Biotech Guest post Research integrity

Fake data and real pomegranate juice in Nobelist Lois Ignarro’s papers

Louis J. Ignarro knew how to monetize his 1998 Nobel Prize for discovery of nitric oxide as molecular cell signalling agent. He made many millions selling dietary supplement for Herbalife and pomegranate juice for POM Wonderful Company. Some of that found its way (without proper conflict of interest declaration) into Ignarro's peer reviewed papers. Those, done in collaboration with certain Photoshop artists like Claudio Napoli, contain clearly fabricated data.

The Nobel Prize is the highest award there is in science, a genius certificate which makes the recipient omniscient and puts him (usually it is a he) on a God-like moral level, ie. above and free to do anything. Not all Nobel Prizes were deserved, scientifically or ethically, with James Watson being the prime example for both. Examples of Nobelists venturing into silly science or even quackery are not that rare. Linus Pauling used his Nobel intellect to find a cure for virtually everything with Vitamin C,  Luc Montagnier discovered the “memory of water”, commonly known as homeopathy, to which Montagnier soon added anti-vax conspiracies. Other Nobelists prefer to destroy their academic credibility more subtly: by selling out, admittedly for some really serious money. After all, you cannot expect a genius to survive on elite professor salary and the $1 mn Nobel Prize money alone. This is why no less than 7 Nobelists joined the board of Elysium, a fountain-of-youth-peddling biotech company founded by one, let’s say controversial, MIT researcher Leonard Guarente. Fun fact: the billionaire biotech enterpreneur Graig Venter blocked me for my comment on this related tweet:

Which brings us to another Nobelist who knows how best to monetize his Prize: Louis J. Ignarro, 1998 awarded for the discovery of nitric oxide as molecular cell signalling agent. The story is presented to you by my regular contributor Smut Clyde. Ignarro did two interesting things: for one, he went on to sell dietary supplement for the company Herbalife (where he and his consulting firm were paid $15 mn  already by 2012), as well as pomegranate juice for the POM Wonderful Company. Some of that salesman activity found its way (without proper conflict of interest declaration) into Ignarro’s scientific research output, in form of peer reviewed papers. Which brings us to another impressive collaboration of his: Ignarro co-authored a number of publications with clearly fabricated data, in collaboration with certain Photoshop artists like Claudio Napoli. Smut Clyde will guide you through the maze of duplicated images and gel bands.

It is up to the scientific community and journals to figure out if a Nobel Prize winner can ever be author of fraudulent papers, and if yes, how one is best to discreetly ignore the evidence as not to damage the venerated institution of the Nobel Prize.

Ignarro God

Image Enhancement Software: Just say NO; by Smut Clyde

A Nobel Prize in the sciences is a gratifying adornment to one’s career, and few people turn one down when it is offered, but sometimes events remind us that they are awarded for participation in a notable advance in chemistry or physics or biology as the case may be… not for infallibility, or all-round perfection of character. Nobel laureates can be just as gullible, just as venal, just as easily flattered as (say) science journalists.

This is easy for non-laureates to say, but the perspective of the laureates themselves is different; to them, it is obvious that the visit to Stockholm for the presentation of a large golden gong does make them omniscient on questions of science, and impervious to temptations and conflicts of interest. Specifically, they can safely receive funding from corporate sources without it swaying their independent judgments or their disinterested, objective research into the corporations’ products. There is no reason why they should even mention the funding, since the possibility of a “conflict of interest” is so absurd.

The topic of the Biblical theme of ‘temptation’ invites a Garden-of-Eden-themed digression: Most Western Christian traditions interpret the Fruit of the Knowledge of Good & Evil from Genesis as an apple, but this is simply a pun on the similarity of ‘apple’ and ‘evil’ in Latin. Indeed, it runs up against the obstacle that apples were not cultivated in the Middle East at the time when the myth was codified (not that bronze-ago horticulture is my area of expertise). More scholarly exegeses suggest grapes, or figs, but my favourite Forbidden Fruit is the pomegranate. What with the whole Persephone myth, and their appearances in paintings from Botticelli to Dali, pomegranates have more than their fair share of cultural resonance.

image-1535161616278
Source: “Inducible nitric-oxide synthase and nitric oxide production in human fetal astrocytes and microglia. A kinetic analysis” (Ding, .. Ignarro & Merrill: JBC, 1997)

We’ll come back to that. Right now it is time to meet Louis J. Ignarro, co-recipient of the 1998 Nobel Prize in Physiology / Medicine, for “discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system“. Also, having successfully monetarized those discoveries, recipient of “more than $15 million since 2004” from the diet-supplement company Herbalife.

