The week in Structures – 8th May 2013 #PDB

Week #2: Trying to jumpstart our immune system to fight off HIV. 

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Fighting the good fight against HIV.

In this weeks PDB releases, we see a couple of HIV-antibody structures, continuing the work of the Kwong and Mascola labs in the NIH. In 2011, these labs released a paper (published in Science) studying various antibodies that recognises and neutralise HIV-1. If you can recall your high school biology classes, antibody recognition of an invading pathogen is the first step in activating the immune system that hopefully results in successfully fighting off the infection. This work has been further developed with papers this year in Cell and Nature - Prolific and important work!

The structure shown below contains the “FAb fragment” (gold & bronze cartoon) of the antibody bound to gp120 (the big blue blob).

Antibody recognition of HIV gp120 – a surface marker of the HIV virus. After PDB # 4JB9

Glycoprotein 120 (gp120) is a component of the HIV surface spikes – which recognise a range of different molecules on the surface of our cells and initiate infection. It is because of the this that gp120 is an attractive vaccine candidate – however, recombinant gp120 has failed deliver results in clinical trials, and it is hoped that by studying antibody-gp120 interactions and perhaps by designing a novel gp120-dervied antigen, an effective HIV vaccine can be developed.

Presumably the authors discuss the relevance of their findings in the paper (not released at the time of writing here) in the context of HIV treatment and rational vaccine design. (PDB codes 4JB9 and 4J6R). (EDIT: Other work in the same edition of Science discuss similar topics, here and an article “Rational HIV Immunogen design to targets specific germline B-cell receptors.”)

Other structures of note this week at the excellently named “star domain of quaking protein in complex with RNA” and some work looking at the mechanism by which angiopoietin 1 and 2 interact with the Tie2 receptor tyrosine kinase ([paper] | [PDB] ), which might lead to therapeutics that prevent tumours growing their own blood supplies (angiogenesis blockers).

On branded drugs.

I went shopping yesterday…

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On the left, a box of 14 generic allergy relief tablets, each containing 10mg Cetirizine HCl and Lactose – costing £1.

On the right, a box of 14 branded allergy relief tablets, each containing 10mg Cetirizine HCl and Lactose – costing £5.67. In a sale. Down from £7.57.

The active ingredients are  identical. The evidence for the efficacy of the active ingredients is identical. So how can companies justify charging 7.5 times more? I understand and acknowledge that effective marketing and other psychological factors might lead to a more effective placebo component of any clinical effect ^[1][2] – but a 7.5 fold increase in effectiveness?

The chemical structure of cetirizine. (Photo credit: Wikipedia)

If we assume that the sellers make some sort of profit on the generic, then someone must be making a huge profit on the branded anti-allergy meds. Which seems a little immoral. If you can offer people relief for 7.1p per day, why charge as much as 54p per day?

Whether or not this pricing is down to the pharmaceutical industry I cannot be sure – but big pharma don’t have the greatest public image at the moment – and examples like this sat on the shelf of your local supermarket perhaps serve as another example of why.

The week in Structures – 1st May 2013 #PDB

The first in a (hopefully) weekly series of reviews of interesting structures in latest PDB release**.

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High-resolution* Cryo-EM models of Human and Drosophilia Ribosomes!

Proteins are the major molecular players in life as we know it – they are the chemists, the engineers, the messengers and the defenders of life. They make new chemical compounds that we need, they break old compounds down so that we might re-use them – they transmit messages between different cells and tissues and help identify invading pathogens. In short – proteins do pretty much everything, and as such are the subject of intense study and scrutiny.

What better subject for the first of my PDB release highlight posts, than the Ribosome – a vast (at least at a molecular level) and ancient molecular machines that are responsible for the synthesis of proteins in our cells. The (perhaps outdated) “central dogma” of biology is that DNA is transcribed to mRNA, which is translated into protein, and the proteins then go and do everything. Anger et al have released 5Å resolution Cryo-EM-derived models of human and drosophila (Fruit Fly) ribosomes – they are so vast that they have to be split across several PDB files each…

Human (3J3A, 3J3D, 3J3B, 3J3F)

Drosophila (3J38, 3J3C, 3J39, 3J3E)

… and so complex as to defy creation of a clear and crisp picture.

