Return to the Stamataki Lab

I’ve kicked off this summer with a trip to Birmingham to meet with Dr Zania Stamataki. During my prior work experience here, I improved my wet lab skills by learning essential techniques such as splitting cell lines, performing serial dilutions, and imaging plates with confocal microscopy.  This year, I chose to hone my computational biology skills to best prepare me for a future in research; Dr Scott Davies introduced me to bioimage informatics through training me to work with the ZEISS Efficient Navigation (ZEN) microscopy software.

The ZEN software can be used to process the plethora of data that a single imaged experiment can yield. I worked with data from an experiment in which Huh-7 hepatocytes (from an immortalised liver carcinoma cell line) are co-cultured with live T-cells and imaged using confocal microscopy.

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I personally found this sophisticated mode of imaging fascinating so I did a little more research into the bioengineering responsible for the high resolution and vibrancy of the  cellular interactions captured by the confocal microscope. Unlike the humble optical microscopes of my school, which only need light and a pair of eyes to visualise cells, confocal microscopes require far more exotic elements: lasers and fluorescent dyes.

 

GFP

One of the most widely used fluorescent biomarkers is green fluorescent protein (GFP) which is used in this experiment to highlight the cell membranes. GFP is a very important tool in research as it enables us to look beyond the cell surface and visualise organelles and protein movements. GFP has a unique sequence of the amino acids serine, tyrosine, and glycine which can be triggered to undergo chemical transformations in the presence of UV light. This special amino acid sequence is buried deep within the molecule’s barrel structure of intertwined protein sheets.

 

When activated, a cyclisation reaction occurs in which glycine forms a chemical bond with serine. This new closed amino acid ring spontaneously dehydrates and oxygen surrounding the GFP molecule forms a new double bond with the tyrosine – creating the fluorescent chromophore. In this way, GFP automatically assembles its own chromophore which can be used by researchers as tracking dyes. For instacnce, through genetic engineering, the GFP protein can be incorporated into the genome of specific cells or be used to label proteins of interest.  It is especially perfect for studying live cells since the alternative small fluorescent molecules (ex. fluorescein isothiocyanate) are highly phototoxic and inflict more damage upon live cells during imaging.

In the microscope images I worked with, GFP had been used to label the hepatocyte cell membranes. The T-cells were stained a vibrant blue whilst a cytoplasmic dye had rendered the hepatocyte cytoplasm red. The smaller dark regions enclosed within  cytoplasm are the hepatocyte nuclei.

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Over the course of the week, I became proficient in producing orthogonal videos, which showcase the full depth of the hepatocytes, from multiple micrographs in a time lapse. Whilst processing these videos, I witnessed countless interesting phenomena; with Dr Stamataki’s permission, I’d like to share a few of my favourites with you.

Binucleate Hepatocyte Division

When I first starting working with the experiment data, Zania pointed this event out to me and I was both riveted and shaken to my core; this cell defied everything textbooks had me believe about cell biology! In this orthogonal video, a binucleate cell successfully undergoes mitosis to produce not two but three daughter cells.

Binucleate cells can be caused by cytokinesis failure in which the cell membrane fails to form a cleavage furrow so two daughter nuclei are trapped in the same cell. Alternatively, during mitosis, sometimes multiple centrioles form making the cell multipolar- pulling  the chromosomes in several directions and producing several nuclei in a single cell. Judging by how the binucleate cell proceeds to divide into an odd number of daughter cells, its original two nuclei probably arose from the latter mechanism.

Binucleation in vivo is pretty rare and, in a healthy human liver, such cells are incapable of dividing again, instead forced to remain in interphase indefinitely. Binucleate cancer cells, however, have a far different fate; more than 95% can undergo mitosis with a higher rate of chromosomal disjunction error – producing daughter cells with even more mutations. Despite binucleation rarely arising in healthy tissue, this phenomena is less of a rarity in cancer cells due to one of their unique quirks. A prominent biomarker of cancer cells is their multipolar spindles which increase the risk of binucleation and other mitosis abnormalities – leading to an accumulation of mutations in subsequent daughter cells in the cancer tissue.

