Flaw cytometry, FACsimiles and fly-speck thunderclouds

This post was earlier cross-posted at Leonid Schneider's site, hence the unfrivolous tone. The version there is improved by Leonid's editing, background details and frame-story. OTOH, it does not have any references to the role of fly-specks in literature... in particular, the opening chapters of Osudy dobrého vojáka Švejka za světové války when Palivec (hapless landlord of The Chalice) is arrested on suspicion of treason because flies had shat upon the pub's mandatory portrait of Emperor Franz-Joseph.

The post is based on Elizabeth Bik's examination of some papers by Academician Xuetao Cao, as a follow-up / elaboration on Leonid's own summary of Bik's concerns. The story was picked up by other media and became a cause célèbre, with the Chinese academic establishment promising to fully exonerate investigate the adumbrated Xuetao Cao. Also, Paris, Rome, Munich and New York are burning.

When a paper has five diagrams to illustrate the various techniques that were used (and to reassure the readers that experiments did actually take place), is it the same paper when four of those Figures are replaced with less problematic versions? This is a new phrasing of the conundrum of continuity and identity over change, taking its place in the menagerie of Metaphysics alongside the Ship of Theseus, Trigger's Broom and Locke's Sock. And evidently the paper's Conclusions at least are unchanged by the substitution of data, at least in the view of the editors of Clinical Cancer Research. The resulting spectacular megacorrection (Wang et al. 2005 [3]) featured in a recent post at Science Integrity Digest.

[Figure 3B is no longer operative]

[Figure 3D]

One of these now-deprecated Figures consisted of Flow Cytometry plots, which have come up before. Here, measurements of surface protein expression in a succession of cells become log-scale coordinates of a FACS plot. There are lasers involved, and fluorescent antibody markers, but all that concerns us now is that a raw data file of sequential cell measurements can be run through the plotting software repeatedly, while twiddling the settings of the gating parameters that define which cells to plot and which to omit (as outliers or irrelevant). Thus multiple slightly-different FACSimiles can be produced, and if the researchers are sufficiently sloppy in how they file the printouts then these might turn up in a paper mistakenly presented as different experiments.

Alternatively, one might fake a FACSimile in 'Visual Enhancement Software' by cut-&-pasting groups of points, though the results can leave the impression that a child ran amok with a potato stamp. If one is caught out it is hard to blame sloppy filing, and it is time to find a junior author or graduate student to through to the wolves. Or under the bus. Or to the wolves living under the bus.

Now FACSimiles and "flaw cytometry' feature heavily in the oeuvre of Dr Cao across the 16-year span 2003-2019, at least in the fraction of that oeuvre that has so far provoked discussion in threads at PubPeer. In many cases the use of a duplicate or almost-duplicate plot was an innocent mistake, quickly corrected (Qian et al, 2013 [12]) or subsequently acknowledged (Liu et al., 2018 [15]).



Also Wang et al. (2014) [14], Fig 5 from Liu et al. (2018) [16], and probably more by now, for this is a developing story.

In other cases, concerns about copy-pasted images were conveyed to journal editors five years ago but progressed no further (Wang et al. 2004 [2]; Li et al. 2007 [5]). Perhaps the Journal of Biological Chemistry at the time had not tasked any staff to find gambling going on here ensure image integrity; alternatively, the staff-member in question saw all the glitches and many-colored boxes in the emailed notifications and thought that they were invitations to an art exhibition... perhaps a Frank Stella collaboration with Nam June Paik.

[Thunderclouds with color-coded rainfall]

I should emphasise at this point that these are all multi-author papers, and it is above my non-existent pay-grade to single out any one person as the originator of the fabrications. Dr Cao was the principal author of many but not all of them, but he is clearly a man of many roles and responsibilities competing for his time and attention. In addition, email addresses for Dr Cao were only recently uploaded to the PubPeer database to ensure that he received invitations to take part in the discussions. Now that he is aware of the concerns being raised and has responded in some cases, and given his role as China's Integrity Watchdog, there is every reason to believe that he or the corresponding authors will correct or retract other papers.