The Paper concerning these is due to be published in Nature, but is not available at the time of writing (EDIT – Now it is – here (£)). Presumably, along with the methods  employed in what must be a massive modelling task, there will be some discussion of the differences between these two eukaryotic ribosomes, and the prokaryotic ribosome structures that were the subject of the 2009 Nobel Prize for Chemistry.

Other highlights in this weeks crop of 287 PDB releases include yet more BACE-inhibitor complexes (potential lead compounds for Altzheimer‘s treatments) and CDK2 and CDK8 -inhibitor complexes, which may be lead to novel anti-cancer therapies.

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*ok – 5Å might not be great for crystallography, but for our Cryo-EM-based friends, this is pushing the limits and is clearly the result of a great deal of hard work and a massive amount of particle picking.

** The PDB is the “Protein Data Bank” – all structures of proteins/DNA and related molecules are deposited into the PDB and then made available to everyone (for free) prior to or at the time of publication of the paper that describes the work pertaining to the structure. The PDB issues new releases every Wednesday morning (UK time).

Like science?

Like science and the progress and prosperity it brings the UK? Then sign the pants off this bad boy. http://ow.ly/kyyQ7 #sivpoint8

POP QUIZ – What’s this?

What the hell is this thing?

A little game – What the hell is this thing? Take a guess in the comments below – the red blobs are me deleting labels that might have given it away.

The prize is kudos, the admiration of your peers, and a life time subscription to this blog.

[Journal Club] – “Identification of a central role for complement in osteoarthritis”

In a little experiment, I’m going to do a journal club style blog post. I’m going to look at a paper, explain why I like it, and how I think it could be improved. If you like it, “like it” and I may do more. 

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The paper I am going to look at is “Identification of a central role for complement in osteoarthritis” by Wang et al, published in Nature Medicine in December 2011. [PUBMED|PDF|F1000] I chose this because it is a pretty big paper in my field, and it is a damn good paper (but it is not without flaws).

The paper explores the hypothesis that the complement immune system is involved in the development of osteoarthritis (OA) (a degenerative disease of our joints).

The authors show that the presence of markers of complement activation in samples from (human) patients, such as “C3a des arg” and “C5b-9″ (the membrane attack complex), correlate with osteoarthritis (fig1 b&c).

The membrane attack complex (or MAC) is the final step of the complement immune cascade, and is a complex of proteins that in high levels punches holes in cells and kills them.

They also show that patients with osteoarthritis make more complement activating proteins and less complement inhibiting protein in the synovium of their joints when compared to healthy patients (fig1 e).

Having demonstrated a correlation between complement activation and osteoarthritis in human samples, Wang et al go on to explore this in mouse models of arthritis.

They show that if you knockout the genes for complement components C5 and C6 in mice (preventing the formation of the MAC), these mice develop less severe arthritis (as measured by cartilage degradation) in surgically-induced arthritis (fig2).

The also show that in mice lacking CD59, a natural inhibitor of complement, cartilage degradation is more severe.

These genetic models support the authors’ hypothesis that complement activation might enhance osteoarthritis progression/severity.

•Mice that can’t ACTIVATE MAC have LESS cartilage damage in OA models

•Mice that can’t REGULATE MAC have MORE cartilage damage in OA models

They then show in human chondrocytes (cartilage producing cells)(in cell culture) that extracts of arthritic cartilage or certain proteins (such as fibromodulin) known to be over-produced in arthritis can induce the production of the MAC.

And they show that low (“sub-lytic”) levels of MAC can make normal chondrocytes (but not C5 deficient chondrocytes) produce proteins known to degrade cartilage, such as ADAMTS-4 and -5, but also some Matrix MetalloProteases (MMPs).

Again, this observation supports the hypothesis that complement activation (that culminates in MAC formation) can induce osteoarthritis.

All in all, a very comprehensive paper, and a fantastic piece of work, and I’ve deliberately ignored some parts of the paper to simplify the story.

There are however, a few chinks in the armour.

1) All mice models of arthritis were surgical models – i.e surgery was performed on the mice that caused them to develop arthritis. No “sham-surgery” controls were performed (or if they were, they weren’t mentioned, which seems odd).

Given that it is known that surgery can induce complement activation, this control absolutely should have been done.

Other models of arthritis (such as proteoglycan induced arthritis) were not explored.

2) They should have looked for the products of ADAMTS induced cartilage degradation in the arthritis mice. Antibodies that recognise the breakdown products (so-called ”NITEGE”) are available and could be used in immuno-histochemistry to see if/where ADAMTS-mediated cartilage damage is occurring. (H/T to Jen in our lab who pointed this out).

