Singapore: SMS alert #7
Example 1
Kannaiyan R, Manu KA, Chen L, Li F, Rajendran P, Subramaniam A, Lam P, Kumar AP, Sethi G.
Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/Akt signaling pathways.
Apoptosis. 2011 Oct;16(10):1028-41.
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore
Fig. 2
A Fifty μg of wholecell extracts were resolved on 10% SDS-PAGE gel, electrotransferred onto a nitrocellulose membrane, and probed for cyclin D1, cyclin E, p27, and p21. The same blots were stripped and reprobed with anti-actin antibody to show equal protein loading (lower panel). B Whole-cell extracts were prepared, and
50 μg of the whole-cell lysate was analyzed by Western blotting
using antibodies against XIAP, cFLIP, Bcl-2, Bcl-xL, and survivin as
indicated. C Whole-cell extracts were prepared, and 50 μg of the whole-cell lysate was analyzed by Western blotting using antibodies against c-Myc and VEGF. The same blots were stripped and reprobed with anti-b-actin antibody to show equal protein loading.
Fig. 4
“Celastrol induces caspase activation and PARP cleavage.
Cells were treated with 1 μM celastrol for the indicated times. Wholecell
extracts were prepared and subjected to Western blot analysis
using antibodies against A caspase-8, Bax, Bak, and Bid, B caspase-9,
C caspase-3 and PARP. The same blots were stripped and reprobed
with anti-actin antibody to show equal protein loading. The results
shown are representative of two independent experiments.”
Fig. 2 and 4 depict five apparently identical loading control panels for β-actin. However, according to my understanding of Western blotting, a loading control is representative of a single blot.
My question to the authors: How many different Western blots have been performed to acquire the data shown in the above figures?
Possibly, this example might be just another case of sloppiness or ignorance. But isn’t it fair to ask what NUS is going to do against the alleged inflation of sloppiness from its own ranks.
Please remember, that in a recent posting, the potentially flawed use of loading controls by the Halliwell lab has also been documented.
Example 2
Kannaiyan R, Hay HS, Rajendran P, Li F, Shanmugam MK, Vali S, Abbasi T, Kapoor S, Sharma A, Kumar AP, Chng WJ, Sethi G
Celastrol inhibits proliferation and induces chemosensitization through down-regulation of NF-?B and STAT3 regulated gene products in multiple myeloma cells.
Br J Pharmacol. 2011 Nov;164(5):1506-21
National University of Singapore
Fig. 3
Fig. 4
Apparently identical loading controls in Fig. 3 and 4 are boxed in red.
Same issue, same questions.
Fig. 2
Apparently identical panels are marked in red.
A bit too many replicats for my taste, but hey, better safe than sorry.
Let me make this point clear: These findings are examples of presumably sloppy science. I do not insinuate intentional deception. Nevertheless, it is the obligation of NUS to look into the original data of these suspect figures to make sure the appropriate original data are indeed available.
I agree with you on the red boxes marking the same data.
Reuse of data looks very much like it happens more than once in the papers above.
We can all make mistakes, that is only human, but it is more often than just the slip of the pen, or in the modern world the computer key.
Some people say that the modern electronic methods make it easier to make such mistakes, and that perhaps it was better in the old days. More like those practical lessons for children including pieces of paper, scissors, glue and string.
Nostalgia is not the answer though.
In fact a few more comments on the blog administrator.. He is very quick to point the blame at the lead author on these papers. Yet the papers contain multiple authors each of whom according to the journals i used to publish in “assume equal responsibility for the data presented.” In fact, some journals even go as far as requiring that all authors sign off for such and will not publish the article without the signed consent of all authors. Given that in the majority of cases, it is not the lead author who performs the experiments, nor prepares the figures, then the responsibility is indeed shared by all authors. As anyone who has observed the workload of a Professor would know, they actually spend most of their time in lecturing, tutoring, teaching, administrative duties, grant writing and other paperwork. The Professor considers him/herself lucky if he/she can go to the lab to find out what is going on and talk to his/her people.
Dr Zwirner has also commented on numerous occasions that it is not appropriate to publish errors and later correct them. I assume the vast majority of people would not knowingly publish an error and would prefer to correct it before publication. It is an honest but difficult act to publish corrections to published errors once they are noticed.