Much of the time, alas, any error was unlikely to be innocent, given the levels of shenanigans and jiggery-pokery. Non artifact, sed artifice. In Wang et al. (2014) [13], Figures 2G and 2H are rife with refracted, 90°-rotated constellations, like star-gazing through a kaleidoscope:

Figure 4C (below at right) is a Transwell migration assay of some kind, but it has kaleidoscope problems of its own.



Moving along to Han et al. (2009) [10], Figure 4 is a patchwork:

One of the squares from the patchwork, having appeared three times in 4A, makes a fourth reappearance in Figure 7B of Li et al. (2009) [11].



Now [11] is a kettle of red herrings of another color. It is hard to know where to start. Reused FACS confections form a delicate web that binds all the illustrations together. The rubber-stamping is subtle (except when it's not)...



...so an easy way to demonstrate how patches of random half-tone are replicated between Figures is to overlay them, with one image black/white reversed, to see if and where patterns of identical dots cancel out.



Two more examples because why not? Figure 6C from Yang et al (2004) [1]...

...and Figure 1B from Wang et al. (2006) [4].

Here is one that was Withdrawn (not retracted) in 2015: Wang et al (2008) [9].

VOLUME 283 (2008) PAGES 12076–12084

This article has been withdrawn by the authors.

The Withdrawal notice is terse and cryptic, but it sounds as if someone on the authorship list belatedly paid more attention than the editors and reviewers had, and decided not to enrich the scientific literature with legacies like these:

Yes, I know... this is a case where a Western Blot was caught in the matter-transporter malfunction, rather than a FACSimile, for this diagrammatic corpus is versatile and not exclusively flow-cytometric. Here are some more Western Blot examples. Figure 2 from [5], which we just encountered:

And returning to the beginning of our time-line, [1], Figure 2:

At left, Fig 4A/B from He et al (2007) [6], with a procession of Loch Ness monsters. At right, tying together Sun et al. (2008a) [7] and Sun et al. (2008b) [8].



Of course readers are really here for the flyspeck thunderclouds. Fortunately those last two examples provide a segue back to the main theme. So here are are Dr Bik's findings in Figures 3B and 6 of [7], before she ran out of colors:

Figure 2E of [8] has a little less going on.

Now the lower-left frame of 2E is not one of the "twenty-seven 8 x 10 colored glossy photographs with circles and arrows and a paragraph on the back of each one explainin' what each one was", but all the same it is scaring me, because what are those TWO BLACK-RINGED EYES creeping down from above into the cut-off frame?

Perhaps it is a hommage to a 1991 Rik Mayall movie.

Even more than usual, I should credit Dr Elisabeth Bik and other less-nonymous contributors to PubPeer for providing all the images I am curating here. Interested readers should visit the source material to see how much I left out!

SOURCES:

[1]. "Cyclin L2, a novel RNA polymerase II-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells", Lianjun Yang, Nan Li, Chunmei Wang, Yizhi Yu, Liang Yuan, Minghui Zhang, Xuetao Cao (2004)
Journal of Biological Chemistry [PubPeer].

[2]."A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization", Xiaojian Wang, Nan Li, Bin Liu, Hongying Sun, Taoyong Chen, Hongzhe Li, Jianming Qiu, Lihuang Zhang, Tao Wan, Xuetao Cao (2004).
Journal of Biological Chemistry doi: 10.1074/jbc.m405147200 [PubPeer].

[3]. "Silencing of human phosphatidylethanolamine-binding protein 4 sensitizes breast cancer cells to tumor necrosis factor-alpha-induced apoptosis and cell growth arrest", Xiaojian Wang, Nan Li, Hongzhe Li, Bin Liu, Jianming Qiu, Taoyong Chen, Xuetao Cao (2005).
Clinical Cancer Research doi: 10.1158/1078-0432.ccr-05-0879 [PubPeer].