Recall that in the biomedical literature, it is the custom to illustrate one’s method and evidence by displaying compass-themed electrophoresis gels – Western Blots and such – testifying to the authors’ good faith as well as to the authenticity of the experiments they report. Ignarro’s papers are no exception in this regard. As is also the custom, these papers have come under the scrutiny of the vast and cool and unsympathetic intellects who comment at the PubPeer discussion forum… often using pseudonyms to disguise the truth that they are in fact rogue AIs (or so I choose to believe).

Their observations suggest that Dr Ignarro has been unfortunate in his choice of colleagues, assuming that the cavalier attitude towards theory-validating “beautiful evidence” (Tufte 2006) is not his own. Beginning with an early example: “Antisense knockdown of inducible nitric oxide synthase inhibits induction of experimental autoimmune encephalomyelitis in SJL/J mice” (Ding, Zhang, Wong, Rogers, Ignarro & Voskuhl: Journal of Immunology, 1998). It is not ideal for three out of four lanes in a Northern Blot to be identical.

inos

In a 2001 report, there is a surprising degree of repetition between the six bands of Figure 2, i.e. the expression of six different mRNAs under five different conditions (the lanes). Did anyone actually reviewAn NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity” (Fiorucci, Mencarelli, Palazzetti, Del Soldato, Morelli & Ignarro: PNAS, 2001)?!

Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis” (de Nigris… Ignarro & Napoli: Cancer Research, 2008) and “CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy” (de Nigris, … Ignarro & Napoli: PNAS, 2010) are related in terms of content, so we treat them as a pair. Fragments of Blot proliferate and reverberate within the Figures, like frames from a Nam June Paik video, or as if they tripped and fell into a malfunctioning matter transporter.

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“Unregistered Submission” sums it up: “In lane 8 the profile of a snowman with a cap, scarf, and coat has something hovering above its head.”

Even tissue sections of tumor metastases in mouse lungs overlap more than they should. I include Figure 4B mainly so that you can share my theory that any mentality capable of straightaway seeing the disguised and recycled images is not entirely human.

kio7pog

A couple of papers are especially intriguing because they were subsequently revised with Corrigenda. After investigations by a journalist for Bloomberg, the journal editors belatedly persuaded Dr Ignarro that even potential or merely perceptible Conflicts of Interest deserve to be recognised. No aspersions were cast on the findings of health benefits from amino-acid / vitamin diet supplements, but nevertheless readers were informed of links between Ignarro and the companies producing and distributing that supplement.

So “Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice” (Napoli, … & Ignarro: PNAS, 2004), and “Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress” (de Nigris, … , Ignarro & Napoli: PNAS, 2003), both acquired this Erratum:

Ignarro notified the journal editor that he thought that disclosing his financial connection to Herbalife was unnecessary because (a) the study was conceived before he teamed up with Herbalife and (b) he had only played a minor role as a reviewer of the article [9].

 

 

So “Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice” (Napoli, … & Ignarro: PNAS, 2004), and “Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress” (de Nigris, … , Ignarro & Napoli: PNAS, 2003), both acquired this Erratum:

“the authors should have noted that Louis J. Ignarro developed and markets Niteworks, a dietary supplement of L-arginine, L-citrulline, vitamin E, and vitamin C. Dr. Ignarro is also a member of the Scientific Advisory Board of Herbalife, the distributor of Niteworks.”

The 2004 paper contains a three-fold appearance of a specific protein trace, spread across two slices of Western blot.

The 2003 paper applied a “shear stress” paradigm, cultivating coronary epithelial cells and subjecting them to three levels of shear “in a cone-and-plate viscometer” to simulate the risk factors of atherosclerosis and to trigger the NO response. Two features commend it to our attention. First, the Figures show individual Western-blot bands excised from the context of their original gel(s) and lined up neatly in grids, like a postage-stamp collection, or like a comparative display of liver flukes in a medical museum. The information density of these grids increases their persuasive impact. However, their evidential value is reduced by the discovery of nigh-identical blots, appearing in another paper from 2005, albeit in different arrangements and illustrating a different application of the shear-stress procedure.

For by 2005 Ignarro’s team had moved on to researching pomegranate juice, as a more natural dietary enhancement for the beneficial effect of those NO-mechanisms. The title says it all: “Beneficial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress” (de Nigris,… Ignarro & Napoli: PNAS, 2005).

Two more papers attested to the cardioprotective benefits of pomegranation: “Pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide” (Ignarro… & Napoli: Nitric Oxide, 2006) (yes, there are journals devoted to studying NO); and “Effects of a Pomegranate Fruit Extract rich in punicalagin on oxidation-sensitive genes and eNOS activity at sites of perturbed shear stress and atherogenesis” (de Nigris,… Ignarro & Napoli: Cardiovascular Research, 2007). If ‘pomegranation’ is not a word, it should be. The authors had become scrupulous about explicit Financial Disclosures, so later corrections were not required:

This work was funded partially by a grant from the Lynda and Stewart Resnick Revocable Trust to L.J.I and C.N. Lynda and Stewart Resnick own the POM Wonderful Company, for which L.J.I. is a consultant.

