"Out, Damned Spot!" The Western Blots of Dr. Wang!
"Out, damned spot!" is the plea of Lady Macbeth as she imagines a spot of the dead king's blood on her hand - her guilt in his murder is driving her mad. Likewise, the western blots of Dr. Wang are driving some Twitter PhDs mad. I am writing this article as an experienced MSE. I’ve had extensive materials characterization experience via university relations and part ownership of a laboratory outside of the US. I have great experience in X-ray Diffraction and many of its techniques including small angle x-ray scattering (SAXS), atomic Pair Distribution Function (aPDF) and residual stress analysis. Other techniques I’ve worked with include Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Fluorescence (XRF, both ED and WD), Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), Thermal Gravimetric Analysis (TGA), Atomic Absorption and many other materials characterization techniques when working in conjunction with cross-functional teams. In addition to this, I have experience as an FSE and understand the systems and underlying principles of many of the analytical techniques used. Due to the broadness of MSE and it’s integration into every field, I’ve garnered a wide network of professionals whom I consult with including but not limited to: Molecular biologists, Neuroscientists, Biochemists, Materials Scientists focused on organic analytical techniques, such as protein crystallography, and fields as varied as law and regulatory. I currently work in the medical field and continue to have professional relations with other laboratories and experts in specific fields, some of which are highly relevant to the Cassava case.
There have been many allegations raised regarding the validity of Western Blot experiments performed by Wang, et al. This analysis analyzes the techniques used to form conclusions as well as final remarks regarding what is important.
There are typically two ways to record WB’s. One is use of CCD (charge coupled detectors), which you see commonly in many cameras and the second is use of X-Ray film (think of developing a photograph in a dark room).
For the latter, many variables come into play such as pipetting technique, exposure times, quality and type of the membrane, medium in which the experiment takes place, the stripping of color and processing of the image and much more. Therefore, the images produced vary in background, grey value density, grey value gradients, contrast between blots and membrane and more.
Due to the nature of the technique, it is true that WB’s can be manipulated. However, it is also true that images that seem manipulated are actually not. In order to establish whether the technique of analyzing a screen shot image, via alteration of contrast/brilliance and/or other parameters, holds ground to establish a case for fraud, I treated dozens of x-ray based film western blots obtained from various sources, none of which were originally created by me.
What I found is that the grand majority of these images contain aberrations such as smudges, grey value gradients, rectangles, halos and splices. See Appendix 1 for an example of the first dozen images. The images analyzed vary from Nature papers, commercial data sheets, other publications and dissertations. As you can see, you will find aberrations in every image. Aberrations, as mentioned before, can be caused by the experimental conditions, the materials used, exposure times as well as the conversion of the image into a digital format. It stands to follow that should one accuse another for manipulation due to aberrations in the images, then the technique used should be verified to work in all instances; it must be able to filter between manipulated and valid data with accuracy. I do not believe this is the case, or subsequently, one would have to prove that all the images listed in Appendix I, derived from various journals and commercial products have manipulated data. Ergo, the technique used to draw conclusions in this matter can be erroneous and may lead to false-positives.
The second argument I’ve seen is that there is no MW (Molecular Weight) markers in the raw images. There are many laboratories that use some form of invisible ink, such as horseradish peroxidase conjugate, onto the nitrocellulose membrane (as used by Wang, et al) to mark the MW’s. This ink is only revealed when the membrane is treated with chemicals such as NBT (Nitro Blue Tetrazolium). Such use is explained in basic and advanced textbooks on Western Blotting. Such textbooks such as: Kurien, Biji T. Western Blotting for the Non-Expert. 1st ed., Springer 2021 and Kurien, Biji T., and R. Hal Scofield. Western Blotting: Methods and Protocols. Humana Press, 2015.
This is a common procedure specially in labs without the financial resources for radiography pens, which are not that great in the first place. As you can see, there are very logical reasons for why the MW many not initially be seen. One must always take care to remove sentiment from any analysis such that one can capture and envision all possibilities.
For the reasons presented above, it is irresponsible to reach any sort of conclusion utilizing the data presented. Fraud is a very serious accusation and it is not taken lightly by any scientific institution. In a bona fide, good-faith critique of data, in order to establish that any particular image is manipulated, one must first establish that the analytical techniques used will be able to distinguish as close to 100% between correct and incorrect WB’s. Given the data in Appendix I, I do not believe this is the case, for to prove that the WB’s in Wang’s paper are falsified, one must also prove that every image contained in Appendix I, and many more, are also falsified. This is not correct scientific investigative procedure. One does not publicly accuse anyone of fraud without the full data and assurance of utilizing correct investigative procedures, and specially not when there can be, implicitly or explicitly, influence from the financial sector. If this is the future of scientific integrity, it is a sad future indeed.