Lamellipodia Network Formation

Inside the body, liver cells are always in contact with their neighbours with junctions between their lateral faces. In a lab culture, however, liver cells are seen tightly adhered to the plastic that they are grown on, practically clinging to the plate for dear life! The in vitro hepatocytes achieve this through multiple membrane projections known as lamellipodia.

Lamellipodia are made of the cytoskeletal protein actin and, in the video above, can be seen stretching out to not only tether carefully to the confluent monolayer but to also engulf some extracellular material. I’m mesmerised by the rapid yet tentative movement of these hepatocytes’ extensive threadlike network.

Another Kind of ‘Nuclear Fission’

The earlier orthogonal video of two nuclei becoming three was very surprising. In contrast, the phenomena below is absolutely incredible.  Two neighbouring T cells apply a mechanical stress on the hepatocyte’s nuclear envelope great enough to break the nucleus into two ‘daughter’ nuclei without the hepatocyte undergoing mitosis! I find it truly fascinating that the nucleus is not as static an organelle as it is so often portrayed; instead, it is dynamic and capable of distorting and even bifurcating in response to changing cytoplasmic pressures. 

 

 

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As always, I’ve really enjoyed my time at the Centre for Liver Research this year and I’m so grateful to Zania for allowing me to learn so much about the cell biology and immunology of the liver and participate in her current work. Her team is currently in the midst of publishing some amazing cutting-edge research on a cellular mechanism similar to entosis; as soon as it is available to the public, I’ll share it here and, hopefully, write a review article about this interesting hepatocyte process!

WordPress does not support video uploads for this blog so the orthogonal videos in this post were compressed into gifs, hence losing resolution and quality. If you would like to see the original videos, I’ve also uploaded them to my YouTube channel:

• Binucleate Hepatocyte – Abnormal Mitosis
• Extensive Hepatocyte Lamellipodia Network
•  Nuclear Split

Bibliography

Kim, Dong-Hwee et al. “Volume regulation and shape bifurcation in the cell nucleus.” Journal of cell science vol. 128,18 (2015): 3375-85. doi:10.1242/jcs.166330

Shi, Qinghua; Randall W. King (13 October 2005). “Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines” (PDF). Nature. 437 (7061): 1038–42. doi:10.1038/nature03958. PMID 16222248

Yang, F.; Moss, L.G.; Phillips Jr., G.N. “STRUCTURE OF GREEN FLUORESCENT PROTEIN.” Nat Biotechnol vol. 14 1246-1251 (1996). doi: 10.1038/nbt1096-1246. PMID 9631087

 

 

 

The Shape Of Water

Water is the most abundant molecule in cells, organism, and on earth and it is so vital to life due to a combination of its unique chemical and physical properties.

Let’s first take a closer look at the structure of this essential molecule; water is polar (meaning that it has small charges on the individual atoms in the molecule which let it bond to other water molecules). This is so important and influences all other properties of water. Because water is polar, it can interact with other substances (such as salt) to dissolve them. In our bodies, all the chemicals are dissolved in water in our cells (in the form of the cytoplasm): water allows us to exist at a cellular level.

 

The solid form of water (ice) demonstrates another cool property of water as ice is actually less dense than its liquid form. We probably all take this surprising physical property of water for granted, however, for all other substances, the opposite is true. Ice is less dense than water because when the liquid cools down and molecules lose energy, more hydrogen bonds form between the molecules- forcing the molecules into a more spread out lattice to accommodate for the maximum amount of hydrogen bonds.

This unique physical property ensures that, even when its really cold, bodies of water such as lakes and oceans freeze from top-down – insulating the water below the surface and protecting aquatic life. This special property of water might have prevent organisms from going extinct in the past.

Water is so important to life that it is no wonder that we are almost constantly told to remain hydrated, especially in the summer. Naturally, in order to transport water, the majority of us use plastic water bottles: they’re convenient, lightweight, and cheap. But their hidden cost comes in the form of the damage it does to our health and the health of the planet.

