It’s not pretty! The print is abysmally small, the margins are tiny, long stretches of text are common (it abhors paragraphs), the images are jammed together, and the formatting is designed to make you work and is decades out of date, but the fact is that when you’re one of the top research journals in the world, you can make your own rules.

PNAS or The Proceedings of the National Academy of Sciences of the United States of America – the official publication of the National Academy of Sciences – has been described as “prestigious”, “renowned”, “high impact” and even “sedate” (which in the research world is not necessarily a pejorative term).

PNAS, which touts itself as the second most cited scientific journal in the world, publishes a wide variety of scientific content. One statistical analysis concluded that, “Three journals have by far and away the most overall influence on science: Nature, PNAS, and Science, closely followed by the Journal of Biological Chemistry. ” Nobody else was even close.

PNAS may not be pretty – it’s probably intentionally unpretty – but getting published in it gets the word out. Since Rahim Esfandyarpour (the lead author) and Ron Davis’s nanoneedle paper was published in the most recent edition of PNAS, numerous media outlets have picked up their findings. I got to talk to both of them.

The paper immediately makes clear two things: the lack of a biomarker makes getting diagnosed with chronic fatigue syndrome (ME/CFS) a “lengthy and costly” affair (which, of course, precludes patients from getting treatments when they may help the most – while the disease is still new). From a research standpoint, the paper noted that lack of a biomarker may have tragic consequences if “heterogenous samples of patients” with “only marginally similar conditions” are being included in studies.

Obviously, getting a biomarker would fulfill a huge need, and other possible biomarkers may yet pan out. None, however, potentially tick off all the boxes (potentially very cheap, easy to use and very accurate) as well as Ron Davis and Rahim Esfandyarpour’s nanoneedle.

The first nanoneedle papers in 2013 indicated that the needle was originally designed as an “ultra-sensitive, real time” biosensor designed to cheaply detect biomarkers such as proteins inside (or outside) cells at a far more sensitive level than had ever been done before. One very promising feature of the nanoneedle was its ability to quickly identify biomolecules without needing to label them first (and then use a tool to detect them).

By 2016, a redesigned and improved nanoneedle was presented as a rapid, inexpensive, high throughput, real-time, label-free, highly sensitive alternative to the expensive or less sensitive devices available.

Then came chronic fatigue syndrome (ME/CFS). Focusing on the unusual problem of energy depletion following exertion (post-exertional malaise (PEM)) in ME/CFS, Davis and Esfandyarpour added a salt stressor to ME/CFS samples and used the nanoneedle to measure electrical impedance (a measure of electrical resistance). (The salt solution was used because it requires cells to generate a lot of energy (ATP) to maintain the appropriate salt concentrations inside and out.)

The increasing electrical impedance or resistance seen in the ME/CFS but not in the healthy control samples suggested Davis indeed induced a state of post-exertional malaise in his ME/CFS samples.

That was not surprising; what was surprisingly was how definitively he has done it. In one of the most evocative images yet to come out of the ME/CFS scientific literature, the graph below shows a dramatic separation between patients and controls. With the needle packing tens of thousands of biosensors, adding up to millions of data points, Davis has every reason to believe that the finding is robust, indeed.

Causes

A major question involves determining what biological process is producing the rise in impedance in the ME/CFS patients’ samples. Esfandyarpour pointed out that while answering this question is obviously biologically important, it’s not critical for diagnostics or drug screening to know what’s going on. Many drugs work, after all, without our knowing exactly why. Similarly, all a good diagnostic test really requires is that it be specific to a disease.

(Esfandyarpour said “many” different drugs, inhibitors, enzymes, and substrates involved in cellular energy production have been tested to see if they can return ME/CFS samples to normal functioning in the nanoneedle. Two thus far (he didn’t say which ones) have returned the ME/CFS samples to normal functioning.)

Still, learning what the nanoneedle is showing should give us an idea of what has gone wrong in ME/CFS. There are several ways to do this – some of which do not require the nanoneedle. Different kits could be used to measure how the cytokines, etc. in the sample change following the introduction of a salt stressor. Adding an inhibitor to the sample that affects certain parts of the cells and assessing how the sample responds could provide clues as well.

