DNA in different cells, preventing autolysis and foetal cells in vaccine production

Continuing on the theme from my last post, here's a selection of my answers to recent questions that I saw on Reddit which I thought were interesting.

Is every cell [in our body] carrying the same DNA?

"The standard answer is usually yes, apart from...

• Mutations are the most obvious differences between cells, which usually comes up when this question gets asked. This happens in non- and pre-cancerous cells, but as genetic instability is a common property of cancer it tends to be much worse in cancer cells. It's also worth remembering that doesn't just mean the wrong base of DNA at a position, but can include insertions, deletions and duplications, not just of a base or two but potentially up to whole or huge chunks of chromosomes, even fusion between different chromosomes (which can make fusion proteins with novel functions). This is why you sometimes see aneuploidy (an atypical number of chromosomes) in some cancer cells.

• Gametes (i.e. sperm and egg cells, and their precursors). These germline cells aren't always grouped in when people ask this question, but they are definitely 'in our body' so I am! Not only are these cells haploid (only containing one copy of each chromosome) but during meiosis (the kind of cell division that produces them) the chromosomes undergo recombination, so the pairs of each chromosome will swap bits; this means that the gametes you produce won't have the same versions of the chromosomes that you inherited from your parents, but something in between. This helps keep our gene pool diversified.

• My particular favourite, as it's what I work on - adaptive immune cells. There are potentially infinite different kinds of viruses, bacteria and fungi etc which could infect us and do us harm, which we need to protect ourselves against. This is pretty hard to do with a finite, static genome, as the pathogens could quickly evolve around it. What we evolved is a branch of immunity - our adaptive immunity - which anticipates this huge diversity of infectious agents and responds in kind, by pre-emptively shuffling bits of DNA around to make millions of different receptors, to try to recognise as many different (non-self) things as possible. This happens in developing B-cells and T-cells, which is used to make B-cell receptors (which when released in a soluble form become antibodies) and T-cell receptors (BCRs and TCRs). This is acheived through a process called VDJ recombination, named after the segments of DNA which get recombined together to form a new gene. This provides the basis for how our immune systems learn - if you get infected with something that a particular TCR can bind say, that T-cell will divide and differentiate, which means that the next time you get infected with it those T-cells are already in place, waiting to go and fight it off.

• Microchimerism. In biology a chimera is an organism that contains cells from more than one zygote (fertilised egg). This happens in labs lot for various reasons (which is why you get mice like the one on the right, made up of cells from black-furred and white-furred mice zygotes), but it also happens naturally at some rate (with only a few cells making it microchimerism). The most common example we know of (at least for us placental mammals) is foetal chimerism, where cells from a developing foetus pass through the placenta and establish themselves - sometimes permanently - in the mother (which may help prevent her immune system rejecting the foetus). There are case reports where cells can go the other way (so say cells from the mother can be detected in the circulation of her child), although I think this mostly occurs when one or other seems to have some genetic condition. There also of course exist actual human chimeras - anyone who's ever had an organ or bone marrow transplant will have a large number of cells in which the DNA will be very different (although hopefully not at the MHC alleles, which mediate rejection), as it all came from the donor."

Why aren't cytoplasmic granules of natural killer cells degraded by the potent enzymes that they contain?

"There's a number of different mechanisms by which cytotoxicity is controlled. There's also some disagreement, and a lot we still don't know (which is always a good sign that someone's asking a good question!).

In terms of perforin specifically, there's two factors that come to mind that are probably the best answers to question. Note that they also all largely apply to cytotoxic T-cells as well as NK cells, as they use the same basic cytolytic machinery:

• Perforin requires calcium to form pores and insert to membranes, at concentrations that are typically only found outside (and not inside) cells. This means that the perforin should only work once its been secreted across an immune synapse.

• It also requires a pH above ~6, in order to adopt the correct conformation. Stored lytic granules are pretty acidic, which helps maintain the contents inactive (unless they are release and the acid is diluted out)

These points are covered pretty well in this detailed review, if you're interested. There are a number of other possibilities - like there may exist certain chaperones or regulatory proteins which help keep the perforin inactive, or that cleavable post-translational modifications may help keep in an inactive form. That latter one was quite notable, although it seems opinion has moved towards the glycosylation actually helping guide perforin along through the ER quickly during synthesis, to stop it lingering in calcium-rich/pH neutral compartments where it might do some damage."

