The Positives in the Negative

The Positives in the Negative

NISHTHA

During my PhD days, the majority of my time in the lab went into growing a HUGE number of mammalian cells in dishes. We call it “cell culture”. Usually, small dishes - with bottoms the size of a credit card or a stove knob - are sufficient to conduct experiments because cells pack a lot within themselves. But I was working on things that cells throw out - these ‘vesicles’ (little bags of molecules) are a thousand times smaller than the cells, and I needed to collect a LOT of them to be able to study them. We call them small extracellular vesicles (sEV) - small because they are small, extracellular means outside of the cells, and vesicles is just a scientific name for these “bags” (literally, “blisters” in Latin). These sEVs play an important role in intracellular signalling, conferring protection during cellular stress (for instance, when a cell is subjected to heat shock).

Both Microvesicles and exosomes are types of extracellular vesicles.

Illustration by Ipshita

I needed culture dishes the size of a shoe. And at least 4 of them. Because we had a shared culture facility (the super-clean room where culturing is done), and since many trainees used the same setup and not all users followed rules to the T; my huge cultures would end up getting contaminated with bacteria or even yeast!

Contamination - a destroyer of experiments - and spirits

Each time my cell cultures got contaminated, it cost two weeks and a ton of material. Throw these away and start anew! And I had to repeat, helpless. Cells with bacteria growing in them don’t show us the real pictures because they’re sick with infection! There is no way around it. All of us who cultured cells dreaded this one singular thing. It's a coveted art to keep your cultures clean.

Not just human or animal cells in dishes, even bacterial cell cultures can get contaminated. Essentially, anything other than what the researcher intends to grow in the dish is a ‘contamination’. Since microbes and fungal spores are all around us - even in the air - they can enter our culture dishes and we do everything possible to keep them at bay. So, bacterial cultures can get contaminated with bacteria other than intended, and even fungus! Argh! The immediate response - throw them and start afresh. The disgust is almost visceral.

Contamination has raised its ugly head ever since biologists started culturing cells in dishes. In another story from the 1920s, a biologist was trying to grow staphylococcus bacteria (call it Staph) - that causes skin infections - in his dishes. These things take time to grow and sometimes we just forget them in the incubators (sorry). This guy returned from a vacation, only to find fungus growing in the bacterial plates! Argh!! Contamination!!! If it were me, I would huff and stomp, throw those damn dishes and get back to step 1 - material preparation.

Gabain, Ethel Leontine, Public domain, via Wikimedia Commons

But he did what we often forget to do in the 21st century cutting-edge science - he observed the fungus. Wherever it grew, the bacteria couldn’t grow in that area of the dish! He got fascinated, experimented more, and found out - the fungus oozes out a chemical that prevents bacterial growth! Penicillin - the first antibiotic - was discovered. ‘This guy’ was Alexander Fleming. And he was called a ‘tardy technician’, just so you know.

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One sometimes finds what one is not looking for... I certainly didn't plan to revolutionise all medicine." Fleming said. Would Fleming have made this discovery without peering into the 'useless'?

Maybe my contaminated cultures hold significant discoveries like Fleming’s, or maybe they don’t, but when you are doing 21st century science, and you get data that does not align with what we’re trying to prove to be true (our beloved hypothesis), we just bin it and chalk up a new scheme. We call it ‘negative data’ - data that does not support our hypothesis. It belongs in the trash - not in our thesis, not in our papers. And the race to the perceived finish line continues. Churn what is acceptable, what is logical, what makes sense to us.

Many such trashed possibilities filled a good portion of my PhD days. Let me give you an example from my own research on small extracellular vesicles (sEVs).

Lost opportunities with Small extracellular vesicles

In my host lab, we were tasked with finding out how unhealthy cells can be helped to regain their health. Our focus was on the kind of sickness that makes the proteins in the cells lose their 3D shape, become dysfunctional and get tangled up. This happens not just in sickness, but also under stresses like high temperature. The cells have ways to resolve the problem and gain normalcy soon after the stress is gone. The lab’s aim was to find ways to leverage and enhance this capacity, in the most natural way possible, to make cells fitter or more resistant in the face of stresses.