[4]. "Induction of allospecific tolerance by immature dendritic cells genetically modified to express soluble TNF receptor", Quanxing Wang, Yushan Liu, Jianli Wang, Guoshan Ding, Weiping Zhang, Guoyou Chen, Minghui Zhang, Shusen Zheng, Xuetao Cao (2006).

Journal of Immunology [PubPeer].

[5]. "hPEBP4 resists TRAIL-induced apoptosis of human prostate cancer cells by activating Akt and deactivating ERK1/2 pathways, Hongzhe Li, Xiaojian Wang, Nan Li, Jianming Qiu, Yuanyuan Zhang, Xuetao Cao (2007).
Journal of Biological Chemistry doi: 10.1074/jbc.m609494200 [PubPeer].

[6] "TLR4 signaling promotes immune escape of human lung cancer cells by inducing immunosuppressive cytokines and apoptosis resistance", Weigang He, Qiuyan Liu, Li Wang, Wei Chen, Nan Li, Xuetao Cao (2007).
Molecular Immunology doi: 10.1016/j.molimm.2007.01.022 [PubPeer]

[7]. "Rapamycin suppresses TLR4-triggered IL-6 and PGE(2) production of colon cancer cells by inhibiting TLR4 expression and NF-kappaB activation", Qiaoling Sun, Qiuyang Liu, Yuanyuan Zheng, Xuetao Cao (2008a).
Molecular Immunology doi: 10.1016/j.molimm.2008.01.025 [PubPeer].

[8]. "Rapamycin reverses TLR4 signaling-triggered tumor apoptosis resistance by disrupting Akt-mediated Bcl-xL upregulation", Qiaoling Sun, Yuanyuan Zheng, Qiuyan Liu, Xuetao Cao (2008).

International Immunopharmacology doi: 10.1016/j.intimp.2008.08.009 [PubPeer].

[9]. "IPP5, a novel protein inhibitor of protein phosphatase 1, promotes G1/S progression in a Thr-40-dependent manner", Xiaojian Wang, Bin Liu, Nan Li, Hongzhe Li, Jianming Qiu, Yuanyuan Zhang, Xuetao Cao (2008).
Journal of Biological Chemistry doi: 10.1074/jbc.m801571200 [PubPeer].

[10]. "CD69+ CD4+ CD25- T cells, a new subset of regulatory T cells, suppress T cell proliferation through membrane-bound TGF-beta 1", Yanmei Han, Qiuli Guo, Minggang Zhang, Zhubo Chen, Xuetao Cao (2009).
Journal of Immunology doi: 10.4049/jimmunol.182.1.111 [PubPeer].

[11]. "Cancer-expanded myeloid-derived suppressor cells induce anergy of NK cells through membrane-bound TGF-beta 1", Hequan Li, Yanmei Han, Qiuli Guo, Minggang Zhang, Xuetao Cao (2009).
Journal of Immunology doi: 10.4049/jimmunol.182.1.240 [PubPeer].

[12]. "Fas signal promotes the immunosuppressive function of regulatory dendritic cells via the ERK/β-catenin pathway", Cheng Qian, Li Qian, Yizhi Yu, Huazhang An, Zhenhong Guo, Yanmei Han, Yongjian Chen, Yi Bai, Qingqing Wang, Xuetao Cao (2013).
Journal of Biological Chemistry doi: 10.1074/jbc.m112.425751 [PubPeer].

[13]. "Intracellular NAMPT–NAD+–SIRT1 cascade improves post-ischaemic vascular repair by modulating Notch signalling in endothelial progenitors", Pei Wang, Hui Du, Can-Can Zhou, Jie Song, Xingguang Liu, Xuetao Cao, Jawahar L. Mehta, Yi Shi, Ding-Feng Su, Chao-Yu Miao (2014).
Cardiovascular Research 10.1093/cvr/cvu220 [PubPeer].