Actually, the internet which unfortunately this blog is using in attempts to discredit people, actually allows the publsihed scientific literature to be more fluid than in the past, and identified errors can be corrected quickly and linked with the original article. While science in any case is self correcting (any important erroneous published data will eventually be corrected in the course of further research) I applaud the capacity of the authors to be able to correct published errors to their own manuscripts.
With this in mind, a pubmed search of Zwirner, without looking into the content of his publications, shows that errata have actually been published on papers that he himself has co-authored. I understand from reading elsewhere that these errata were to remove his name from papers where he thought the data was not correct. However, according to his near perfectionist philosophy that he is preaching on this blog, should not he have detected these errors and corrected them before publication ? He has therefore exhibited the same behaviour that he is now criticizing and demonizing others for. After all, co-authors “assume equal responsibility for the data presented”. According to his approach use don this blog, it was also his responsibility to ensure that the published data was valid before publication and not simply issue a correction afterward.
Thus his approach seems somewhat duplicitous and self serving and I can only surmise he is simply a disgruntled scientist (after all he did leave Science) seeking self aggrandisment. As has been noticed by other blog posters, he could simply contact the authors if he is serious about improving the quality of science.
However, before he publicly criticizes others, his own record should be squeaky clean, which it is not, according to his own criteria. It is extremely easy to be a critic. It is extremely difficult to make a real contribution.
“Science and pseudoscience differs not in that one contains fraud and bad practices and the other does not, but in that in science there is at least a willingness to clear up the mess sometimes. ”
A quotation from
Gabor Hrasko
President – Hungarian Skeptic Society
puts it well.
My comments.
1.If the authors correct the mistakes it would go a long way to reassuring people that it was science.
2. An erratum is not the same as a condemnation, but a sign that a mistake has been made.
Professor Zwirner, as you write, did give the reasons for the errata.
To quote yourself “I understand from reading elsewhere that these errata were to remove his name from papers where he thought the data was not correct”. Do you have a problem with this?
I have no problem with erratum at all… as long as it is not a condemnation… and it is not construed as an error with ill conception or intent… I said that in my previous post…
Please send me these examples for closer scrutiny.
If as you say “As anyone who has observed the workload of a Professor would know, they actually spend most of their time in lecturing, tutoring, teaching, administrative duties, grant writing and other paperwork. The Professor considers him/herself lucky if he/she can go to the lab to find out what is going on and talk to his/her people.” then should they be taking on the responsibility to mentor and supervise grad students / post docs? If they do not have time and have not been involved then what right do they have to be an author on the publication? The lead author may not have done the experiments or made the figures but by definition lead author takes the overall responsibility for the manuscript. Before submitting it is the lead author’s responsibility to check that everything is correct.
As for some one asking for their name to be deleted as they do not think that the data is correct is a commendable act and not an act that needs to be criticized.
The quality of western blots are terrible, looks like Sethi haven’t learnt well to do westerns in BBA lab. I wonder how he managed to publish papers in BBA lab, did they really do western blot? Respective loading control are vital to interpreted the data, its not very difficult job to show equal loading using different blot. Science is certainly screwed up by the product of BBA’s. They run sweat shop rather than doing original research and advancing the knowledge.
I feel for the people who suffer because of all these issues. These things might end many good careers and happy lives. Seems that people are settling even their personal scores. My advice to affected people is to take a break from work and reflect back on their approach to science and life in general. If you can’t be honest and devoted to your work, how can you be honest to your relationships and life in general. You guys lack substance in character and need a serious effort to build (not repair) it from the scratch. Even if some mistakes are not frauds or manipulations, it seems that these people were surrounded by friends in life who never told them that they were wrong. These fair-weather friends can laugh and cry with you but they can’t give you a character and life full of self-pride. Good friends are the one who dares to tell you that you are wrong when you are. Good friends would scold you but save you from embarrassments and downfall.
Here in USA, everyday, I see many friends around me talking about science, workplace, achievements and weather. They go together and club, they go and drink, they go church and pray, they cook and eat. They just live and no one want to talk about character and values.
