Sulfate or Phosphate?

Some months before @robField’s tweet setting off the train that led to the Eu sensor that discriminates PAP/PAPS, Ed Yates and myself were having a curry with Dulce Papy-Garcia from UPEC, who had examined one of our PhD students. A matter we discussed at length was ‘why sulfate’. That is, why does biology use both sulfate and phosphate to modify post synthesis proteins, polysaccharides and other molecules. We didn’t come up with an answer,  but the conversation led Ed and myself to consider that the question merited exploration. 

This we thought would be a simple matter. 

It turned out to be one of the most difficult papers Ed and myself have written, to the extent that after N drafts (where N is a significantly larger number than either of us had experienced in any previous writing exercise) and too many summers we still had nothing satisfactory. So, we cunningly inveigled two colleagues, Tim Rudd from NIBSC and Marcelo Lima from Keele to join us on what we advertised as the sunny beach of sulfate and phosphate, but which in reality was a rather dank quagmire. There is though something about strength in numbers, and with very helpful input from Steve Butler in Loughborough, we arrived at what we considered a satisfactory synthesis. Happily, the reviewers concurred, and the paper is now published at Royal Society Interfaces, “Phosphorylation and sulfation share a common biosynthetic pathway, but extend biochemical and evolutionary diversity of biological macromolecules in distinct ways”.

This is by no means the last word on the matter, but along with some previous thoughtful papers we cite (if we have missed one, please let me know) it provides some ideas that may help us to understand why biology co-opted particular elements from the inorganic world to perform groups of functions vital to life as we know it now.

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Real-time sulfotransferase assay

More sulfation

Earlier this year Simon Wheeler (who now has a well deserved substantive position, congratulations!) and Steve Butler published the first output from the BBSRC TDRI awarded to Steve, with myself and Ed Yates in supporting roles. It is always nice to collaborate with real chemists, as it reminds me I am very much a pseudo chemist, and I learn a lot. After what I would consider a quite heroic effort on the synthesis front, Simon and Steve pulled out a very useful sensor, based on a europium complex. The Eu sensor has good selectivity for PAP over PAPS, the universal sulfate donor. The assay works well and is very amenable to high throughput 384 well format assays (= more papers on the way). So we can now measure sulfotransferase activity in realt-ime independently of the acceptor for pretty much any enzyme-substrate combination. This represents an important tool for the wider sulfotransferase community. 

The paper also demonstrates the importance of social media in science, as a means to access in a non-direct manner new information that sets off an innovative project. I saw tweet from @Fieldlab highlighting a paper from Steve’s lab on lanthanide sensors able to discriminate nucleotide phosphates and read the paper. Naively I thought PAP/PAPS sensing using such compounds should be easy, so I contacted Steve. After some preliminary tests with PAP and PAPS on his side, we wrote the grant – another lesson here, as the application neared final from I went over to Loughborough for a meeting, which allowed us to iron out a few problems far more effectively than by electronic communication. The work was, as hinted above, far from straightforward, but like everything that is new, very rewarding and continues to be so.

I have just moved from the bird site to the proboscidean one and things look like there will be even more of such ‘random access’ of information there, so let’s see what turns up!

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An Open Letter to my Representatives

I have written the following to my Brexit Party Representatives in the European Parliament. At the heart of parliamentary democracy is the idea that our representatives do indeed represent the interests of their constituents, regardless of Party politics. Of course interests have to be balanced but when these are win-win, there can be no reason for not representing a particular interest. As the matter is not a personal one, then I have also put it on my blog, since there is no reason for secrecy.

I will, of course, post any further correspondence, unless it is confidential for some reason.

“Dear Ms Fox (& cc’ed to Mr Nielsen and Mr Bull)

I am writing to you as one of your constituents regarding an issue which affects me personally and the region. As a University Professor I have over the years been awarded research funds from various Framework Programmes. Most recently, I am part of the €4M FET-OPEN programme “ArrestAD”. This aims to test a new paradigm for Alzheimer’s Disease screening and lay the foundation for a new class of drugs that would arrest the disease.

FET-OPEN projects are very much blue skies and in our case we appear to have hit the jackpot. The trials of the diagnostic in Paris and Warsaw  are quite spectacular and our own work has shown that the targets which ArrestAD has proposed are eminently druggable.

ArrestAD is a 4 year programme. As in any blue skies research, towards the end of the penultimate year a decision is made by the research team whether we should apply for a new, larger project, under one of the translational programmes available under H2020, or to can the idea, in the event it isn’t going to deliver.

Since ArrestAD is delivering its promise, the team will be going forward and applying for a translational programme. This will involve further clinical centres and greater industry participation, since we need more patients and, for drug development, far greater resource.

Alzheimer’s being what it is, ArrestAD obviously impacts widely and not just on myself: there are substantial social and economic ramifications for our region, the UK, and beyond.

The problem we face is that with a so-called No Deal Brexit, the UK loses access to funding from H2020 and the future framework programme. There is no  legislation in the UK Parliament that would guarantee funding as a 3^rdcountry. The upshot is that Brexit will prevent my continued contribution to this likely life-changing research programme. Importantly, it will prevent the UK from reaping economic benefit (clinical trials, pharmaceutical industry).

As my representative in the European Parliament, it is imperative that you work to find a solution, which ensures that the drug development arm of this project remains based in the UK, and that the UK is able to participate fully in the wider clinical trials of the diagnostic. I think you would agree that given the impact of this dreadful disease, this is in everyone’s interests.”

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Helium shortage

For those who follow the news, though helium is rather common in the solar system, it is rare on earth. The shortages were predicted some years ago, and were put off only by the Pentagon agreeing to put some of its strategic reserve into the market.