The fact that plastic poses a risk to the environment probably doesn’t come as a shock to you. Despite this, you would probably still be forced to use plastic bottles since there are virtually no other cheap, lightweight alternatives that can even hold a candle to plastic. Until now…

Introducing Ooho! A water bottle that is completely edible and 100% biodegradable. Developed by Skipping Rocks Lab and inspired by the natural membranes found in eggshells, these orbs of water are encased in a calcium alginate gel layer and formed through a fascinating process known as spherification which was developed, not by chemists, but by chefs.

The process of spherification was pioneered in the 1950s by Unilever and it involves the reaction of two common chemicals which can form a tough membrane on the outside of the water- essentially, the water became its own water bottle through polymerisation. The first ingredient of the edible ‘water bottle’ is Sodium Alginate, usually derived from seaweed. This is a long-chain carbohydrate that is soluble in water. In its original plant, it is used to store sugars created by photosynthesis. When Sodium Alginate is dissolved in water, these long-chain carbohydrates float around on their own but they don’t connect to each other. That’s because poking out from these long chains is a branch of carbon and oxygen atoms, which chemists call an anionic group which has a slight negative charge. The sodium ions are attracted to this, because they have a positive charge. But sodium is monovalent, meaning that it wants to bond to just one of these carbon and oxygen branches at a time.

However, if you add calcium ions to the solution, the structure changes. Calcium ions have two positive charges, so they want to bond onto two of these branches at a time, meaning that it can connect two alginate molecules together. So the calcium ions replace the sodium and create cross-linkages between the long-chain carbohydrates. When enough of these chains are connected together, this 3D matrix of connected alginate chains forms a semi-solid impermeable membrane that the water can’t pass through -creating a sphere that surrounds the water. The fully formed water sphere is made from nothing but water and the two chemicals!

As part of the session, we experimented with sodium alginate and calcium lactate to design our own edible ‘water bottles’. We first dissolved one gram of sodium alginate powder into 250 ml of water to create sodium alginate solution, Then we dissolved five grams of calcium lactate powder into 1250 ml of water to create a calcium lactate bath. We then gently set a tablespoon of the sodium alginate solution into the calcium lactate bath- allowing the spoon to become completely immersed before tipping it over to pour out the sodium alginate solution.

The main difficulty was estimating when to remove the water orbs from the calcium lactate bath; we came to the conclusion that 20 minutes is the minimum amount of time needed for the membrane to solidify properly whereas leaving the ‘water bottles’ submerged for an hour ensured that the membrane remained flexible but strong.

Avenues for further investigation would be to freeze the sodium alginate into spheres and then submerge the solid spheres into the calcium lactate bath to produce ‘water bottles’ with a more defined shape and to experiment with calcium chloride which is said to produce a stronger membrane. Overall, however, I feel the experiment was fairly successful and we were able to sample numerous ‘water bottles’!

This technique is causing a bit of a stir in manufacturing circles as it could provide a waste-free way to contain and transport water as the membrane is both edible and biodegradable. It is still a long way from replacing the water bottle on store shelves, however, as the gel membrane breaks down over time and isn’t as tough as plastic.

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This blog post is based on the course content I created for a series of sessions on the biochemistry of water as part of the Exploring Everyday (Bio)Chemistry Society which I run at The Tiffin Girls’ School. The sessions focus on connecting chemistry with everyday products and phenomena to encourage KS3 engagement with science and they have been a great success so far. More blog posts based on this society can be found here.

In The Lab: Synthesising Polymers

In one of the earlier sessions of Exploring Everyday (Bio)Chemistry Society, we discussed the properties of natural polymers (including spider silk). Our focus now moves on to artificial polymers (including rayon and nylon). During our experiment based sessions  of this term, we replicated one of the earliest methods of synthesising rayon (a semi-synthetic fibre designed 125 years ago by the English chemist, Charles Frederick Cross).

First, a solution of tetra-amine-copper(II) ions must be prepared; I measured out 10 grams of basic copper carbonate and, working under a fume hood, added 100 cm³ of 880 ammonia solution. Despite operating in one of the best-ventilated labs, the choking scent of ammonia wrapped its tendrils around my airways almost immediately- suffocating me. I can safely say that the outcome of this experiment was still definitely worth it!

