An Update on ME/CFS Research with Ronald W. Davis, PhD

[Janet Dafoe]

(Ron Davis's) nanoneedle biosensor technology used in this study

Obviously this study has huge potential. First, there's developing a model for this illlness at the cellular level, getting white blood cells to show stress-based changes mirroring PEM experienced in patients. Then there's the finding that the problem is in the serum not the cells (wow) - and the work underway to find out what in the serum is the problem Because the study is so important, I'd love to know a bit more about what the change in impedance actually means in terms of what's happening to cells.Here's what I found out - more answers very welcome.

Basic impedance approaches go back decades, measuring the impedance of the broth that bacteria are cultured in: metabolising bacteria chuck out metabolites that tend to be charged molecules that conduct better than water, so impedance (electrical resistance) falls.

This still from the video (transcript, thanks!) shows the technology used here is nanoneedle biosensors, which is a Stanford Genome Technology center nanobiotechnology project.

View attachment 19790

It was apparently developed by bioengineer Rahim Esfandyarpour, with Ron Davis as the senior author on the 2013 paper announcing it. Here's what I understand of it:

Nanoneedle: basically a tiny rod with 2 electrodes that measures impedance. Cells and medium/serum accumulate on the nanoneedle (not sure how many, small number I think) and it's the impedance of that mix that's measured.

Biosensor: normally this means it's 'tuned' to detect impedance from a particular molecule which could even be an antibody. Here it might be a a specific metabolite or metabolites. OR maybe they've chose to measure impedance more generally.

Here's a bit more on the mechanism from another 2013 paper
Unfortunately that doesn't mean a whole lot to me! Answers welcome.

I'd also be interested to know they cells are 'stressed' as it's the all-important step distinguishing mecfs cells from healthy ones (or healthy cells in mecfs serum from healthies).

I sent an email a few days ago to Janet and Ron asking for more info (thanks for providing that, @Rose49). I'm know they are both very busy but will post here if I hear anything back.

I can't find that email. Maybe you don't have correct address ? Can you pm me?

2500 sensors. 200 measurements per
Sec. The measurements are time averaged so it gives a very stable signal. 1/10 drop of blood with red cells taken out . I'll ask Ron more and correct this and/or add to it if I can.

Edit from Ron:
The stress is adding sodium chloride which cells have to pump out, which requires ATP.
This biosensor is not sensing a specific metabolite or protein, but it is sensing the overall impedance of the cell.

The explanation of that paragraph that you didn't understand involves a big mathematical explanation that involves imaginary numbers and Ron didn't think he could do it here very well.

 

[Kati]

I can't find that email. Maybe you don't have correct address ? Can you pm me?

2500 sensors. 200 measurements per
Sec. The measurements are time averaged so it gives a very stable signal. 1/10 drop of blood with red cells taken out . I'll ask Ron more and correct this and/or add to it if I can.

Edit from Ron:
Thanks stress is adding sodium chloride which cells have to pump out, which requires ATP.
This biosensor is not sensing a specific metabolite or protein, but it is sensing the overall impedance of the cell.

The explanation of that paragraph that you didn't understand involves a big mathematical explanation that involves imaginary numbers and Ron didn't think he could do it here very well.

Imaginary numbers! It's ok Ron, i'll take your word for it.

 

[BruceInOz]

Imaginary numbers! It's ok Ron, i'll take your word for it.

@Barry53 gave a wonderful analogy of a capacitor in an electrical circuit in terms of a diaphragm in a water pipe. When an alternating voltage is applied to such a circuit, electrons will flow in the circuit producing an alternating current. There are two important characteristics relating the applied voltage to the current. One is how much the circuit resists the current flow. The other is how much the current flowing is in step or in phase with the applied voltage.

Complex numbers (with so called real and imaginary parts) are just a useful tool to describe these two features in a nice compact mathematical notation.

Do I take all this to mean that when Ron shows the graph of one quantity - impedance - versus time, that this is the magnitude of the impedance and the phase shows nothing interesting?