3) None of the subjective cartilage degradation scoring was blinded. This is a potential source of bias.

I still think that the hypothesis explored in this paper is a good one, and that complement activation is almost certainly part of the complicated puzzle of osteoarthritis pathology, but ideally I’d like to see a follow-up paper that included some of these extra experiments.

[PODCAST] – Me on Dessert Lionel Discs

Damn good coffee, and hot! (Photo credit: photojenni)

Self promotion, dead ahead!

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I have been fortunate enough to have been interviewed by @Astrondrew of  Sound of Science for episode 5 of the Dessert Lionel Discs podcast

In it I talk about how I got into science, who inspired me to do science, and what exactly I do as part of my day job. I also talk Pink Floyd, Crystals, Twin Peaks and damn fine coffee.

Please listen, and I hope you enjoy!

[TALK] Studying Life at a Molecular Level!

Caffeine. One of nature’s many triumphs.

ATTENTION GOOD PEOPLE OF LIVERPOOL!

I am talking about how we understand what we understand about the molecular nature of life!

It will be just like “Wonders of Life” but with less silhouetted  pointing up a mountain, and more pictures of molecules and crystals and me.

The talk is on Tuesday 12th March, 2013 at the Hope Street Hotel, on Hope Street, in Liverpool – doors open at 730pm.

There is a facebook event page here. See you there.

Definition: “Sequence Gazing”

Sequence gazing (vb):

A technique similar to meditation, often practised by molecular biologists and biochemists. To gaze intently at amino acid and DNA sequences, and multiple alignments thereof, until either “the answer” or a testable hypothesis presents itself.

On not getting the joke…

A quick rant about some negative responses to #overlyhonestmethods

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For those not on Twitter, #overlyhonestmethods was a twitter hashtag, instigated by @Dr_Leigh, in which Scientists posted snippets from well, Overly honest methods sections.

IMO, it was mostly very funny, and the response was very positive – showing that scientists have a sense of humour whilst de-mystifying and humanising a profession which is occasionally seen by some as stuffy and aloof.

However, as someone who has occasionally tangled with those that might be considered as anti-science, it was quite clear to me that those with such an anti-science agenda might seek to use #overlyhonestmethods to have a pop at science.

If you scan #overlyhonestmethods, you’ll find them.

To those people:

1. Not every experiment ends up in a paper. I would consider myself very efficient if 20% of mine did, although, I suspect this figure might vary massively from discipline to discipline. Personally, I do both “hypothesis testing” experiments, where I test a specific question, in what might be considered the traditional scientific method, and “what if” experiments, where I just test something out, play a hunch, follow a lead from another experiment, etc. Both approaches are equally valid, but it goes without saying that positive “what if” experiments invariably lead to more focused “hypothesis testing” experiments as ideas and hypotheses develop.
2. Never under-estimate the role that serendipity plays in science. In one of my papers, the straw that broke the camel’s back was someone forgetting to add a particular reagent to an assay. Turns out that this reagent was inhibiting the enzyme in question, and this was a physiologically relevant discovery. If you fail to do an experiment “by the book” it is often worth completing it just to see “what happens if…” It is also worth occasionally deviating from the published protocols as well, to ask “what if…” Will I get better results or a more obvious outcome if I add more X, or do the incubation at a different temperature, etc etc.
3. Experiments are repeated. Lots. Generally speaking, experiments are repeated at least 9 times (3x technical replicates, 3x experimental replicates) to ensure that the result seen is not happening by fluke. In reality n numbers are often greater than this. Ignoring duff experiments where something went wrong is annoying but no real hard-ship.
4. IT IS A JOKE. Everyone likes to have a laugh at work. Yes, even stuffy old, lab coat-wearing, mad-haired scientists. If you’ve not put a dry-ice pellet in an Eppendorf in someone’s lab coat pocket without them realising, and then waited for the nature to take its course, then you’ve not lived.

It really is a shame that people will use something which has received overwhelmingly positive feedback, and has been hailed as an excellent piece of spontaneous and anarchic scientific communication, to have a go at science. In conclusion, it is becoming clear that no matter how obvious it is that something is a joke, you will always find someone poe-faced enough to not get it – such is the rich tapestry of humanity.