The nanoneedle salt stress test – too good a clue to leave abandoned on the lab bench?

If the data is that good, will they be publishing it? I guess the lack of specificity is not necessarily what the ME community wants to hear, but been able to distinguish between sick and healthy. It would be quite something.

 

There are a couple of reasons there might be better explanations.
1. I'm not sure how much research was actually done after the work for this research. Throughout, the researchers were hampered by the slow speed of their setup, and manufacturing issues. Hence the work to develop a better system. Does anyone know how much research was done beyond that published here?
2. They applied for an NIH grant for the above more than once. Which is a lot of work.
So they may not have had much other data, and the big effort to design a new system and try to get if funded doesn't obviously fit with believing the Research group not believing in the method.

 

I am still confused. Are these molecules in the plasma of patients or controls, or molecules introduced by the experimental process as an artefact?

If the former, that's still interesting biologically.

If an artefact that could account for the case/control differences, I'm not sure how that would work. The paper says sample prep was identical for cases and controls. There were some differences in blood sample collection. However, all 20 cases and 5 controls were collected at the same time by the same team. There is clear blue water between cases and controls, so how could the artefact explain that, given identical sampling for 20/5 (see this post)?

 

Either? Both? No way to know without doing further experiments to figure it out. Imagine that the points of the needles (nano-scale gold) are acting as catalysts. They could react with something in either samples, changing the cells, the chemicals in the serum, the electrodes, or the microchannel itself.

It does mean that there is a difference between the two groups. However, is the difference due to ME, or the difference between healthy individuals going to a lab and unhealthy people for whom the trip is a challenging task? Maybe the HCs were people who worked in the building or nearby, while the patients had to travel much further.

I agree that such a dramatic difference is something potentially worth following up on. However, I feel that it's so worthwhile that the researchers should have followed up on it (major prestige and funding!) ... unless they looked at it further and decided they'd made an embarrassing mistake and not worth following up on and hoping it will just fade into obscurity. Personally, if I was in that situation, I'd probably publish it with the mistake clearly explained (forgot about nano-scale gold being a catalyst). Publishing that sort of educational paper (how experiments can go wrong) should be worth some professional credit.

Lack of funding shouldn't be a valid excuse for something as valuable as a clinical test for ME. Someone would provide the funding if you could convince them that the results really were that dramatic and reliable.

 

Not sure. Live cells might change volume but lysed cells would I think be unchanged in size - just with holes in. Traditionally viability is tested with something like dye exclusion (trypan blue maybe).

 

You are being generous, I think, @Simon. If I had this result and believed in it I would have replicated it, and I never had very big grants or fancy equipment. If you believe in something in something like this in science you can get it done.

If ME cells behaved as differently as they appeared to in terms of some strange impedance measure nobody can quite understand the meaning of then they would almost certainly show similar results with standard tests that you could put a PhD student on to at barely any cost. If standard metabolic/respiratory tests did not show change it would be hard to argue the nano needle result meant much.

The equipment seems to be expensive because it had vast arrays of electrodes for mass use that are irrelevant to checking this result.

And nobody else has repeated it, I suspect because they cannot work out what the results were supposed to be measuring.

 

I seem to remember it being because Ron's student R Esfandyarpour - the first author on the nanoneedle paper - was starting his own lab and needed the NIH grants to get set upright. As you say Ron mentioned they applied for multiple NIH grants and were repeatedly rejected. Would also make sense that Ron would entirely give the nanoneedle project to him if it was R Esfandyarpour's baby and he wanted to base his career off of that. Also I think Ron's attitude has been to try and find a cure as fast as possible which might explain his shift to the metabolic trap hypothesis - which I've never really understood since it doesn't seem to be based off of any actual evidence base.

I think it's possible it flopped but also possible politics has just gotten in the way.

I think Ron might have had a problem with manpower, a PhD student themselves will cost at least £100K for three years in the UK. Do you have respiratory assays in mind that don't cost much and also wouldn't take a large amount of time? The Clarke electrode throughput is low and the seahorse respirometer gets pretty pricey and even that would be probably months of work to do a solid replication (not to mention this machine is notoriously unstable). All worth doing just not completely trivial.

 

I just do not see this as a realistic objection.

When I decided that rituximab might be beneficial for my RA patients I knew I would get no funding - because it would have to come from the drug company and they had said no - so in the middle of my career I spent hours by patient's bedsides, making solutions, putting up IVs, monitoring pulse and blood pressure and so on. Does Ron know nobody sufficiently interested to make that sort of commitment? Nobody prepared to loan a bit of time on a machine that is probably lying around unused somewhere in the same building? The use of rituximab in SLE was set up by Maria Leandro who worked for free because she had moved to the UK with her husband and wanted to get some research experience.

