Saturday, 28 January 2017

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."

Thursday, 12 January 2017

Can gut bacteria cause bad breath?

I know that I've been a bit remiss on the blogging lately, for all the usual reasons (busy at work, holidays, writing papers, general life admin etc). However I've noticed that instead I've been answering more technical and scientific questions on Reddit - it scratches the same itch as when I blog, but it's a shorter format that takes less effort, and someone else has thought up the question so I get the pleasure of consider something that I didn't think up in the first place.

I think I might occasionally repost some of the answers I posted there, to here, if only because I sometimes want to refer back to answers I gave and this blog moves (a lot) slower than Reddit.

Today, I just answered the question: can bacteria in the small intestine cause breath to stink?

To which I answered:

"In the case of people with "healthy" gastrointestinal tracts, these sphincters are one of the ways by which we keep the proper direction of flow (of food, which later becomes faeces along with all the associated gases), which in turn (usually) keeps bacteria where they're supposed to be.
The fact that the food that leaves the stomach (in a kind of watery paste called chyme) is still very acidic means it's still relatively inhospitable for bacteria, and has had less time for bacteria to enter/grow, which is why the small intestine ordinarily has much less bacteria (fewer species and fewer cells) than the large intestine (which is where the digesting stuff actually becomes faeces). Peristalsis (the movement of your intenstines that pushes everything along) drives the flow, making sure faeces and flatulence goes out the right hole. This movement is why you can still sometimes fart even when constipated (unless you're really impacted).
All these mechanisms keep the 'lower gases' coming out of the mouth - which is why in the vast majority of cases of bad breath originates from bacteria in the mouth. However IBS and constipation are obviously cases where all the digestive stuff isn't working like normal.
People with severe IBS and/or constipation can sometimes get so backed up (past the valve that you mentioned) that the small intestine gets way more bacteria then normal - this is called small intestine bacterial overgrowth (SIBO). In this situation, gases produced by bacteria in the small intestine can work their way up and out through the mouth (presumably bubbling up through the stomach as its sphincters open and close to let food through/burps out), including hydrogen sulphide, which is one of the classic halitosis related molecules. Checking someone's breath for volatile compounds produced by bacteria is actually one of the major ways by which doctors diagnose this condition.
I should say, I am not a gastroenterologist so I don't know whether these volatiles are frequently present at levels detectable to the human nose, which isn't something that seems to get measured a lot in the scientific literature, however I did find a couple of non-peer-reviewed reports like this that say bad breath can be a symptom of SIBO. In fact, some people even think that SIBO might be a/the cause of IBS (because if you treat it, you can reduce IBS symptoms in some people), but this is contentious and still being worked on.
So to answer your general question - it's plausible, although without testing it's impossible for anyone to say whether it's actually the case for you. Generally maintaining good oral hygiene is the best recipe to prevent bad breath, but in some cases there might be deeper medical issues, so it might be worth seeking the advice of a gastroenterologist."

Thursday, 10 November 2016

Installing Trinity on Mac OS X via homebrew -- update

A couple of months ago I wrote a short post about how I managed to install the RNA assembler Trinity on Mac OS X (El Capitan), on the off-chance it would be useful to someone else.

This morning I received an email from my friend Mazlina, who I worked with in London, saying she had been trying to do just that and had coincidentally stumbled on my post*. However it hadn't worked out quite as easily for her as it had for me.

It turned out to be due to a Java version problem. While 1.8 was installed, brew --config claimed that only 1.6 was, which is insufficient for Trinity installation.

Here's how she solved it, quoting from her email:

"First, I did
brew doctor
and just cleared up whatever it told me to [...]

$ brew doctor
Your system is ready to brew.

So by running
brew cask search java
it lists down the available java versions (the one you have, 8, is just java), and I went with 9-beta because that was the only one I could download at that time. And when I ran 

$ brew cask install java9-beta
$ brew install trinity
worked like a charm.

Not sure if that's exciting enough to go on the blog, but it solves it anyway."

(For reference, as I told her, given the average excitement level of the blog this should fit right in!)

* I'm never really sure whether these posts are read or not (as the stats from Blogger are always inflated by scanning bots) apart from when people let me know they've seen them - so if you ever bump in to me at a conference or something and have found one of them useful or interesting please let me know, I love to hear about it!

Friday, 4 November 2016

Cheap protocol for DNA extraction from agarose

I've just stumbled across this lovely paper from Sun et al., which reports a delightfully cheap and simple technique for extracting DNA from agarose gel slices.

Basically it's straightforward as poking a hole in the bottom of a .5 ml microcentrifuge tube, then nesting that inside a 1.5 ml tube. Some cotton or glass wool then goes in the bottom of the .5 ml tube, with your agarose slice containing your excised DNA band on top, and you just spin it through;  apparently the agarose gets retained on the wool and the aqueous phase gets spun to the lower compartment.

DNA gel extraction kits aren't the most expensive thing you're likely to buy (at about £1.50/$2 per tube), but if you do them occasionally it might be worth trying it out for the time saving: if you are doing lots regularly, it might well save a pretty penny.

Saturday, 29 October 2016

...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

Thursday, 20 October 2016

Freezer box tube storage templates

As I settle in to my new postdoc position, in a relatively newly established lab,  I've been setting up my lab management techniques.

One of the things that's always bothered me is the best way to record what tubes are in which box in the freezer. On one hand, a straight up list is most convenient for typing and copying, while on the other a table showing what's in which position is more intuitive and convenient for printing.

In my previous labs I've always worked with existing templates, or within a particular framework. This time around though it's a reasonably fresh start – and I'm also still in that lag phase where I'm still waiting for most of my reagents to be delivered – so I thought I'd take the opportunity to whip up a nice solution that addresses both issues before I lay down too many tubes.

The fruits of my labour can be downloaded from GitHub

Basically the idea aims to combine the ease of entry of a simple vertical table, with the easy visualisation of a coordinate table system. So you can copy and paste rows of data on the tall table on the left, then the spreadsheet auto-fills in the appropriate cells on the table on the right which can then be selected and printed for pasting into your lab book. 

If you don't like the exact entry fields that I used you can also change the headings of the table on the left. I would however encourage you to use some marker of the appropriate lab book entry, which is something many forms (including my old ones) omit: in an ideal world, given any tube you should be able to find out all it's information (or vice versa), so this information is vital.

There's a 9x9 and a 10x10 format available. If anyone does use it and have any thoughts I'd love to hear about it.