[How is the HepA vaccine ethical when it uses MRC-5 cells?]
(NB: I think that this was probably just an anti-vaxxer account trying to colour people's views against vaccines given that it's the sole post from this user in a sub that gets high traffic from anti-vaccine proponents, but it is a valid question that a quick google doesn't produce many good answers for, so I thought it was worth addressing.)

"I'm presuming the ethical problem you're having is that some people have here is that the vaccine production involves MRC-5 cells, which are derived from an abortus foetus?

First off it's worth correcting one thing - the vaccine won't actually contain MRC-5 cells - it just uses the cells to grow the virus, which will then be inactivated to make the vaccine. Remember that viruses cannot grow on their own, they need to use cells to do so, so it's impossible to make an inactivated viral vaccine without cells. (It's also mostly impossible to make protein-subunit vaccines without cells, although you can use non-mammalian cells like bacteria or yeast in that case.)

However if your issue is with the fact that foetal cells were used at all, that's slightly trickier, and your interpretation of the facts may change depending on your viewpoint.

My view point is that early stage embryos are not sentient, and certainly not sapient, and aren't really 'people' as such (there's actually some great discussion on this in a thread that came up earlier in /r/biology today, which deals with this topic very well). MRC-5 cells came from a 14-week old foetus that was aborted for psychiatric reasons, well before the demonstrably concious stage of development.

Another way to look at it is like organ donation. If a baby died (for whatever reason), would you think it was unethical to transplant any organs from that child to others to save their lives? Despite a tragic thing happening, one, two, maybe even three other lives might have been saved or prolonged. If that's acceptable to you, consider that the foetus cells have basically been donated, more than forty years now, to an effort to protect millions of people from a horrible disease. Over 188 million doses of Hep A vaccines have been given; as just under 1% of infected people would be expected to die, a rough estimate would be that Hep A vaccination has probably saved at least 17 million lives (and prevented a great deal of non-fatal yet horrible disease).

(In fairness I'm not sure how many of those doses used MRC-5 derived vaccines, but then these cells are also used in the production of vaccines for other diseases too.)

From a different perspective, even if you don't accept the evidence that foetuses aren't sentient, even if you don't care and think that humanity begins at conception, even if you don't buy the organ/tissue donation analogy, there's a final pragmatic argument: we have the cells, and they work. If we want to stop people contracting, suffering or dying from preventable diseases, we need to use the tools that we have available. Hep A is a nasty disease, and we have a highly safe and effective vaccine - to my mind, advising people to not get the vaccine (in the absence of an equally good alternative) would be a much more unethical alternative."

...but which pen is mightiest?

Being a lab scientist is a funny business in some respects. You end up knowing and caring about some pretty esoteric stuff, like the infinite grades, purities and types of water, or the slight differences in tactile sensation from pipetting different viscosity liquids.

One such matter that likely preys on the average bench scientist's mind more than the global average is the right choice of marker pen for writing very small on tiny plastic tubes. In my particular case, most of the tubes that I use most frequently come from the Eppendorf DNA LoBind range (on account of the problem with using standard polypropylene tubes for working with DNA).

The particular problem in this case is that whatever it is that's added to the plastic to discourage DNA binding seems to make it particularly reluctant to take the ink of a marker well. Given the importance of getting enough information onto the tube, come hell or high water long term frozen storage or spilt ethanol, this can be a problem. However it's one of those problems that's never really that important to solve – you just keep buying the same markers and fudging along as best you may, right?

Well not this time! Part of the joy of starting a new position is that you get to start doing things from the beginning that you wished you'd been doing earlier towards the end of the last position, so that's what I did regarding pens. I ordered in a selection pack, and tested it alongside the pen my lab was currently stocking (fig. 1).

Figure 1: The contenders. From bottom to top: (A) a Sharpie (ultra fine point, retractable); (B) a Securline Marker II/Superfrost; (C) a StatMark Pen (for microscope slides), and (D) a Securline lab marker. Note different sizes is an illusion; photo was taken at an angle.

First things first, let's compare the ink-to-plastic interfaces, that is to say, the nibs (fig. 2). Three of the tips are pretty similar (A-C), being fine point hard tips. Of these three only the Sharpie (A) stands out as it's a clicky retractable tip, which is convenient as there's no lid to lose. The thick tip lab marker from Securline (D) has a bigger, slightly softer chisel tip (much like the VWR markers I used a lot in London, which periodically seem to disappear from the lists).