I must say, when I joined this forum I never imagined a discussion would lead to complex numbers!!!

 

[Kati]

@Barry53 gave a wonderful analogy of a capacitor in an electrical circuit in terms of a diaphragm in a water pipe. When an alternating voltage is applied to such a circuit, electrons will flow in the circuit producing an alternating current. There are two important characteristics relating the applied voltage to the current. One is how much the circuit resists the current flow. The other is how much the current flowing is in step or in phase with the applied voltage.

Complex numbers (with so called real and imaginary parts) are just a useful tool to describe these two features in a nice compact mathematical notation.

Do I take all this to mean that when Ron shows the graph of one quantity - impedance - versus time, that this is the magnitude of the impedance and the phase shows nothing interesting?

I must say, when I joined this forum I never imagined a discussion would lead to complex numbers!!!

i gather it means that us patients with ME have great natural ressources in our body and could probably power up small appliances just by having this extra proteins that are still nameless?

We are rich my friends.

 

[ash0787]

so the anode / cathodes are fabricated onto the nano needle at diameters slightly larger than the cells diameter and an algorithm determines whether or not a cell lies perfectly positioned between any of the numerous electrodes ?
rather than the entire needle being an electrode and the needles being spaced within the channel at cell diameter distances which is what I originally assumed. I prefer a diagram for this sort of thing rather than just trying to guess by a text description.

I'm not sure the exact structure / design of the thing is that important for us to understand, as long as it functions as stated which I'm sure it does, I am slightly curious though as to how such a thing would be cleaned since it was suggested that it is reusable and its sensitive to microscopic particles, just forcing distilled water down the micro channels ? I suppose thats an entire field of biology though which is well understood to anyone that actually goes to university etc.

I've said this before somewhere but I bet the general electrical impedence concept was already known for a long time and possibly something ron encountered during his time working on yeast, but perhaps nobody thought that it was that useful of a fact or there was no practical way to use it in a test, or perhaps that it was used in a certain way routinely, but previously nobody thought that it might be useful in the context of researching human diseases, tends to be how it goes.

 

[halcyon]

I've said this before somewhere but I bet the general electrical impedence concept was already known for a long time and possibly something ron encountered during his time working on yeast, but perhaps nobody thought that it was that useful of a fact or there was no practical way to use it in a test, or perhaps that it was used in a certain way routinely, but previously nobody thought that it might be useful in the context of researching human diseases, tends to be how it goes.

I'm not clear on the specifics of the technique they're using but it sounds similar to the one described in a 1993 Nature paper, called electric cell-substrate impedance sensing. Sounds familiar:

As the shape of animal cells responds very sensitively to alterations in metabolism as well as chemical, biological or physical stimuli, the ECIS technique is applied in various experimental settings in cell biological research laboratories. It can be used as a sensor in cytotoxicity studies, drug development or as a non-invasive means to follow cell adhesion to in vitro surfaces.[3] Equipments based on the ECIS technique are also dedicated to monitor the chemokinetic activity of adherent cells spread on the electrode surface (micromotion) as well as their chemotactic activities in ECIS-based wound healing assays.

 

[Murph]

I threw the name Ronald W Davis into pubmed the other day just to see if he was publishing anything, despite OMF not having publishing as its main goal.

Short answer: yes he is! Rather a lot actually. Three already in 2017. I guess that's what happens when you're high up in an institute - you are involved in a lot of projects and have authorships out the wazoo.

Many of his publications are about technology. Here's the abstract for a recent paper on the nanoneedle:

Detection of cells is typically performed using optical fluorescence based techniques such as flow cytometry. Here we present the impedance detection of target cells using a nanoelectronic probe we have developed, which we refer to as the nanoneedle biosensor. The nanoneedle consists of a thin film conducting electrode layer at the bottom, an insulative oxide layer above, another conductive electrode layer above, and a protective oxide above.