Wake-up guys, this is the best time for you to get your character peer-reviewed. Dump your friends as they do no good to you. Review your relationships as you might be with someone who can just suck and can not provide what you need. Evaluate your life and find friends who dares to tell you that you are wrong. Get advice from people who have character and who believe in themselves. Look at the history and you would find brave people who fought back during crisis and won their pride. Stop working now and this is your to revive your new life. Find better friends and you will never regret your efforts for a new life and a new meaning to it.
Looks to me like they cut the Western Blot membrane into strips to probe for different proteins and when needed they stripped the membrane if the proteins where close in size. Hence why the B actin is the same and they are showing a representative blot processed this way.
Some people will say that its better to do this, as your sample loading and transfer efficiency will always vary between blots so you can only compare the changes in profiles if its actually on the same blot and companies even sell cutters for this e.g http://www.inotechintl.com/products/stripcutter.php.
And if you run a separate blot for b-actin your loading etc may be incorrect so people tend to lean towards stripping the blot to probe for b-actin so its the same blot.
Its quite a wide practice worldwide in labs and if you google it you will find sites like these:
http://www.protocolpedia.com/forum/4-dna-forum/253-cutting-membrane-after-transfer
http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/western_blotting~application_support~western_blotting_experiments
http://www.gelifesciences.com/APTRIX/upp00919.nsf/content/120F2DA11D5A94F7C1257628001CC336?OpenDocument&Path=Catalog&Hometitle=Catalog&entry=0&newrel&LinkParent=C1256FC4003AED40-F4C02B388883F561C125701900490F87_RelatedLinksNew-C821BEC677D8448BC1256EAE002E3030&newrel&hidesearchbox=yes&moduleid=46950
I think this is an interesting paper that illustrates the different ways of doing Western blots….not just probing one blot once with for one protein which is more traditional. Multistrip Western blotting to increase quantitative data output. Anatoly Kiyatkin, PhD1 and Edita Aksamitiene, PhD1 Methods Mol Biol. Author manuscript; available in PMC 2010 August 18.
or see Sean Gallagher, Scott E. Winston, Steven A. Fuller, and John G.R. Hurrell
Current Protocols in Molecular Biology (2004) 10.8.1-10.8.24,John Wiley & Sons, Inc. which is a chapter on Immunoblotting and details the use of membrane strips for troubleshooting etc.
or Antibodies A Laboratory Manual by E Harlow and D Lane. pp 726. Cold Spring Harbor Laboratory.
The other one that is popular is mutliplexing with primary antibodies for western blots and there are also other techniques such as sequential transfer. I am not at ALL suggesting that the authors did this, but rather would like to also show that that there are lot of different ways of doing Western blots.
In general those of us running such techniques on a daily basis can pretty much figure it out how authors did it based on our working knowledge and standard protocols.
Yes, use of same beta actin for same stripped blots is very commonly used now a days in several publications in US as well and I think blogger is not aware of this experimental strategy. Please see below recent examples from a leading research group and they have published close to 500 papers using the same strategy:
PLoS One. 2011;6(10):e26943. Epub 2011 Oct 31.Fig.1C
J Biol Chem. 2011 Nov 7. [Epub ahead of print] Fig.5A
J Biol Chem. 2011 Nov 7. [Epub ahead of print] Fig. 2C
Cancer Prev Res (Phila). 2010 Nov;3(11):1462-72. Epub 2010 Oct 26.Fig.2A
Carcinogenesis. 2011 Sep;32(9):1372-80. Epub 2011 Feb 16. Fig.2
Dear Smith,
Use of the same photomicrograph is very common in US, especially in the MD Anderson Cancer Center and we are all aware of this experimental strategy. Please see below examples from a leading research group.
http://blog.m3.com/Retraction/20111113/The_University_of_Texas_M.D._Anderson_Cancer_Center_1
http://blog.m3.com/Retraction/20111113/M.D._Anderson_Cancer_Center_2
Also, use of same graphs for cell proliferation is correct as wild type RPMI-8226 cells are sensitive to various drugs. Please see below example from a paper published in Blood.
Blood. 2007 Mar 15;109(6):2293-302. Epub 2006 Dec 12. Fig.1C and ID.