 

 

 

 

 

 

[Carefully measuring out the copper carbonate and dissolving it in ammonia to form a solution of tetra-amine-copper ions]

Next, I added 1.5 grams of finely shredded cotton wool to the solution. The cotton wool provides the cellulose (a natural reactant) for the reaction- which is why rayon is only a semi-synthetic polymer.

 

When cotton is dissolved in this solution, the resulting mixture becomes surprisingly viscous (with an almost shower gel-like consistency).

This initially presented a slight problem in achieving complete dissolution- a problem which was solved with the help of an exciting device I’d never had the chance to use before: a magnetic stirrer.

A magnetic stirrer consists of a small bar magnet which is dropped into the solution and a device which creates a rotating magnetic field – forcing the bar magnet to rotate and, hence, stir the solution.

The resulting effect is that the solution appears to stir itself – much to the shock and delight of some of the younger attendees of the session!

 

 

 

The final step is to simply inject a thin stream of the deep blue solution under the surface of some sulfuric acid.  As the solution enters the acid, its pH drops swiftly and the complex compound is converted into insoluble rayon.

 

Although the rayon initially appears deep blue in colour, as the copper (II) ions diffuse out of the rayon (reacting with the sulfuric acid to form copper sulfate solution), the colour quickly fades away- leaving behind only the delicate, translucent fibres.

              

 

 

Keep Calm and Curry On

If you’ve ever had the pleasure of experimenting in the kitchen and making curries, you are probably already familiar with turmeric – you might even have a jar of the golden spice hiding in your kitchen cupboards right now. Although not usually the first substance to come to mind when thinking about chemistry, turmeric (and its active agent curcumin) have some truly fascinating chemical properties. 

As with all spices, turmeric is a mix of chemical compounds, but its instantly recognisable bright yellow colour is due to the presence of a particular compound: curcumin. 

Curcumin is a polyphenol (an organic compound with multiple phenol units).  The phenol units and aromatic structures of curcurim mean that the electrons in the curcumin molecules can absorb (and, hence, gain energy from) UV light. The energy gained promotes them to a higher energy level (an ‘excited state’). This is the same excitation that occurs during flame tests of metal ions. This process doesn’t last long though; they quickly lose some of this energy as vibrational energy, before dropping back down to their original energy level, emitting their excess energy as visible light. This means that curcumin can fluoresce!

In order to demonstrate this amazing ability of curcumin, I swirled turmeric into a beaker of ethanol (as the spice is more soluble in organic solvents) and viewed the solution under a blacklight- the results were almost mesmerising.

Another exciting property of curcumin is its ability to act as a chemical pH indicator (like universal indicator solution or the natural compound anthocyanin). Curcumin changes colour in alkali solutions because of a subtle change in its chemical structure.

Curcumin’s chemical structure contains a sequence of alternating single and double bonds between atoms, what chemists refer to as ‘conjugation’. The length of this sequence of alternating bond types affects the wavelengths of light that the compound absorbs. In curcumin’s case, when it’s added to an alkaline solution, a subtle change in structure alters the length of the conjugated part of the molecule, in turn altering the wavelengths of light that it absorbs. This causes the colour change from yellow to red.

 

I also demonstrated this property by adding turmeric to a beaker of dilute HCl and a beaker of dilute KOH. In a more informal (kitchen) setting, this experiment could also be replicated with lemon juice (which is weakly acidic) and bleach.

Finally, much to the delight of the attendees, we created pH indicator paper from the turmeric-ethanol solution and filter paper; the homemade indicator paper works within seconds, quickly providing answers to some of life’s greatest mysteries such as what is the pH of coffee, seawater, and even saliva!

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Thank you very much to Compound Interest for the brilliant article and presentation which this session of Exploring Everyday Bio(Chemistry) was based on.

Check out their video (with more details of the experiment and great animations) below.

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This blog post is based on the course content I created for a series of sessions on the chemistry of spices as part of the Exploring Everyday (Bio)Chemistry Society which I run at The Tiffin Girls’ School. The sessions focus on connecting chemistry with everyday products and phenomena to encourage KS3 engagement with science and they have been a great success so far. More blog posts based on this society can be found here.

