).

But How, Why…

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Well, Serbia is the country with almost the lowest salary in Europe.
It's also the country with the lifespan among the shortest in Europe.
The country with packs of stray dogs.
And the country where the idiots are poisoning eagles (our national symbol, on the flag!).
And you can build the house illegally in the middle of the national park. Several years later, you will be able to legalize it for several thousand.

Now you understand how someone can become the project leader without competence and HODL the same false project for 30 years. For very small amount of money/ meaningless position, we are constantly ruing the system and making the "negative natural selection".

That's the reason why most of the people from my generation work aboard.
Not because of low salaries, but because of the lack of the system.

My nomination goes to @scienceangel