One might think the Seahorse – a commonly used tool in ME/CFS that assesses cellular energy production – would play a key role, but it doesn’t. Because Seahorse cannot incorporate the potentially ever so important ME/CFS plasma into its testing regimen, if something in the plasma of ME/CFS patients is causing problems with cellular energy production, it will not be able to pick it up.

Esfandyarpour believes something in the plasma may play a critical role in ME/CFS. Davis’s team has been filtering out substances in an effort to learn what factor that might be. Nothing definitive enough to announce has been found yet, but exosomes (small extracellular vesicles involved in communication between cells) are a possibility.

Davis said he was encouraged by the idea that a substance in the blood is causing problems. Given that, so far as we know, this is rare in diseases, if such a substance is present, it may be quite specific to ME/CFS.

They’ll feel confident about such a substance if: a) when they remove it, the response returns to normal; and b) adding that substance to healthy cells causes their impedance levels to rise. If the substance turns out to be exosomes, the next step is to determine what in the exosomes is causing problems.

If something in the plasma is found to be playing a crucial role in the abnormal response of ME/CFS cells, a next step will be to determine which cells it’s affecting.

A Diagnostic Test for ME/CFS?

The nanoneedle passed a big hurdle when it was able to so definitively distinguish ME/CFS patients from healthy controls. The forty people in the study may not sound like a lot, but it’s not the quantity that tells the tale in this case – it’s the consistency of the result.

Probabilities tell the tale in research. The highest probability that is considered significant or meaningful in medical research is a less than one in twenty probability (p<0.05) that a result occurred by chance.

Studies are designed around the need to hit that probability. Miss it even by a small amount, and your results are worthless. Statistical analyses are done to determine how many participants are needed to hit that probability target.

There was no need for that in the nanoneedle study. The statistics indicated that the probability that the study results were due to chance ranged in the billions. That kind of probability is more commonly seen when trolling for a mega lotto win than in biology. Put another way, the nanoneedle study was actually far, far larger statistically than it needed to be to demonstrate that the ME/CFS samples could be differentiated from the healthy control samples.

Davis will continue testing samples, but for him and his long, long acquaintance with statistics – the question as to whether the nanoneedle can differentiate ME/CFS patients from healthy controls has surely been answered.

Differentiating ME/CFS from other diseases is the next step, but the big takeaway is that Davis appears to have produced a test that irrevocably demonstrates that people with ME/CFS have a biological illness. Developing a biological test for ME/CFS, Davis said, was his biggest goal.

Davis’s next task is to differentiate people with other fatiguing illnesses, such as multiple sclerosis (MS), from ME/CFS – a trickier project. Davis noted that some of the first symptoms to show up in MS are similar to those seen in ME/CFS, and that some people diagnosed with MS have been shown to have ME/CFS and vice versa.

It’s also possible that some people with MS will develop ME/CFS at some point. This type of disease convergence is not uncommon with fibromyalgia. Numerous studies indicate that a significant subset of patients with rheumatoid arthritis or other chronic pain disorders also have fibromyalgia. It’s possible that the same is true with ME/CFS and also some very fatiguing autoimmune diseases.

Davis, as Jarred Younger has, brought up the possibility that people with MS and ME/CFS – two illnesses often jumpstarted by an infection (and perhaps the same herpesvirus infection) – may start out on the same pathways but then diverge at some point.

Having some people with MS test similarly to people with ME/CFS on the nanoneedle is not a deal-breaker, though. Far from it. It’s not uncommon for diseases to share diagnostic markers. Doctors get around this by using a series of tests to diagnose patients. If some people with MS look like ME/CFS patients using the nanoneedle, then a brain scan which shows neuron damage in the MS patients will be able to differentiate the two diseases. People with MS and ME/CFS will be positive on both tests, while people with ME/CFS without MS will test positive only with the nanoneedle.

An even better testing regimen would use another anomaly more commonly found in ME/CFS, such as the red blood cell (RBC) deformity work the Open Medicine Foundation is funding. Since problems with RBC deformity probably do not occur in MS, a positive nanoneedle and RBC deformability result should be enough to differentiate someone with ME/CFS from someone with MS or many other diseases.