This absolves the papers from the potential accusation that they’re essentially advertisements for the POM Wonderful Company. As the name suggests, it markets pomegranate juice and pomegranate extracts:

2.1. Pomegranate juice processing

Pomegranate juice concentrate (Wonderful variety, POM Wonderful, LLC, Los Angeles, CA) and Pomegranate Fruit Extract (Trademark POMx) were used in this study. Pomegranates were handpicked, washed, chilled and stored in tanks. The fruit was then crushed, squeezed, and treated enzymatically to yield the juice and the Pomegranate Fruit Extract. Pomegranate Fruit Extract includes not only juice but also the inner and outer peels and the seeds of the pomegranate. Flavonoids constitute 40% (anthocyanins, catechins, and phenols) of total polyphenols in pomegranate juice [20–22]. Both juices were filtered, pasteurized, concentrated, and stored at −20 °C until use. More details on these compounds and preparation should be addressed to http://www.pomwonderful.com.

The Company’s owners have earned an enviable reputation for their skills in product placement and even featured in a documentary on the practice. The company pours sponsorship into pomegranate-related research, and has incurred a series of lawsuits due to its recurring use of that research to claim medical benefits such as treating or preventing “heart disease, prostate cancer, and erectile dysfunction” (h/t Whackyweedia).

u5qn6qtHowever, that does not concern us here. What does concern us is that a single Western blot illustrates two quite different experiments in the 2006 and 2007 papers (though with the eNOS band and the Actin loading control separately rotated through 180° *), and provides two different sets of measurements.**

Moreover, the eNOS protein band had earlier appeared as an Actin control band (re-cut from nine to six lanes). That original paper — “Sp1 transcription factor expression is regulated by estrogen-related receptor alpha1” (Sumi & Ignarro: Biochemical & Biophysical Research Communications, 2005) — is free from pomegranate involvement.

Having covered the pomegranation, we have exhausted the central topic of this post. But I am reluctant to finish yet, so here as coda is another recent collaboration with Filomina de Nigris and Claudio Napoli, co-authors on most of the entries above: “Glycoxydation promotes vascular damage via MAPK-ERK/JNK pathways” (de Nigris et al.: Journal of Cellular Physiology, 2012).

The paper’s noteworthy feature is its reuse of archival gel strips (albeit in modified form) retrieved from a 2000 de Nigris / Napoli paper on a different topic.

7gbmecy

De Nigris and Napoli are PubPeer personalities in their own right, whose oeuvres have attracted a cloud of commentary. De Nigris has even collaborated with Alfredo Fusco, the undisputed Italian master of image-enhancing techniques and subject of an interminable investigation. So we resist the temptation to explore that 2000 paper (although it is a showcase of research thrift, where a small stock of images are modified, retouched and recycled to illustrate different points)… it is a rabbit-hole leading to an entire warren of time-waste where one could easily get lost.

* Research with dietary supplements (“metabolic intervention”) to enhance NO signalling continued into 2006-2007, though those later papers managed to evade the editorial imposition of a Conflict-of-Interest declaration. The same combination of l-arginine and antioxidant vitamins was used in 2006 to reduce ischemic muscle damage in diabetic mice, and in 2007 to reduce ischemic damage in hypercholesterolemic mice: see “Autologous Bone Marrow Cell Therapy and Metabolic Intervention in Ischemia-Induced Angiogenesis in the Diabetic Mouse Hindlimb” (Sica, … Ignarro & Napoli: Cell Cycle) and “Therapeutic effects of concurrent autologous bone marrow cell infusion and metabolic intervention in ischemia-induced angiogenesis in the hypercholesterolemic mouse hindlimb” (de Nigris, … Ignarro & Napoli: Int. J. of Cardiology) respectively.

That opens a whole new can of fish, or kettle of worms as the case may be. For the tissue sections in these two papers illustrating reduced fibrosis after bone-marrow stem-cell treatment with / without the special diet are the same apart from a 180° rotation.

fibrosis

Who would have thought that diabetes and high cholesterol are the same condition, seen from opposite directions?

There were no tissue-sample illustrations in “Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways” (de Nigris, … Ignarro & Napoli: Journal of Cardiovascular Pharmacology, 2007). There was, however, yet another economical 180° rotation.