The way lab science has traditionally operated, in fact, is that you get the crucial experiments done by borrowing resources from other projects and then put in a grant to do an experiment you know will work. It all depends on persuading a few people around you that what you are doing is credible. The nano needle stuff has been shrouded in mystery.

Sadly, 'dramatic' results like this happen all the time in labs. It is just that a few weeks later everyone realises someone screwed up the protocol.

 

Are you saying the cells were already dead at the start of the experiment? Or that they might have been killed by hyperosmotic stress without first enlarging (@chillier or @Creekside said a volume change is normal in cells that are stressed by hyperosmotic conditions)? I assume the authors didn't test for viability because they hadn't seen a volume change.

I'm certainly standing on the sunny side of the street.

Due to regular migraines, I'm not keeping up here and haven't even posted the last thing I composoed - the arguments keep moving on before I can engage. I will post that now, but while I agree it is odd they did not do as you otulined above, why would they push so hard on the NIH grant ( a lot of work) if they thought this a dead duck?

Yes, or even human nature.

 

One more question to address: why hasn't OMF pursued impedance testing?

1. The nanoneedle is cutting edge and proprietary technology, and unfortunately its inventor has no particular interest in ME. He left Stanford, and is apparently now leads his own research group at University of California Irvine. Presumably, he is pursuing his own interests.

I think OMF said he would work on the nanoneedle for ME if it was backed by an NIH grant, Which would help him get tenure. (My guess is that's why OMF haven't just used their own cash to fund the research.)

2. This begs the question, why haven't they simply used other impedance technology or other approaches instead?

Possibly,
2.1 Ron has always been focused on developing a fast and affordable biomarker test. The other impedance systems I've seen very much lab-based technology. The nanoneedle technology is well suited for a chip-based test that can be manufactured cheaply. So there probably isn't an alternative technology that would give Ron what he was looking for.

Agree, I think that's the question that most people here would ask. It would surely be enormously valuable to demonstrate that there are biological differences behind ME.

But researchers have their own priorities.

Ron is incredibly technology focused. He's invented all kinds of gear, including a lot of the critical gene sequencing kit used for the human genome project, and he is head of the Stanford Genome Technology Centre. Perhaps he wouldn't even consider using a low-tech method for anything?

I've never really understood why OMF has put so much effort into studying metabolic traps. It’s a theory-driven approach with little hard evidence in support. Yet they've made that choice. Researchers will have different information to us and different priorities. I think we should factor that in when trying to make sense of Why things have stalled with the nanoneedle.

Whatever the reason, I still like the idea of pursuing the salt stress test in other ways. Maybe the simplest way of doing that would be using a Laboratory based impedance system. Or other simple approaches suggested by @Jonathan Edwards .

It is possible that OMF is just quietly dropping the whole idea because they’ve had undeclared disappointing results. Most big results don't check out, even published ones (eg XMRV (poor methods) and Rituximab open-label ( (just science doing its thing)) But I think there are good reasons to believe what OMF say, that they would like to pursue nanoneedle research.

 

Would hyperosmolar challenge lead to swelling? If anything I would expect the cells to shrink initially. They might swell if their osmotic control failed and they died slowly I suppose.

People put in grants on the basis that they think they can sell some early data. The aim is to get money, however you can. I have put in grants for stuff I din't believe in - in fact those are usually the grants you get funded in this game.

 

Why on earth measure impedance? If cells are sensitive to salt stress there are a dozen more conventional things to look at. Even if you are interested in the membrane it makes sense to look at transport across it rather than tangential impedance which seems to me the most indirect and uninterpretable measure of anything one can think of. I may have missed the point but nobody has explained to me otherwise.

 

My mistake - I meant shrink (I'm going to blame my migraine for that one!)

Except OMF are in the happy situation of having lots of funds. They say they are applying for NIH funds as Esfandyarpour's condition to work on the project. The money will go to him. So I don't see the benefit to OMF unless they mean it when they say they want the research to take place.

I don't have a strong opinion on this. My thinking had been that impedance does seem to pick up a broad range of issues, so it might be a good catch-all approach, as well as using the same general method as the nanoneedle. But I would be happy to with any approaches that looked at the impact of the salt stress test., though I think it would help to use more than one method.

 

I think that's a case of "Here's a new tool. I wonder if it will show a difference between ME and controls." Hammer, meet nail.

 

Fast and affordable are worthy goals, but I think the most important goal right now is a genuine marker for ME. Faster and cheaper can be developed later. A slow, expensive test for ME would be way more useful than a fast, cheap test that doesn't work for ME.

 

Does the Bioimpedence spectroscopy thread more or less kill this thread? Only 4 samples, but it didn't confirm Ron's finding. It did find something else, but a 4-sample finding isn't much to get excited about. It does show that fancy nanoneedle equipment isn't necessary for this sort of experiment, so "We can't do further studies because the nanoneedle equipment isn't available" isn't a valid excuse.