Figure 2: The nibs of the different markers.

The first test: how well do they actually write? Fig. 3 shows the results of writing the same message on four different LoBind tubes. All three of the fine tips have pretty reasonable contrast, although I think the StatMark may have gone on slightly easier. The chisel tipped Securline produced the thickest yet faintest text.

Figure 3: The results of writing the same test message on four different LoBind tubes.

After writing on the tubes, I wanted to test the ability of the text to stand up to the solvent that's most likely to be a problem in the setting of my work: alcohol. Each tube top received a 15 ul drop of 70% ethanol in the middle, before giving it a couple of firm wipes with a paper towel, in order to model the kinds of exposure a tube might receive say mid-purification. The results are shown in fig. 4, revealing that only the two Securline markers pass the test (which isn't so suprising, given that they are marketed specifically as solvent resistant).

 

Figure 4: The ethanol test. Top panel shows the pre-exposure tube, bottom panel shows post-ethanol. A = Sharpie, B = Securline Superfrost, C = StatMarker, D = Securline lab marker. Note that the top panel was taken about five minutes after the shots in fig. 1 and provides relative contrast in a single frame. Shame about the photo in the second panel.

The last remaining test is the smudge test, as anyone who has had to label 50 different tubes by hand in a hurry can attest that things can get a bit on the messy side. In this test, I simply wrote 'smudge' on the side of the tube (à la Misery) and immediately gave it a quick wipe with a gloved thumb to see how well the ink had set. Fig. 5 reveals that in this test it's the standard Securline lab marker that did best, with the StatMarker coming in second.

Figure 5: Results of the smudge test.

This is by no means a rigorous assessment – it's all incredibly qualitative, the types of tubes tested being one, and there's a complete lack of technical repeats* – but it's certainly the most thorough investigation into lab marker suitability I've done. For what it's worth, these data have informed my labpenmanship in the following ways:

• Due to it's ease of writing, clarity and durable contrast, I'm going to write on the tops of my tubes with the StatMark. This should make them easier to read in a freezer box.
• However, due to it's lack of solvent resistance, I need some backup labelling on the side, which I'll do with the Securline Marker II/Superfrost, as it's decent to write with and should hold up well in the event of rogue wash getting splashed around.
• The other markers still have a place though: the thick tip Securline is perfect for labelling larger, Falcon-style tubes, while the Sharpie is good for annotating the gels in my labbook (which means I can leave the tube-labelling markers in my clean PCR hood and keep everything gloriously separate).

I hope it might be useful for others, and would be interested to know if anyone has had success with other markers, or with these markers on tubes other than the DNA LoBinds.

*Having gone to this effort I briefly toyed with the idea of writing this up as a tongue in piece manuscript, but then I thought of the reviewer comments that even I would give this so I passed

PhD thesis writing advice

When I sat down to write this post, I had an opening line in mind that was going to bemoan me being a bit remiss with my blogging of late. That was before I checked my last post and realised it had been ten months, and 'remiss' feels a bit inadequate: I basically stopped.

I had a pretty good reason, in that I needed to finish my PhD. Those ten months have basically been filled with write paper, submit paper, thesis thesis thesis, do a talk, thesis thesis thesis, get paper rejections, rewrite, resubmit, thesis thesis thesis … then finally viva (and a couple of more paper submissions). It is exhausting, and frankly having either a thesis or a paper to be writing at any one given time takes the shine off blogging somewhat.

Now that it's done and out of the way (no corrections, wahey!), I begin to turn my mind to getting the blog up and running again. What have I been up to lately that I could write about, I asked myself. Well, there's always this big blue beast.

Having been the major task of the last year of my life, I've spent more than a little time thinking about the process of thesis writing, so I thought I'd share some pointers and thoughts, maybe being the usual and the obvious.

1) Everything takes longer than expected.

This is the best advice I got going in, and is always the first thing I say to anyone who asks. In a perfect example of Hofstader's Law, no matter how much time you think a given task should take, in reality it will take longer.

2) Be consistent.