 

Wake Up And Smell The Solvents

A thick blanket of petroleum ether fumes had descended upon the lab- interspersed with the acrid scent were traces of delicate citrus notes. This was the surprising result of my attempt to extract essential oil from oranges via expression and solvent extraction.  Despite producing an essential oil that didn’t quite resemble the uplifting citrusy scent of oranges, I left the lab with a smile that stretched for miles – ecstatic about having designed and carried out a complete experiment entirely single-handedly as part of an exciting club I have recently set up at school.

The idea of organising a club had been slowly growing in my mind since the start of the summer holidays. Having just finished all my GCSE exams, it was almost expected that I would want to completely avoid any curricular activities; however, I remained passionately interested in the subjects I chose to study at Sixth Form and started really looking forward to studying A-Level Chemistry in particular. Curious as to what the A-Level course would be like, I came across the FutureLearn course on ‘Exploring Everyday Chemistry’ run by the University of York. In six weeks, I was exposed to the fascinating organic chemistry and biochemistry that underpinned so many commonplace products and concepts- ranging from fragrances to beverages.

https://www.futurelearn.com/courses/everyday-chemistry

 

My mind started brimming with terms such as ‘limonene’, ‘enantiomers’, and ‘vibrational bond energy’. I decided on modifying the content of the course to start up our school’s very own Exploring Everyday Chemistry Society; the concept of connecting chemistry with everyday products is a brilliant one that would encourage engagement with science (particularly for younger pupils in KS3 and 4).

What makes this proposed club unique is that, each half-term, I planned on carrying out and leading experiments to enhance the course content and help younger students (and myself) develop vital practical skills.

Which brings us to this afternoon; I had the wonderful experience of demonstrating the extraction limonene essential oil from orange peel to Year 9 student.

 

First, working over a 250 cm3 beaker, we used the grater to remove only the outer, coloured rind from the oranges and scraped the material from the grater into the beaker with a spatula.

Next, the gratings were gathered into the garlic press and squeezed into a conical flask through a strainer.

 

 

20 cm3 of distilled water was then added to the flask and the liquid was transferred to a separating funnel. We then carefully added 10 cm3 of solvent (petroleum ether) and shook the separating funnel before leaving the emulsion to separate out into two layers.

The bottom layer (mostly water and debris) was run off and discarded and the top layer (consisting of petroleum ether and dissolved essential oil) was transferred into a water bath to evaporate off the petroleum.

 

 

Due to time limitations and using excess petroleum ether, the final collected essential oil still contained a slight amount of solvent.

I did find that allowing the liquid to evaporate at room temperature reduced the intensity of the petrol vapours. However, when I repeat this experiment with the Year 10 students next week- I’ll ensure to use less solvent and boil off the solvent thoroughly.

 

Overall, this experiment is a great success to me and has been an experience I enjoyed tremendously – we produced 16 little vials of essential oil to take home and treasure.

(I still wouldn’t use them as perfume though…)

Dissection : A Tour of the Eye

The eye has been beautifully and carefully designed for its function; this post will explore the anatomy of the eye (accompanied with images from a dissection).

Picture this: sitting before you on the lab worktop is an eye which gazes into yours as you stare with fascination. You pick it up (gingerly since this is your first dissection and you’re accustomed to seeing eyes in the context of a human face) and notice that it is cool to touch, chilling your fingers through the nitrile gloves.

The human eye is around 24 mm in diameter and roughly spherical, slightly reminding you of a marble. Observe the front of the eye where the cornea, sclera, and fatty tissue can be identified. Turning the eye reveals the extrinsic muscle bundles (a human eye would have 6, however, since you are dissecting a sheep eye, you can only see 4) and the optic nerve.

Place the eye in a dissection pan and rotate it so that the cornea is pointing to the left and the optic nerve is on your right. Using a mounting needle, create a small puncture in the sclera midway between the cornea and the optic nerve – with the puncture as a starting point, you can now use the dissecting scissors to cut the eye into two hemispheres.

As you do so, you notice just how tough the sclera is. You also feel a strange fluid oozing out of the incision and trickling between your fingers; this is the vitreous humour. Along with the aqueous humour (found behind the cornea) it helps maintain the shape of the eye.