The point is that a diagnosis often does not rely on a single test: a single definitive test isn’t needed to diagnose a disease.

A handful of tests could enable ME/CFS to be clearly differentiated from other diseases in the fairly near future. It won’t take thousands of patients to show that either. So long as you have well-defined patients in studies produced by good researchers which produce good results (i.e. good probability stats), neither the number of studies or the number of patients need to be particularly large – the results simply need to be trustworthy.

Metabolic Trap

Davis has begun to separate out dendritic cells and test them to see if the metabolic trap is present. He brought up an interesting possibility – emphasizing that it was only a possibility – that if the metabolic trap is present, it could lay the groundwork for an autoimmune process to manifest itself. This is because dendritic cells are responsible for shutting down autoimmune producing B-cells.

If the metabolic trap is present in dendritic cells, it’s probably mucking up their ability to knock out bad B-cells. Furthermore, because the IDO1 pathway plays a role in immune system regulation, it wouldn’t be odd at all for pathogens to take a swing at knocking it out in order to evade capture. That – plus a possible genetic predisposition to problems with this pathway – could tell the tale in ME/CFS. That said – at this point it’s just a possibility (but it’s nice to have possibilities) – these are tangible ways of potentially explaining this disease.

The NIH Attempts to Take Credit

Director Francis Collins immediately jumped on the nanoneedle finding to trumpet the NIH’s role in funding the breakthrough. That was, in part, true – the NIH did provide funding for the early development of the nanoneedle – but then there is a second part to the story. The NIH later dropped its funding for the needle and it did so just about the time it was starting to be used on ME/CFS.

In 2016, citing a renewed emphasis on ROI grants, the NIH jerked Davis’s longstanding technology grant, undercutting his nanoneedle and other research. Ironically, for all its emphasis on accelerating ME/CFS research, the NIH, inadvertently it must be said, pulled the plug on funding for one of our most important ME/CFS efforts. In a recent conversation, Davis looked back:

“I used to have experts in almost all areas. We were on a roll developing all sorts of things. I don’t have that anymore because the NIH terminated my big grant on technology development.” Ron Davis

It turned out that the NIH no longer shared Davis’s emphasis on developing cost-effective tools that help people and reduce the cost of health care.

So when Francis Collins himself tweeted the good news that the NIH had funded Davis’s effort to develop a new test for ME/CFS…

“In @stanfordmed study, researchers developed new blood-based test that positively identified participants w/#MECFS. If findings can be validated in larger study, it may provide a diagnostic tool for clinicians & a target for new ME/CFS Txs. #NIH-funded https://stan.md/2IU7nYN“ Francis Collin

He crossed a line for Janet Dafoe and patients who went on something of a twitter rampage at the director’s gall. One wonders how much further we would be if the NIH had continued their support. Janet pointed out that the NIH hadn’t funded any of Davis’s ME/CFS nanoneedle work – which was, of course, what the paper was on; everything was funded through patient donations to the Open Medicine Foundation.

“That NIH grant was TERMINATED about 2 years ago because NIH said they didn’t want to fund that technology anymore. That grant had funded the initial development of the nanoneedle on cancer cells & bacteria. All the ME/CFS work started AFTER that & was FUNDED BY PATIENTS@OpenMedF. You don’t get credit for this good news for ME/CFS” Janet Dafoe

Then she pointed out that the NIH had muffed another opportunity to fund ME/CFS nanoneedle work when NIH reviewers savaged Davis’s NIH application, in part because they objected to the new technology he was using.

“In fact, Ron Davis applied to NIH for the nanoneedle work and was turned down! Reviewers were very critical and said it wouldn’t work, in spite of good preliminary data. STOP saying you don’t fund ME/CFS because you don’t get good grant applications! Patients funded this!” @OMF Janet Dafoe

Attacking the competence of Ron Davis – who’s been called a “frequent provider of disruptive core technologies“, and played a key role in the Genome project, etc. etc. was probably not the brainiest move by the NIH reviewers, who are now presumably busy wiping the egg off their faces.