The longer a given document is, the more chance there is that inconsistencies will creep in. Theses are long documents that are invariably only read by accomplished academics (or those in training), many of whom have a keen eye for finding such consistencies. 'n=5' on one line and 'n = 4' on another. 'Fig1: Blah', 'Figure 2 – Blah blah' and 'Fig. 3 Even more blah'. These might not seem like big deals, but added up they can make the document feel less professional. Choice of spelling (e.g. UK or US English), where you put your spaces, how you refer to and describe your figures and references, all of these types of things: it doesn't matter so much how you choose to do them, as long as you do them the same each time.

On a related note, many technical notations – such as writing gene, protein or chemical names – are covered by exhaustive committee-approved nomenclatures. Use them, or justify why you're not using them, but again, be consistent.

3) Make it easy for yourself: get into good habits.

Writing long documents is difficult, so don't make it hard on yourself. Start as you mean to go on, and go on like that – I made a rather foolish decision to change how I formatted my figures one chapter in to my writing, and going back to re-format the old stuff was time I could have been spent writing*. Doing something right the first time is much more efficient than re-doing it two or three times.

Make sure you make full use of reference manager software. This will sound obvious to people that use them, but I am consistently surprised by the number of PhD students I meet who write their bibliographies and citations manually. I personally use Mendeley, which operates perfectly well and has a nice reading interface as well, although there are plenty of others. You are probably going to have a lot (hundreds) of references, and even more citations: doing them manually is a recipe for disaster.

Similarly, don't do any manual cross-referencing if you can avoid it – the document as you write it will likely be entirely fluid and subject to change for months, so any 'hard' references you put in could well end up needing to be changed, which not only takes time but increases the risk of you missing something and carrying an error along to your finished PhD.

If you have the time, I would recommend trying to get into LaTeX (with the 'X' pronounced as a 'K'), which is a free, open-source code-based type-setting program. It's a bit of a steep learning curve, but there are plenty of good templates and once you've got a grasp of the basic commands it's incredibly powerful. Crucially, as your file is just a text document (which effectively just 'links' to pictures when you compile your PDF) it remains small in size, and therefore easy to load, backup and play with. It also makes referencing, cross-referencing, and generally producing beautiful looking text a lot easier then most word processors.

Theses are often full of technical words and abbreviations, and it's entirely likely your examiners won't know them all beforehand – therefore they need to be defined the first time they're used. However, if you're moving chunks of text around (sometimes whole chapters), how do you know which one is the first time? My tactic was to not define anything while writing, but whenever I did use a new phrase I added it to a spreadsheet, along with its definition. Then once everything was set in place I worked through that spreadsheet and used 'find' to add the appropriate definition to the first instance of every term. What's more, that spreadsheet was then easily alphabetised and converted straight into a convenient glossary!

4) Be prepared to get sick of it ...

You will spend an unhealthy amount of time doing one thing: working on this one document. It will bore you, and it will make you boring, as it will take over your time, thoughts and life**. It is basically guaranteed that you will bone-weary of sitting down to your computer and working on it again. It's relentless, it just keeps going on and on and on, to the point where you forget that your life hasn't and will not always just be thesis-writing.

5) ... but remember it will end.

It might not seem like it at the time, but it will. You will finish writing, you will finish checking, you will hand it in. You'll then find some errors, but that's OK, your examiners are never going to read it as closely as you do when you check it. Remember that your supervisor(s)/thesis committees/whoever shouldn't let you submit unless they think you're ready, so the fact you're submitting means you're probably going to be fine!

* For what it's worth, the final way I did my figures was better, I should just have thought about it and done it first. Basically I outputted my plots from Python and R as SVG files and compiled them into whole figures in Inkscape (which is also great for making schematics) and saving these as PDFs. A word of warning thought – certain lines/boxes in occasional Python-saved SVGs failed to print (apparently something to do with the way fancy printers flatten the layers), so it is probably worth keeping backup EPS or non-vector versions of your Inkscape files on hand.

** Look at me, I've just closed my file for the last time (before uploading to university servers) and the first thing I do is go and write a thousand words about it!

Up-goer five PhD description

Someone recently pointed out the Up-Goer Five text editor to me, which only lets you type using the 1,000 top most common words (inspired by this xkcd). The challenge is then to describe your PhD project using just these words. Here's my try:

I study a type of blood cell that helps keep us safe and well, called 'T_cells'. These cells search the body for signs of problems or things that shouldn't be there, and fix them if they find any. They can look for many different kinds of problems; taken as a group, they can look for far more problem signs than almost any other cell type. They feel the face of other cells as they move around the body, each one looking for different signs that something isn't right. I'm working on checking to see what 'T_cells' people have, to see if we can learn why some people get sick more easily than others.