Now that you have separated the two hemispheres, you can easily identify the iridescent tapetum lucidum, the dark choroid, and the transparent retina which line the posterior of the eye – with forceps, try peeling away these layers for closer observation.

The retina is a thin layer of tissue that is responsible for converting light into neural signals for visual recognition by the brain. It has layers of photoreceptor cells that detect the colour and intensity of the light that hits them. The choroid contains an extensive network of blood vessels to support the retina. Its dark colour absorbs light so that it is not reflected around the eye.

The bluish, glittering tapetum lucidum (not present in the human eye) reflects light on to the retina and helps with night vision as it can reflect light even at very low intensities. Cats are famously known for the way their eyes shine eerily in the dark; they, too, have a tapetum lucidum layer which helps them detect prey even on the darkest of nights.

Posterior hemisphere of eye: retina, choroid, tapetum lucidum & sclera

The front hemisphere of the eye contains the lens, cilliary body, and suspensory ligaments. You carefully pry the lens from the eye.

You are initially slightly surprised by the fact that the lens is cloudy; this cloudiness is a cataract and is normal in ageing and dead specimens. In a healthy and alive creature, the lens is completely transparent. The lens’ function is to focus light onto the retina and (with the help of the cilliary body) can change its surface and shape to adapt to seeing object that are very near or very far. Gently place the lens on some newspaper and you can directly observe its magnifying capabilities.

 

Now that the lens is removed, an opening reveals itself: the pupil. Found in the centre of the iris, the pupil allows light to enter the eye and can change shape to suit the environment (with the help of two muscle layers of the iris). In the dark, the pupil dilates to maximise the light entering the eye. In intense light, however, the pupil constricts to prevent damage to the delicate photosensitive cells of the retina.

Front hemisphere of eye: iris, pupil, cornea & sclera (lens removed)

In humans, this variation in pupil size often goes unnoticed, but gaze into the eyes of the cat and you cannot possibly miss this protective mechanism of the iris. Cats’ pupils are roundest at daybreak and sunset to maximise vision at these fairly low light levels yet they reduce to a needle-like slit at midday.

This concludes your tour of the eye; you gently place the remnants of the sheep’s eye back onto the dissection pan.

 

I do hope this virtual dissection has helped you to gain a newfound admiration for this extremely well-designed organ.

Dissection : A Tour of the Brain

The human brain, despite its vast complexities, has a relatively straightforward architecture. This post will explore the structure of one of the most important organs of the body.

Imagine that you are looking at a human brain sitting on the lab worktop before you.

At first sight, the brain appears to consist entirely of a large mass of spongy tissue. These deeply folded outer layers of the brain make up the cerebral cortex and take up nearly two thirds of the entire volume of the brain.

Trace the seemingly erratic pattern of curved  grooves on the surface (the sulci) and you will be able to identify the major divisions of the cerebral cortex. Divided into two hemispheres (which are bridged by a bundle of fibres called the corpus callosum), the cerebral cortex houses the majority of the ‘grey matter’ and is separated into a few important lobes.

The first lobes you notice are the frontal lobes. They are aptly named considering that they are found at the front of the brain (near the forehead). The lobes (one in each hemisphere) are responsible for decision making, planning, memory, voluntary action, and even personality. Next, you spot the parietal lobes which are located at the crown of the head. These lobes are heavily involved in perception and interpretation of all sensory information. They are also necessary for spatial awareness and attention. Below the parietal lobes lie the occipital lobes. Found at the back of the brain, the occipital lobes are mainly involved in vision. Damage to the occipital lobes often leads to blindness as well as other vision-related defects.

Now imagine picking up the brain (whilst wearing latex gloves- naturally). Raise it above eye level and you will see the prominent brain stem emerging from the base of the brain. In vivo, this would be connected to the spinal cord- linking the brain to the body. Gently place the brain back down to observe the cerebellum.

Our last stop on this tour is the cerebellum. Nestled just behind the brain stem, the cauliflower-shaped brain structure plays an important role in not only movement, fine motor control, and posture but also in memory, mood, and language processing as well as Pavlovian learning.