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Finally, “Therapeutic targeting of the stem cell niche in experimental hindlimb ischemia” (Napoli, … & Ignarro: Nature Clinical Practice, 2008) does not mention dietary supplements at all. Yet the more-or-less fibrotic tissue sections are back… recut in the manner of a contraband gemstone, and in one case rotated through 90°. For the sake of variety.

fibrosis2

* For another economical 180° rotation, see “Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways” (de Nigris, … Ignarro & Napoli: Journal of Cardiovascular Pharmacology, 2007).

** The 2007 paper also has a minor issue with tissue sections which overlap, despite purportedly coming from differently-dosed mice.

 

 


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Ignarro, with his Nobel vehicle, as he arrived to 2018 Lindau Nobel meeting

 

 

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6 comments on “Fake data and real pomegranate juice in Nobelist Lois Ignarro’s papers

  1. jimwoodgett

    Must be something about Nitric Oxide research and nutritional additives since co-laureate Ferid Murad was involved in an enterprise too: https://www.nutra-creations.com/dr-ferid-murad/

    Like

  2. 2010 Ferid Murad paper on Nitric Oxide retracted in 2017.

    http://www.jbc.org/content/285/10/6980.long
    J Biol Chem. 2010 Mar 5;285(10):6980-6. doi: 10.1074/jbc.M109.065987. Epub 2009 Dec 18.
    Stimulation of inducible nitric oxide by hepatitis B virus transactivator protein HBx requires MTA1 coregulator.
    Tri M. Bui-Nguyen‡, Suresh B. Pakala‡, Divijendranatha Reddy Sirigiri‡, Emil Martin§, Ferid Murad§ and Rakesh Kumar‡1
    – Author Affiliations

    From the ‡Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, D. C. 20037 and
    the §Institute of Molecular Medicine, University of Texas Health Science Center, Houston, Texas 77030.

    2017 retraction.
    http://www.jbc.org/content/292/11/4765

    VOLUME 285 (2010) PAGES 6980–6986

    This article has been withdrawn by the authors. Upon becoming aware of concerns raised regarding errors with respect to Fig. 3B, the authors are withdrawing the paper and apologize for these errors. The senior author states that the experiments and the final assembly of Fig. 3B were performed by specific co-authors from his laboratory. The authors state that the potential issues raised with Fig. 3B do not affect the scientific conclusions of this work.

    Like

  3. FASEB J. 2012 Jun;26(6):2558-68. doi: 10.1096/fj.11-199604. Epub 2012 Mar 5.
    2 comments on PubPeer (by: Peer 1, Hoya Camphorifolia)

    Sensing endoplasmic reticulum stress by protein kinase RNA-like endoplasmic reticulum kinase promotes adaptive mitochondrial DNA biogenesis and cell survival via heme oxygenase-1/carbon monoxide activity.
    Zheng M1, Kim SK, Joe Y, Back SH, Cho HR, Kim HP, Ignarro LJ, Chung HT.
    Author information
    1
    Department of Surgery, Ulsan University Hospital and School of Medicine, University of Ulsan, Ulsan, Korea.

    https://pubpeer.com/publications/AA5FEAE8EC26B08FC3896D253D65E8

    Like

  4. Proc Natl Acad Sci U S A. 2010 Aug 10;107(32):14484-9. doi: 10.1073/pnas.1008256107. Epub 2010 Jul 26.

    CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy.
    de Nigris F1, Crudele V, Giovane A, Casamassimi A, Giordano A, Garban HJ, Cacciatore F, Pentimalli F, Marquez-Garban DC, Petrillo A, Cito L, Sommese L, Fiore A, Petrillo M, Siani A, Barbieri A, Arra C, Rengo F, Hayashi T, Al-Omran M, Ignarro LJ, Napoli C.
    Author information
    1
    Division of Clinical Pathology, Department of General Pathology, School of Medicine, Second University of Naples, 80138 Naples, Italy.

    https://pubpeer.com/publications/0688752CBEFFC922B6D8B62802B5E2

    Like

  5. Cell Cycle. 2008 Jan 1;7(1):81-8. Epub 2007 Sep 28.
    NCX-4016, a nitro-derivative of aspirin, inhibits EGFR and STAT3 signaling and modulates Bcl-2 proteins in cisplatin-resistant human ovarian cancer cells and xenografts.
    Selvendiran K1, Bratasz A, Tong L, Ignarro LJ, Kuppusamy P.
    Author information
    1
    Davis Heart and Lung Research Institute and Comprehensive Cancer Center, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA.

    Figures 1B and 3A.

    https://imgur.com/KZp0eNr

    Figures 4 and 5B.
    https://pubpeer.com/publications/DF8CFF856F96199AD8FA16A70755E4#2

    Like

  6. Pingback: Ciarlatanerie da premio Nobel - Ocasapiens - Blog - Repubblica.it

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