In the legend to fig. 2 it is clearly stated that “doxorubicin-sensitive and doxorubicin-resistant, melphalan-sensitive and melphalan-resistant, and bortezomib-sensitive and bortezomib-resistant RPMI 8226 cells were plated in triplicate…. to analyse proliferation of cells”
From this information the reader could deduce with good reason that these were independent experiments with different cell types which would lead to different results and graphs. It is not at all outlined in the text that the “sensitive” cells represented the same cell type. Even in that case, however, the results and graphs should slightly vary since, according to the figure legend, they are supposed to represent independent experiments. This is apparently not the case, since the graphs (Doc-S, Mel-S, Bor-S RPMI 8226 cells) appear to be identical.
I acknowledge that the authors might have acted in good faith since they relied on the methodology and experimental description of the Blood 2007 article (Gautam Sethi coauthored both articles) which, in my view, was equally misleading .
Does this mean this is the case of Self-plagiarism? Dr. Zwirner: Smith says it is common practice in the USA and gives examples of a single professor who seems to be the former mentor of the corresponding author of papers listed above in Singapore: SMS alert #7. Does he mean to say that all 500 papers had such instances? this will be another pandora’s box if this is true..Are there any other examples from other researchers practicing this in the USA? Is this really accepted everywhere now?
Similar case is being discussed heavily on Retraction Watch: http://retractionwatch.wordpress.com/2011/10/27/cancer-journal-retracts-herbal-medicine-paper-citing-misconduct-probe/ . This incidence has attracted record number of comments.
It does not matter how many papers they have published. Misconduct is misconduct.
Take a look of this!
http://blog.m3.com/Retraction/20111113/The_University_of_Texas_M.D._Anderson_Cancer_Center_1
Here is another example from the same group.
http://blog.m3.com/Retraction/20111113/M.D._Anderson_Cancer_Center_2
There is one common link between these papers. Does he have the answer?
Yes, they could have indicated in the figure legend that all three drugs sensitive RPMI-8226 cells used are same but I guess most of the people working in this field actually are aware of this fact and hence no one was misled!
Authorities should investigate these issues as soon as possible and bring it to a closure
The reuse of western blots of loading controls is not good practice, and has led to retractions.
Retraction
DNA-PKcs Is Required for Activation
ofInnateImmunitybyImmunostimulatoryDNA
Wen-Ming Chu,* Xing Gong, Zhi-Wei Li, Kenji Takabayashi, Hong-Hai Ouyang, Yi Chen, Augusto Lois, David J. Chen,
Gloria C. Li, Michael Karin, and Eyal Raz*
*Correspondence: wmchu@ucsd.edu (W.-M.C.), eraz@ucsd.edu (E.R.)
DOI 10.1016/j.cell.2009.01.022
(Cell 103, 909–918; December 8, 2000)
We realized that the anti-IKKa (IB) loading controls presented in Figures 3A, 3B, 3C, and 4C are duplicate presentations of the same
gel lanes and do not represent the correct controls for the individual experiments. In addition, the anti-IKKa (IB) loading control in the
right panel of Figure 4C is an inadvertent duplication of the DNA-PKcs (IB) data in the left panel of Figure 5F. These errors in figure
preparation limit the interpretability of the related experimental data in these figures, which are an essential component of the support
for the main conclusions of the paper regarding the activation of IKK and NF-kB. We are therefore retracting this paper. We apologize
for these errors and for any inconvenience they may have caused. Despite these errors, we stand by the reproducibility of the experimental
data and the conclusion, which has been reached by numerous subsequent studies, that IKK and NF-kB are required for
activation of innate immunity.
Augusto Lois, a coauthor on the original manuscript, was not reachable via any of the available contact information and therefore has
not seen or agreed to the text of this Retraction.
you are right. Even smallest mistakes should not go unnoticed. Researchers get taxpayers support to do work and should show professionalism in science. I don’t know what Singapore would do but this problem is bigger than Singapore’s research problems. Many researchers move to other countries after publishing their work in a country and their problems remain unnoticed. There should be a way by which these people could be legally questioned wherever they are (I don’t know if there is any)