There is no need to reassess, re-run or reanalyze the WB images if we can validate or invalidate the data more accurately with better techniques. However, a system such as a BiaCORE can be expensive and only well funded labs, big pharma or government labs would have one in-house.
Why does this matter? Let’s summarize:
1. The image forensic techniques used to critique the WB technique in this situation seem to be inappropriate due to the inability to distinguish between true aberration and manipulations; this problem arises inherently due to the technique, i.e, Western Blotting.
2. There are new, better techniques available to validate the old data.
3. PTI-125 is available for research purposes
Let’s extrapolate to the real world:
1. Big labs and Pharma can test the validity of Cassava’s claims in a private setting much more accurately.
2. If anyone had questions of my material I’d simply tell them to “test it”. I’d establish IP protections with interested businesses and make it available. This indeed seems to be the case with PTI-125 (Simufilam). Should anyone wish to test PTI-125 in a research setting, they can. There is no obfuscation when the substance is available for testing. But don’t think labs are compelled or required to release any information whatsoever.
3. We can safely assume that any partnership conversations would integrate data from more well established techniques to wash away the uncertainty. No astute individual bases their decisions on incomplete data when gathering more is at their grasp.
A Final and Most Important Consideration
By now, there are many families experiencing the effects of Simufilam on their loved ones. There are four situations that may occur.
In the case that Simufilam causes detrimental health effects, families report such effects to the proper authorities, file a Citizens Petition due to citing detrimental health effects and this event must be reported by Cassava. This, of course, has not occurred.
There is no cognitive effect of Simufilam and thus the study continues with the progressive neurological decline characteristic of AD. Such decline would become evident with ADAS-Cog tests.
Families notice an improvement in the quality of life for the individual. ADAS-Cog tests show improvement in cognition. In this case, the filed CP meant to halt the Phase 3 trials would place an undue burden on the families who now fear losing access to a medicine that has improved their way of life. The undue burden, in turn, is exacerbated by public claims that lack real scientific rigor, and establishment of neutrality.
Should patients show improvement in a phase 3 study, there will be a case to end the placebos and have patients begin taking Simufilam or humane reasons. The families who have benefitted and have now come to depend on Simufilam will be both psychologically and physiologically detrimentally affected due to the behavior of those who wish to capitalize on the downfall of stock prices. Understand that regardless of method of action, financial tactics, CP’s filed, it is the patients who dictate whether this has definitively improve their way of life – to their Pursuit of Happiness. Never forget that everyone deserves this opportunity. Specially those who, for so long, have lost all hope.
Figure 1: original (left), Altered (right)
Figure 2: Here the original seems to have a rectangle, no need for alteration/smudge
Figure 3: original (left), Altered (right). Changes in background density are visible, smudges and rectangles as well.
Figure 4: Here the original contains splice looking rectangles.
Figure 5: The original (left) contains rectangles and the altered (right) contain additional aberrations, including sharp changes in background density.
Figure 6: The original (left) seems normal. The altered (right) contains changes in grey value densities, a rectangular or band like structure, and halos.
Figure 7: Original (left), Altered (right). Notice the altered image has splices, halos and rectangular background across the the blots.
Figure 8: Original (left), Altered images (center, right). Notice how different rectangles (vertical and horizontal) can be brought about in different conditions.
Figure 9: Original (left), Altered (Right). Notice the occurrence of halos and the stark changes in background density. The third blot appears to have a vertical rectangle as well.
Figure 10: Original (left), Altered (Right). Observe how the altered image has a rectangle across the first y blots and about halfway to the 7th. In addition to gray value gradients.
Figure 11: Original (left), Altered (Right). Again, halos, rectangles and splice looking artefacts are visible in the altered image.
Figure 12: Original (left), Altered (Right). Observe the marks on the bottom to lanes of the altered version. One would assume they were pasted on there or manipulated should one desire to take such opinion
Pseudo-professionals may be meowing alarm and may be making much ado about nothing. Like Lady Macbeth, Twitter PhDs may be shouting, "OUT, DAMN SPOT" and imagining things that maybe are not even really there. Professional, expert judgement, such as Journal of Neuroscience, are the only ones who can evaluate the issue without bias.
This post is my opinion and it is not investing advice or medical advice.