We now face the first self-inflicted shortage: helium is now rationed in the UK. Self-inflicted, because we waste it, e.g., balloons and no recovery of the gas at point of use. This of course is all down to cost, and testimony, in a small way, to the failure of applying market principles across the board without any strategic consideration.

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A modest proposal or ‘where’s the money?”

Universities are always cash strapped, and over the last few decades there has been a drop  in the number of PhDs funded by the UK government. In essence stipends have been increased (absolutely necessary and credit to The Wellcome Trust for pushing this), but funding has not increased commensurately. So numbers have dropped.

The response of institutions has been to push for more overseas students and to develop, from internal monies various funding  schemes. However, generally this has only served to replace losses in numbers funded by government. Gone are the days when every PI expected a new PhD student every year. This has a detrimental effect on research culture, as it leads to centralisation, a reduction in diversity (in the broadest sense of the word) and fewer trained scientists. The UK relies extensively on scientists trained elsewhere for its R&D and we need to do more in relation to training. Granted science is a mobile and international profession, but without contributing proportionally to the global talent pool, our R&D may wither in the long run.

The problem is money: institutions have limited funds they can use for PhD studentships. Our European model, where PhD students are fully funded for a set number of years (usually 3-4, sometimes a few more) is in my view preferable to the US one, where students work to support themselves. This is for the simple reason that the latter model can lead to feudalism and abuse of power, which is well documented.

There is money available for institutions willing to take leadership on the Open Access Agenda.

An aggressive pursuit of an Open Access agenda, as has been done in Germany, The Netherlands and Sweden, and most recently UC, means cancelling subscriptions to the journals of the Big Four. This frees up a substantial budget, with no ill effect on research and scholarship. A portion of the funds would, of course, need to be used to hire librarians supporting document sourcing by UG and researchers and the balance to fund PhD students. I note that teaching UGs these skills is important, since most STEM workplaces (industry, where many STEM graduates and postgraduates work) do not have large libraries. One advantage of using internal funds is that one can select and so only take the very best candidates, rather than restricting enrolment to those with access to funding. While such proposals will likely be met with horror by a good many academic staff, once in place it appears that no one notices much change and continues to work productively. This is the experience of countries and institutions that have cancelled subscriptions to one or more of the Big Four.

Sadly, with my University running workshops on “How to get published in Nature”, it appears that we are 20 years behind the times and this simple and effective means to improve teaching and research (PhD students being an engine room for research) is unlikely to see the light of day here.

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Positions open

Two postdoc positions are available in my lab.

Both are part of the larger, European Commission-funded FET-Open programme, ArrestAD, which has recently been funded.

Position 1 aims to characterise heparin-binding proteins in Alzhiemer’s disease.

Position 2 aims to develop inhibitors to Golgi sulfotransferases.

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Of nanoparticles, cells and polyanions

Of nanoparticles, cells and polyanions

It is the end of semester 2 so it’s marking season. Since we double mark (a good thing), the final year research projects are marked by both supervisor and an assessor, a member of staff who is not involved in the project. One of the projects I marked was Gemma Carolan’s on “How do SmartFlares RNA detection probes reach the cytosol? Available are the PDF of report, and posts here and here.

I had a sense of déjà vu while reading the project – the clear endosomal location of the SmartFlares, regardless of the DNA sequences brought me back to the days when antisense was the technology of the future for medicine.

While evaluating new technology it is useful to go back and look at other high flying technology. The reality is that it takes decades before we know whether the promise (and hype) were justified; this is true for any hot topic from stem cells to nanoparticles and graphene.

Antisense effects can be mediated by RNAse H, an enzyme that specifically cleaves RNA-DNA duplexes and which protects our cells from RNA viruses. There are other mechanisms, e.g., interference with splicing or translation, but the RNAse-H mediated transcript degradation should be central to many antisense effects. There were many papers reporting specific effects (evidenced by differences between sense, antisense and scrambled oligonucleotides sequences). These certainly contributed to success of individuals and of institutions, e.g., in UK Research Assessment Exercise and grant awards.

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Sphingomyelin-targetted gold nanoparticles

David Paramelle’s paper on using gold nanoparticles stoichiometrically functionalised with a peptide that recognises sphingolipids has just been published in Advanced Healthcare Materials (Publisher’s site; Pubmed)

The paper is the classic “Sunday afternoon” project, which arose through discussions with Rachel Kraut at NTU.

As ever, a lot more than Sunday afternoons ended up being put into the paper, because David had to develop some new approaches. Particularly nice was the purification of nanoparticles functionalised with the sphingomyelin-binding peptide (called “SBD”) from non-functionalised nanoparticles. This is a key step for the preparation of nanoparticles carrying just one functional peptide or group. Hitherto, we have happily had affinity tags as the functional group, which allows for affinity chromatography (examples here, here and here).

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A prize worth winning: How many extracellular proteins bind to heparan sulfate?

There are many prizes for cultural activities, of which science is one. This week has seen the announcement of the Nobel prizes, a little earlier the IgNobels were awarded. There are, of course many other prizes. I have decided to set up my own.
A question that bugs me and which loomed large while I read the excellent review by Ding Xu and Jeff Esko from UCSD on “Demystifying Heparan Sulfate–Protein Interactions” is how many extracellular proteins are there?

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Covalent conjugation of proteins and sugars to nanoparticles

Dan Nieves’ paper on an easy and accessible method to covalently conjugate proteins, sugars and indeed pretty much any biomoleucle onto nanoparticles has just come out in Chem. Commun.

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