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This is post is part of the Neuroscience Crash Course and Dissection Series and is  supplementary to the more detailed tour . I also recommend exploring the interactive 3D model of the brain (powered by the Wellcome Trust).

 

 

 

 

Work Experience: Centre for Liver Research, University of Birmingham

Over the course of the two weeks of my work experience, I had the opportunity to try out new laboratory techniques and to explore various aspects of the research being done at the University of Birmingham, Centre for Liver Research. In this account I will focus solely on the major highlights (despite each day having been amazing and enriching in its own right).

On the first day, I was greeted warmly by Dr Zania Stamataki and given a tour of the Medical School building and the IBR building. We discussed about the research being done by her team concerning hepatocytes and their interaction with the immune system (mainly CD4+ T-cells). Having completed a FutureLearn course on the liver (Liver Disease: Looking after Your Liver – University of Birmingham) provided me with a decent background to understanding the fascinating concepts Zania introduced me to. I learnt the basics of cell-in-cell structures and the vital efferocytosis that hepatocytes perform and what these processes mean for inflammation and the development of cancer and metastasis.

In the afternoon, Janine Fear lead an induction in which I was exposed to the many fascinating laboratories and ‘behind-the-scene’ areas such as the waste disposal and incineration systems, the autoclave room, and the liquid nitrogen cooling systems. One of the many surprising things I was shown was the autoclave tape – I couldn’t help but marvel at the simple yet ingenious concept of tape that changes colour when exposed to the high temperatures of the sterilisation process. Janine Fear also informed me of the lab safety procedures and that the labs I would be working in are classed as Category 2 and that the majority of the labs are fitted with Class II biosafety cabinets. As part of the induction, Janine also taught me basic skills (such as how to use a micropipette and a pipette gun) which I later utilised when performing experiments.

After the induction, I was introduced to Adam McGuinness (a PhD student) who showed me several culture flasks (containing hepatocytes and T-cells) under a light microscope. He also explained that the nutrient medium provided to the cells contains an indicator which changes to yellow as the solution becomes acidic (because CO2 is produced as the cells metabolise) – indicating that the medium should be changed. I recalled that a similar process was used by the BactAlert machines in the serology lab in Mumbai (where I shadowed Dr Salunke last year); the machine detected low pH of blood cultures (which indicates sepsis: bacteria in the blood produce CO2 – making the blood acidic). The hepatocytes cultured in this lab were from a cancer cell line (Huh7) – I learnt that cell lines are ideal for research as they can survive and replicate better than primary cells in vitro.

On day two, Zania and I discussed the role of a researcher and the many facets of the job (including the process of writing and revising publications and some aspects of what makes for a high-impact publication worthy of a renowned journal such as Cell or Nature). Our discussion then drifted back to Zania’s current research and she explained to me that the host cells of cell-in-cell structures can fail to divide as the cell inside them becomes a physical obstruction to cytokinesis. Fascinated by this concept, I spent the rest of the morning reading publications* on cell-in-cell structures which Zania recommended to me (including one written by her former PhD student, Alex Wilkinson).

In the afternoon, I observed Adam performing an experiment to image a host cell hepatocyte failing to divide due to a T-cell inside it. He explained the various fluorescent cell tracker dyes and cytoplasm stains which would be used to image the experiment. We then visited Dr Omar Qureshi who introduced us to the impressive Celldiscoverer 7 (ZEISS) capable of fluorescence microscopy and performing time-lapse imaging.

The next day,  imaging experts from ZEISS explained the process of analysing the images produced by the Celldiscoverer 7; the analysis software can be configured to automatically count the number of cell-in-cell structures and identify any host cells which failed to divide.

Afterwards, I followed Adam to a meeting with the rest of the students who are taking the same PhD course (Sci-Phy). One of the major advantages of shadowing both a student and a PhD supervisor was that I gained exposure to the many aspects of completing a PhD course (such as writing a thesis and preparing for a viva) from different perspectives. During this meeting, Adam presented his plans for a mini-project which involved using machine learning to identify and quantify cell-in-cell structures.

In the afternoon, I attended an interesting lecture (presented by Alejandra Tomas from Imperial College) with Zania regarding current research into improving pancreatic beta cell function.