The NIH is soon going to get another opportunity to fund nanoneedle work in ME/CFS. With the PNAS paper under their belts, Ron Davis and Rahim Esfandyarpour will be applying for another NIH-funded nanoneedle grant this June. Perhaps the third time will be the charm for the nanoneedle and the NIH.

Resources, Resources, Resources

With its potential ability to serve as a diagnostic tool, a drug screening tool, a tool to help determine if something in ME/CFS patients’ plasma is knocking their immune cells for a loop, a tool that could greatly enhance research studies (by ensuring that only ME/CFS patients are in them), the nanoneedle obviously presents immense possibilities.

The work seemed to be going slowly, though, and I was curious to find out why. Asking if the funding or resources were holding the work back elicited a big laugh from Rahim. Funding, not technology is the limiting factor, he said.

It turns out that as sophisticated as the nanoneedle is, in some ways it’s rather primitive. In its current iteration, the absolute fastest it can work is to test two samples over a couple of hours. That’s not great in an instrument that needs to be involved in testing other diseases, in drug testing, in assessing plasma factors, in assessing the pathophysiology behind the reaction (mitochondria, membrane problems, immune issues, etc.). The drug testing alone is incredibly complex: Davis needs to test different drugs, different doses of drugs, drugs in combination, etc. in different patient samples. With its thousands of sensors, the nanoneedle may be a technological work of art, but it’s a sludge when it comes to processing samples.

Fortunately, a solution is present. Unfortunately, it will take something the ME/CFS field hasn’t had a lot of – resources. Developing a high throughput system that’s able to test dozens of samples at once is, Davis said, a top priority.

There’s apparently no mystery to developing that system. It’s simply a matter of resources. When asked, Esfandyarpour said that designing a high throughput system presented no technical challenges – the only challenge is funding – and not necessarily that much funding. When I pressed him, Esfandyarpour would not say how much money was needed, but when I asked if it was $1 million, he said no, it’s not nearly that.

Developing a high throughput system is the first and necessary step to maximizing the potential of the nanoneedle, but it’s only the first step. The logical conclusion to all of this is the transformation of the lab instrument into a portable one that can be used in a doctor’s office or medical lab. Davis said that doesn’t present any technical challenges either (!). If everything works out, a portable testing unit could be ready within two to three years.

The fact that what is holding us back is a matter of resources makes the NIH’s decision to end Davis’s ongoing technology grant sting all that more. In the end, though, the fact that its resources – not some technological breakthrough that’s needed – is actually very good news.

The resources are out there. I know of very wealthy people with family members with ME/CFS who could easily fund this or other research efforts and aren’t. I don’t know why. It doesn’t make sense to me, but the fact is that the more we spread the word, the more we get our story out, the more people we contact, the more people with resources who do want to contribute will show up.

The PNAS article will surely help with that, and it will help with the NIH. Davis now has a publication in an excellent journal to back him up. He didn’t have that before. “Our goal,” he said, “is to keep making it harder and harder for the NIH to ignore us.” The PNAS study just made it that much harder.

Resources are a problem, but what’s worse is a lack of possibilities. Thankfully, we’re not lacking in possibilities right now. The nanoneedle is just one of an array of possibilities the Open Medicine Foundation is funding under Ron Davis’s direction. These include the muscle biopsy work at the new Collaborative ME/CFS Research Center at Harvard, the spinal fluid work at the new Uppsala Research Center, the red blood cell work at Stanford and San Jose State University, the work on the metabolic hypothesis, and genetics and the severely ill patient study work going on at Stanford.

All are important. (If you want your donation dollars to specifically go to the nanoneedle project, though, – which to me at least seems to hold a special promise – note that with your donation.)

Rahim ended our talk by thanking the patients. He said, “I think we have very promising results and do not lose hope”. Then he said something interesting. Many diseases have had to fight for recognition over time, and in many of them, something happened to flip the research community from apathy to action. When it did, the resources of the research community were brought to bear.

He believes that will happen with ME/CFS. I couldn’t help thinking that the nanoneedle could – if everything works out – provide one of those tipping points for this disease.

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