In my project, we were figuring out if the sEVs from heat-stressed cells could in turn improve the stress-resistance of cells that received them - in some way signalling SOS to them. But we were blocked at the very first step - getting the sEVs from the liquid ‘medium’ that the cells are grown in. Without being able to do that, how would we give them to the recipient cells?

Illustration by Ipshita and Nishtha

Image credits to Nishtha

So, we tried a proxy - picking up the entire medium from donor cells and putting them on to recipients - bypassing the need to fetch only the sEV. And you know what? It did lend some resistance - but only very minimal. Now, at this stage, maybe we should have tweaked our experimental setup and changed our measure of stress resistance (till now, less cells dying after stress was the measure - a rather extreme one) to find out what could be going on. Instead, we rejected this data - the effect was hardly any - so it must not be important, or in other words, statistically insignificant, and hence, negative data. Not sufficient to prove our point. There could well have been other ways of measuring the resistance of these cells, for instance, increase in the levels of specific kinds of proteins that alleviate the stress, but we didn’t explore that.

I still wonder sometimes what it would have led us to if we followed that tiny lead.

Another question that I ponder about is what could have been the mechanism behind the minimal resistance that we got. Cells would throw out all sorts of things in the medium - even toxic products (cellular crap). So in the whole medium, certain parts may cancel each other’s effects out. And that could be the reason we initially saw only a minimal increase in stress resistance. But there are ways to split the medium into identifiable parts. We could (and perhaps, should?) have separately tested those parts out on recipient cells. Sure, it would have been time-consuming, but what if one of those parts was potent enough to make the recipient cells heat stress-resistant?!

By then, in parallel, probably we’d have figured out how to fish-out the sEVs specifically too! And then, maybe we could have found more than one parts of the medium that were helpful - maybe even more powerful if put together! Whoa, that would be some interesting biological mechanism! We could use combinations to figure out what would work in favour of the recipient cells’ fitness under heat.

I did try something else too – to take the recipient cells out of the picture - and conduct a test in a tube with just the proteins and the liquid medium from heat-stressed cells alone - an approximate test of the ability of the sEV to ‘protect’. Proteins lose their 3D shape and become tangled under stress; if the medium can prevent this from happening, or undo it, that would mean it contains something that can improve stress resistance. We tried with one protein - nothing happened. And we never tried again with another protein, or in any other way. Maybe it would have worked for some other protein - and that would have told us a lot about the composition of those particles too! Alas, we ran out of time and resources, with COVID stealing some time too.

But now, I will never know. At least not until someone else does the task and publishes the findings.

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Honestly, the real thrill of conducting research, and what makes it worth persevering despite the gruelling nature of it is that biology, to quote Barbara McClintock, "never ceases to amaze us."

While standard protocols and set pipelines are important for scientific rigour, and making research reproducible, does it leave room for creativity? Imagine the stories we miss as we disregard negative data!

After all, more often than not, science (especially biology) has benefitted from serendipity. And one of the crucial factors responsible for that? Be a keen observer. Like Fleming.

Looking into the negative data may even have more perks. Not only does it hold potential to unlock the ‘amazing’, it is a great antidote to the frustrations of research!

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What a delightful thrill it is to be the only one on earth to know something brand new, even if for just a moment. This is a powerful source of fuelling our own motivation to dig and prod more, find more and be blown away."

At least I was in it for that awe, that thrill. Not anymore.

About Nishtha

Nishtha Bhargava is a life science researcher exploring ways to communicate science to the public. She left a permanent job in a public sector bank to pursue a doctorate in life science. Through the course of her PhD it became clear to her that she has a knack for making scientific concepts easy to understand, which she put to use in outreach activities on campus, and then, in writing. She supports equal rights, inclusivity, diversity and good mental health practices, believing that empathy-driven rigorous science has the power to change the world.