On the fourth day, I had the very exciting chance to do some ‘hands-on’ work; under the direction of Zania, I ‘split’ a cell culture of hepatocytes. Since the hepatocytes are from a cancer cell line, they can be repeatedly ‘split’ to allow them to proliferate and be used for various experiments. It was delightful to have this opportunity which enabled me to sharpen my skills in handling pipettes and working with the hepatocytes. I also learnt how to care for the surfaces and machines in the lab; I cleaned all the worktops in the labs with 70% IMS (Industrial Methylated Spirit) and Trigene before commencing my work, which ensures that the surfaces are sterile.

The afternoon provided another opportunity to immerse myself further in the topic of cell-in-cell structures and I read two absorbing publications: ‘Cell-in-cell phenomena, cannibalism, and autophagy’ and ‘Entosis enables a population response to starvation’. I found the latter article intriguing as it introduced the idea of host cells obtaining nutrients and biomass through phagocytosis of neighbouring cells as part of a coordinated response to long-term glucose starvation.

On day five, I observed Adam perform another experiment which explored the relationship between the number of necrotic T-cells provided to hepatocytes and the number of phagocytosis events (in which cell-in-cell structures are formed). The experiment used serial dilutions of necrotic T-cells; hepatocytes can engulf dead T-cells easily (even in low quantities).  Following this, we imaged the experiment using the smaller, yet equally impressive, CX5 fluorescence microscope.

In the afternoon, we analysed the images from this experiment to yield averages for the number of cell-in-cell structures observed with the different concentrations of T-cells. I performed some manual counts with the help of an image editing software (Image J) whilst Adam began to use Matlab (a programming language) to automate this process.

On the first day of my second week with the University of Birmingham, I attended a conference with Zania (who chaired the third session) on stromal microenvironments in health and disease. A variety of speakers from the University of Birmingham (and a plenary speaker from Barts Cancer Institute) presented their research on a vast range of topics connected to stromal cells (cells that make up the supporting tissue of organs) from potential cures for rheumatoid arthritis (presented by Andrew Filer) to identifying indicators of myeloma and hallmarks of cancer (presented by Dan Tenant). These presentations helped to further enhance my understanding of the incredible research undertaken at the University of Birmingham.

Two days later, I had the pleasure of meeting visiting Professor Lucie Peduto from the Pasteur Institute. Her research lab has a strong focus on stroma which relates to the liver stromal microenvironment researched here. In the afternoon, I attended her seminar on the relationship between stromal cells, fibrosis, and cancer; having attended the conference on stromal microenvironments earlier that week allowed me to gain a better understanding of her research.

The penultimate two days of my work experience were by far my favourite; I had the exciting opportunity to perform an entire experiment single-handedly. My detailed observation of the experiments performed by Adam proved very useful as I performed the same experiment he did on day five (which explored the relationship between the number of necrotic T-cells provided to hepatocytes and the number of phagocytosis events).  It felt like a culmination of all my training and learning from the past two weeks and I utilised all the skills I had learnt by shadowing and observing Adam performing experiments. In the morning of my ninth day at the University of Birmingham, I observed the cells I had split and cultured the previous Thursday and was pleasantly surprised to find that they were thriving and ready to use for the experiment. Completing this experiment helped me to develop my organisational skills as I carefully followed the protocol Adam had created and balanced different tasks simultaneously.

Overall, my work experience at the University of Birmingham has been a fantastic experience and I am extremely grateful to Dr Zania Stamataki and Adam McGuinness for teaching and supporting me throughout my time here.

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* I have provided the list of publications I had the pleasure of reading in case you would also like to immerse yourself into this fascinating field:

• Cell-in-cell phenomena, cannibalism, and autophagy: is there a relationship?
• The cell biology of cell-in-cell structures
• Entosis enables a population response to starvation
• Mechanisms of ploidy increase in human cancers: a new role for cell cannibalism
• To stay young, kill zombie cells

Gram Staining

Whilst observing Sunflower Laboratory, I got the exciting chance to Gram Stain several slides. I have created a little tutorial in which I carry out the procedure (Youtube link)