[14]. "The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation", Pin Wang, Yiquan Xue, Yanmei Han, L. Lin, Cong Wu, Sheng Xu, Zhengping Jiang, Junfang Xu, Qiuyan Liu, Xuetao Cao (2014).
Science doi: 10.1126/science.1251456 [PubPeer].

[15] "HSP70L1-mediated intracellular priming of dendritic cell vaccination induces more potent CTL response against cancer", Shuxun Liu, Lin Yi, Ma Ling, Jinxia Jiang, Lijun Song, Juan Liu, Xuetao Cao (2018).
Cellular and Molecular Immunology doi: 10.1038/cmi.2016.33 [PubPeer]

[16]. "MicroRNA in vivo precipitation identifies miR-151-3p as a computational unpredictable miRNA to target Stat3 and inhibits innate IL-6 production", Xiang Liu, Xiaoping Su, Sheng Xu, Huamin Wang, Dan Han, Jiangxue Li, Mingyan Huang, Xuetao Cao (2018).
Cellular and Molecular Immunology [PubPeer].

Post-Publication Expression of Concern

It appears that the Western Island is so benighted that academics there are unfamiliar with "Killed By Death":

#1 Peer 1 

Does this only work with with the one song from AC/DC or can other music be substituted?

Bryan R. Coad 

#1

In this publication we also investigated a range of monotonous frequencies. Each of these frequencies alone did not not produce as good a coating on the particles as the song. We suspect that the changing frequencies of songs provide chaotic mixing of the particles. We did not investigate other songs besides "Thunderstruck" but it might be possible to optimise the particle tumbling with other songs or frequencies. However, it is difficult to imagine having a song that rocks as much as "Thunderstruck". Dr Thomas Michl conducted this work and would be happy to answer further questions.

Thanks for your question.

Dr Bryan R. Coad, co-author

I suppose "OD'd on Life Itself" would have been inappropriate.

Ha ha, nice try

Some shitweasel at Bentham Open Sores spammed me with an invitation to inform them of any email addresses that are currently not inundated with spam:

This is me shaking my head.

I sent them Thundra's address instead.

You went full Rotwang, man. Never go full Rotwang

"Nanoblade"?! Wait, what, this sounds like a weapon that a post-humanist Ninja character might wield in a really bad Manga.

You look lost. Perhaps I should go back to the cortico-trunco-reticulo-proprio-spinal pathway (CTRPS). Or even further back.

Neuroskeptic was blogging just the other day about Sergio Canavero, who has a vision... a vision of transplanting heads from body to body... or as it may be, of transplanting bodies. But NS buried the lede, which is wasteful. Here at the Riddled Research Laboratory we prefer to stage a fake burial for the lede, in fact keeping it in a locked back room on life support, to use it as a donor for organ transplants.

So Canavero edited a special edition of Surgical Neurology International and went Full-Metal Rotwang, shaking his fists at a disbelieving world and shouting "You fools! I'll show you all!"

"Hysteria", "misbegotten dogma"... this is not the usual register of academic discourse, even in editorial advertisements, but then again, SNI is not your conventional scientific journal. It could best be described as "The kind of journal that invites a total reality-divorcé to edit special issues".

Now this "cortico-trunco-reticulo-proprio-spinal pathway", it is not recognised in textbooks, and indeed seems to be unknown outside the circle of Canavero and his supporters. It is (I gather) a component of the spinal cord. Most of the spine is white matter, afferent and efferent bundles of myelinated nerve axons, analogous to insulation-wrapped wiring, and Canavero does not believe that he can restore their function after joining the two spinal stumps in a head transplant. But that's alright, because the white matter doesn't actually have a function (its supposed importance is a 'previously misbegotten dogma'). It seems that motor and sensory functions are in fact served by the short, unmyelinated neurons of the butterfly-shape of grey matter within the spinal cord; the fools of the neurology establishment regard this as local circuitry, but this is where the CTRPS resides, and where Canavero can graft severed neurons back together.