The electrical impedance is measured between the two electrode layers. Cells captured on the surface of the nanoneedle tip results in a decrease in the impedance across the sensing electrodes. The basic mechanisms behind the electrical response of cells in solution under an applied alternating electrical field stems from modulation of the relative permittivity at the interface. In this paper we discuss, the circuit model, the nanofabrication, and the testing and characterization of the sensor.

We demonstrate proof of concept for detection of yeast cells with specificity. We envision the sensor presented in this paper to be combined with microfluidic pre-concentration technologies to develop low cost point-of-care diagnostic assays for the clinical setting.

If you'd like to see all papers bearing his name, click the link to the paper above, then click his name, then sort by recent. You can also find a paper about another chip he's developed, which may be the 'assay' people talk about.

 

[boolybooly]

I am reminded of the erythrocyte sedimentation test which has anecdotally shown higher blood viscosity in ME patients implying lower levels of hydration and lower blood volume / pressure.

This correlates with the clinical observation that people like myself having a worse phase tend to pee a lot, so I have always accepted it as another inexplicable reality. I tentatively assumed it might be explained because renal reabsorption is an active process and requires energy which might be below normal availability in ME CFS and prone to fluctuation, though an additional hypothesis would be that the immune response might downregulate renal activity in some circumstances, either to save energy or to create favourable conditions for molecular and cellular defences in the blood.

I am wondering how this phenomenon relates to these results and to what extent increased impedance can be explained by lower hydration in the initial blood sample.

 

[Simon]

Edit from Ron:
The stress is adding sodium chloride which cells have to pump out, which requires ATP.

Thanks, Janet.

So adding salt creates an energy demand that stresses the cells and the mecfs ones (or those in mecfs serum) respond differently. Seems pretty good to me: mental or physical activity increases our energy demands, which presumably means more ATP is needed at the cellular level, just like the salt test. (I realise this might be obvious to many but I write stuff out to get it clear in my mind).

This biosensor is not sensing a specific metabolite or protein, but it is sensing the overall impedance of the cell.

The explanation of that paragraph that you didn't understand involves a big mathematical explanation that involves imaginary numbers and Ron didn't think he could do it here very well.

I very much appreciate being spared the imaginary numbers. I'm still intrigued by what's going on in terms of cell biology. Whatever changes the impedance, presumably there's also something going on the cell linked to that, involving molecules rather than imaginary numbers.

For instance, @halcyon noted the 1993 paper

As the shape of animal cells responds very sensitively to alterations in metabolism as well as chemical, biological or physical stimuli, the ECIS technique is applied in various experimental settings in cell biological research laboratories. It can be used in... drug development...

I know that's pre-nanotechnology, but is based on impedance of cells in contact with electrodes. Maybe it's not cell shape that's changing, perhaps it's cell membrane changes eg permeability, or different cell surface molecules, perhaps as a result of signalling cascade intitiated by the stress. OK, I'm guessing wildly here, but I'm hankering for something that would help me understand what might be happinig at the biological level. Or maybe it's basically a black box, where impedance means something is going on, but it's not clear what that is.

I hope Ron will take pity on someone who's happier with molecules than imaginary numbers. And please pass on my thanks for the answers he's already provided.

 

[Sasha]

Here's the abstract for a recent paper on the nanoneedle:

I misread that as "nanonoodle".

I could imagine it would have been very fashionable in nouvelle cuisine.

 

[Barry53]

I misread that as "nanonoodle".

I could imagine it would have been very fashionable in nouvelle cuisine.

Not very filling though.

 

[MeSci]

I am reminded of the erythrocyte sedimentation test which has anecdotally shown higher blood viscosity in ME patients implying lower levels of hydration and lower blood volume / pressure.

This correlates with the clinical observation that people like myself having a worse phase tend to pee a lot, so I have always accepted it as another inexplicable reality. I tentatively assumed it might be explained because renal reabsorption is an active process and requires energy which might be below normal availability in ME CFS and prone to fluctuation, though an additional hypothesis would be that the immune response might downregulate renal activity in some circumstances, either to save energy or to create favourable conditions for molecular and cellular defences in the blood.