Max Perutz 2012 entry

Wow, I hadn't realised how long it's been since my last blog post - I blame the copious amount of food I ate over the holidays, and then the giant pile of work I've been doing since.

So, in order to keep the ball rolling, I thought I'd post a bit of science communication I wrote for my entry in last year's Max Perutz writing competition.

I was going to write an entry for the recent Euro-PMC competition, but I only got as far as deciding on the theme of the pun for my title (I was thinking something cheesy like 'Genius, or geneious?').

Anyway, here's the article as I wrote it last May (I didn't win).

---

Unravelling the secrets of our immune system

Sometimes we only realise how important something is when it goes wrong. In the case of adaptive immunity, things going wrong can be fatal.

Adaptive immunity is one of the systems we have evolved to keep infectious germs at bay. It is our intelligent protection system, a biological firewall, where white blood cells patrol our bodies, keeping us safe from disease. Not only does it stop intruders in their tracks, it remembers the threats it's seen before, so it can defend against them faster the next time they attack.

There are two medical conditions in humans that reveal to us how important it is to have a working adaptive immune system. The first occurs when some children are born without working copies of genes that encode important immune molecules. This means they don't make some of the proteins that are required for adaptive immunity to develop.

Alternatively, people can lose their resistance to microbes later in life. This can happen when untreated HIV positive individuals develop AIDS, or in transplant patients who have taken suppressive drugs to prevent organ rejection.

People without a functioning adaptive immune system are compromised, exposed. They are at risk from any stray infection, vulnerable to all manner of viruses, bacteria and fungi. A simple bug that might not even give you a temperature could spell death to them.

This makes it important for us to know how adaptive immunity works. This is what I do in my research; I look at a particular aspect of this system, to try and understand what a healthy adaptive immune system 'looks' like, and how it goes wrong in disease.

In order to explain my work, you have to know a little bit about how our bodies generate this powerful immunity.

Cells don't have eyes or ears, so they have to use receptor molecules on their surface to detect what's going on around them in their environment. These receptors are proteins, the blueprints for which are encoded in the genes in our DNA.

This means white blood cells could have a receptor that recognises a certain bit of a bacteria say, or the fragment of the outside of a virus. If the receptor finds and binds its target, then that cell can tell that the body is infected with a particular parasite.

The problem lies in that there are far more bugs and germs out there that could potentially infect us then there are genes in our genome. How can we have evolved ways to detect and protect against such a barrage of disease with so few genes?

Maturing adaptive immune cells overcome our finite genomes by shuffling pieces of it around, making unique receptor genes out of genetic building blocks that all our cells contain. These cells, called T cells and B cells, physically loop the DNA over itself, and then cut out the chunks in between.

This means different sections can be moved next to each other, recombining to create new genes. Incidentally, this extraordinary feature makes them some of the only cells in the body that don't share the same genome as all the other cells.

Each developing cell shuffles their DNA around independently, stitching different gene segments together in order to produce its own distinctive receptor. As there are many segments to choose from, the number of different combinations is huge.

Moreover, the DNA sequence at the join sites can be randomly altered, meaning the eventual number of possible different receptor genes is truly phenomenal. The fact that we have millions of white blood cells inside us, each bearing one of the trillions of different potential receptors, has historically kept researchers from measuring this diversity.

My project is to use DNA sequencing technology – developed during the Human Genome Project – to read as many of these uniquely generated adaptive immune genes as we can. By doing this we can characterise a person's immune repertoire, seeing how prepared their body is to fight off infection, and perhaps even see what they’ve been infected by in the past. 

The hope is that we can use this technology to understand what it is that makes a healthy human immune system. Once we know this, we can compare this to the compromised or failing immune systems that we see in infection, cancer and autoimmunity, and maybe get an insight into how to treat or avoid these conditions.

Science has brought a lot of relief to those suffering from disease, and has prevented many more from joining them. However, the challenges faced in curing our ills can only be surmounted by learning as much as possible about the way that our bodies work, as well as the diseases that threaten us. Unravelling the secrets of our immune systems is another step towards that goal.