A 'nanoblade' is required, one honed to nanometric atom-level fineness: sharp enough to bifurcate cells with minimal trauma to their membranes and contents. This may be the same as the GEMINotome "ultra‑sharp nanometer‑grade blade" -- aspirational cutlery for which Canavero's Chinese collaborators have developed the name if not the technology.

A second component of the regeneration protocol is electrical stimulation to help weld together the bisected halves of matching neurons from head and body. Canavero is aware of the parallels with Mary Shelley's novel, but he revels in them, like Gene Wilder's titular character in Young Frankenstein embracing his destiny.

If other laboratories are unable to replicate the results, they had probably tapped into the wrong kind of lightning strike to power the apparatus.

The third crucial component is the 'fusogen' to knit together the ragged edges of the severed membranes of donor and recipient hemi-neurons. This proves to be poly-ethyl glycol, infused with fractal nanoribbon graphene -- so it glows green in the dark, of course. Also to conduct the electricity ("in order to improve and accelerate the recovery of function, we tested PEG enhanced by these electrical conducting nanoribbons. PEG‑GNRs would achieve both membrane fusion, facilitate initial electrical conduction, and then act as a scaffold for sprouting fibers").

So there is re-vivifying electricity, and Daring Experiments that will Confound the Nay-Saying Fools of the Academy, you can see why the Mad Scientist Anti-Defamation League are eager to offer Dr Canavero their assistance. But wait, also there is an underground laboratory, where his South Korean colleague C.-Yoon Kim tested the procedure on rodents and dogs and monkeys!!

Alas, most post-operative rats were drowned "during a storm that filled the underground lab". Presumably the minions were watching for lightning strikes when they should have paid more attention to the water pumps. LESSON: locate next laboratory in a non-flooding mountain-top castle.

Kim's trials were not yet transplants, but proof-of-concept tests for severing and repairing spinal cords. That is to say, Kim claimed to have dissected each laboratory animal down to the neck vertebrae and split open one vertebra, so he could hook up the spinal cord and sever it. Before replacing the stumps in the vertebra, daubed with PEG, with such sub-micron precision that the two halves of each grey-matter neuron were adjacent again and the sliced membranes were contiguous, so that the PEG could fuse them -- allowing full restoration of mobility and sensation within days ("The two stumps of the spinal cord of the rats were kept in mechanical proximity by simple hyperextension of the head").

Details of the surgery ignore the earlier invocations of nanoblades and GEMINotomes, and specify a standard #11 scalpel. Which at the cellular level is about as sharp as a car bumper, crushing cells into oblivion rather than bisecting them; Kim might as well have invited Mrs Spat to sever the rats' spines, or used a cable.* To put it another way: none of this ever happened.

Kim claims affliations to Konkuk University's School of Medicine, and to Seoul National University's College of Veterinary Medicine, but the e-address he uses is vivavets@gmail.com.

* Bonus recapitation:

------------------------------------------------------

If you stare with horrified fascination at Canavero's lurid fantasies and cannot look away, well, there's more!

Imagine what effect might ensue from a young donor body (say, in her 20’s) nourishing with her young blood 24/24, 7/7 the head of an aging body recipient! Yes, life extension on a level that simple, periodic transfusions of young blood have no way to match.
The real concern that needs to be addressed in this letter is whether one day HEAVEN is spun off as a cure for transsexualism (TS). Considering the dearth of donors for many needing new organs, this might seem like pushing the envelope. Yet, it makes sense starting the debate now.
To the casual eye, TSs come in two varieties: male-to-female (MtF) and female-to-male. In this case, common sense would suggest gender reassignment HEAVEN transplant the head of an MtF subject on a female body and vice versa.

No reference here to the pioneering research of Heinlein [1970] and Reiner [1984]; I am disappoint.

"A Call to Arms" is not a treatise on limb transplants (alas), but another SNI Editorial, in which Canavero goes Full Rotwang (again), complains that billionaire philanthropists aren't giving him any money, and speculates on the psychological hang-ups that motivate his critics.