I am wondering how this phenomenon relates to these results and to what extent increased impedance can be explained by lower hydration in the initial blood sample.

How would you explain high blood pressure, yet also a tendency to low blood sodium?

 

[Barry53]

Maybe it's not cell shape that's changing, perhaps it's cell membrane changes eg permeability, or different cell surface molecules, perhaps as a result of signalling cascade intitiated by the stress.

As we are into wild'ish guesses, here is a follow-on one from the above. If we are into something akin to capacitive effects, then maybe we are into the notion of dielectrics, which could very conceivably be (and is typically) a very thin membrane/film of some kind; it is a natural phenomenon. And its dielectric properties and therefore reactance (implying the imaginary component) might well change in sympathy with its biological properties. Is this what Ron Davis and his team are measuring here I wonder? Comparing impedance values for stressed/unstressed samples would potentially give indications of differing membrane dielectic properties.

Probably miles off, but it is all rather fascinating!

Another off-the-wall thought: I wonder what happens if you stimulate the samples with square waves rather than (presumably) sinusoidal ones, being as square waves have a very wide range of frequency components all in one hit - would it reduce the assessment time (yes I know - be quiet and let Ron and his team get on with it! . But it is all so very intriguing!)

 

[Tyto alba]

Another off-the-wall thought: I wonder what happens if you stimulate the samples with square waves rather than (presumably) sinusoidal ones, being as square waves have lots of high frequency components all in one hit

I'd imagine the high frequency harmonics would prevent a clear clean impedance reading(s) if the samples are reacting to a number of different frequencies at one time.

 

[antherder]

Not very filling though.

Unless you are a nanobot.

Or have oodles of nanonoodles.

 

[Barry53]

I'd imagine the high frequency harmonics would prevent a clear clean impedance reading(s) if the samples are reacting to a number of different frequencies at one time.

I was just thinking that the shape of output waveform might give an at-a-glance first-pass indicaation, in case it saved time sorting the wheat from the chaff.

 

[boolybooly]

How would you explain high blood pressure, yet also a tendency to low blood sodium?

I am sorry I don't understand the question.

I could brainstorm a bit but would you kindly explain why I might want to?

Dr Paul Cheney characterised his ME CFIDS patients as having low blood pressure / volume and very unlikely to have high blood pressure. Dr. Nancy Klimas quoted in the link seems to agree.

https://www.healthrising.org/treati...onic-fatigue-syndrome-mecfs-and-fibromyalgia/

So since this matches my own experience I am not aware of the situation you describe being typical for ME CFS patients, is this question related to research findings, or is this how it is for you?

 

[BruceInOz]

As we are into wild'ish guesses, here is a follow-on one from the above. If we are into something akin to capacitive effects, then maybe we are into the notion of dielectrics, which could very conceivably be (and is typically) a very thin membrane/film of some kind; it is a natural phenomenon. And its dielectric properties and therefore reactance (implying the imaginary component) might well change in sympathy with its biological properties. Is this what Ron Davis and his team are measuring here I wonder? Comparing impedance values for stressed/unstressed samples would potentially give indications of differing membrane dielectic properties.

Probably miles off, but it is all rather fascinating!

@Barry53 have you looked inside the paper mentioned here:

Many of his publications are about technology. Here's the abstract for a recent paper on the nanoneedle:

(I found it on scihub)

Since you have an engineering background, you may find this answers some of your questions. Among other things, it includes an equivalent circuit of the sensor.

 

[Barry53]

@Barry53 have you looked inside the paper mentioned here:

(I found it on scihub)

Since you have an engineering background, you may find this answers some of your questions. Among other things, it includes an equivalent circuit of the sensor.

Many thanks. Unfortunately I don't seem to have access to the full article.

 

[arewenearlythereyet]

Unless you are a nanobot.

Or have oodles of nanonoodles.

I hope they're made of rice flour not wheat flour!