Integrity Initiatives in the Südhof Lab

Updated August 8, 2024

Südhof Lab Policies

As science has evolved over the last decades from a paper-based to a web-based endeavor, we have instituted procedures to enable transparent access to all data and tools of the Südhof lab beyond the documentation included in published papers. The overall lab policies are documented in our lab manual.

Current procedures include:

• Full unrestricted access to all published renewable reagents, including mice (via Jackson lab) and plasmids for non-profit research entities
• Access to all experimental protocols
• Public access to software applications (Apps) and algorithms (https://med.stanford.edu/sudhoflab/science-resources/tools.html)
• Deposition of all raw data for published papers in the publicly accessible Stanford Data Repository (as of 2023) or other appropriate databases
• All manuscripts and published papers are screened for copy-paste errors using newly available sophisticated A.I. tools
• Conversion of all lab notebooks to digital form (as of 2023)
• Publishing all papers with an open access CC BY 4.0 license
• Publication of reviews with papers as far as allowed by journals
• Facilitating lab visits to disseminate key technologies

In addition, all experiments in the Südhof lab are required to conform to the following guidelines:

• Experimenters are unaware of (i.e., ‘blinded’) the identity of samples or animal subjects, i.e., these are anonymized whenever possible
• All experiments are carried out in at least three independent replicates (not pseudo-replicates)
• All major conclusions are based on at least two experimentally distinct methods
• Biological sex is considered an independent biological variable whenever appropriate
• All experiments are documented in virtual notebooks stored in Stanford box
• All primary data are reviewed in lab meetings by multiple lab members

With these rules, we aim to promote transparency and reproducibility. Arguably the biggest challenge in maintaining science integrity is not the fabrication of data, but issues in analyzing and interpreting data. The unrestricted access to raw data will greatly help to meet this challenge because it enables others to evaluate interpretations and amend them as necessary.

For our efforts in promoting data availability and transparency, our lab recently received the Stanford University Libraries Data Sharing Prize, an Open Science Award from CORES.

Social Media (PubPeer) Scrutiny of Südhof Lab Work

Websites such as PubPeer provide a valuable service to the scientific community in identifying potential problems with published papers, generally focusing on discrepancies in a paper’s data but not on the reproducibility or validity of conclusions. These posts have helped us to identify problems we overlooked and to correct errors we made.

Regrettably, however, PubPeer and other social media are non-transparent, censor responses, and use anonymous commentators with continuously changing aliases. Moreover, PubPeer posts are communicated to journals, journalists, and university administrators. Many PubPeer comments appear to pursue an agenda unrelated to science integrity. We nevertheless respond to such comments because journals sometimes accept accusations without examining their plausibility. Furthermore, a common strategy by PubPeer commentators is to repeat allegations in different ways to enhance impact, often accompanied by graphs and animations that confer a veneer of seriousness. Even if an accusation is rather implausible, this strategy creates an aura of ‘something is wrong here’ that is difficult to counter.

In addition, many PubPeer commentators maintain commercial websites communicating their findings and have a conflict of interest. Based on PubPeer posts, publications on sites such as ‘ForBetterScience’ and ‘Spectrum’ provide ad hominem criticisms that are impossible to refute. Furthermore, commentators continuously publicize their PubPeer criticisms on ‘X’ (formerly Twitter) and are invited to give paid presentations about science rigor and reproducibility based on their PubPeer posts and social media activities. In particular, critics recently used new sophisticated computational tools to analyze >10,000 images in our published papers and preprints, identifying copy-paste errors in multi-image panels that were previously undetectable (see analysis below). As a result, serious newspapers and YouTube presentations now report that our papers are ‘questioned’ and that there is a scandal without actually examining the controversial nature of these ‘questions’ or the substance of the allegations.

Potential vs. Pitfalls of Computational Error Searches in the Literature

Two principal types of errors in papers are detected by programs such as ‘ImageTwin’ or ‘ProofIG’. First, accidental copy-paste errors of images or data that occur when the copy function fails and a previously copied image or data is inserted twice, leading to image or data duplications. Second, the intentional and inappropriate reuse or manipulation of images or data. Only the second type of error constitutes misconduct. At least two criteria can distinguish these two types of errors. First, unintentional copy-paste errors usually provide no benefit to a paper, i.e. they do not improve the paper. Second, copy-paste errors are generally isolated instances among a large number of images or data in a paper, with many people in a lab committing them inadvertently, whereas data manipulations often recur for the same scientist. Intentional image manipulations are misconduct that include blacking out features of an image or inserting parts of a different image (sometimes referred to as ‘cloning’). The wide availability of programs that enable checking manuscripts for duplications will prevent copy-paste errors in future but the recent availability of image editing software will also, regrettably, facilitate creating fake images that cannot be detected with current programs.

A major problem is that accusations of data duplications or manipulations can be unfounded. For example, when immunoblots are imaged at high resolution but reproduced at low resolution, the bands can assume a ‘halo’ that is not indicative of an intentional insertion of bands (a crude form of misconduct) but simply represents a digital reproduction artifact. Moreover, immunoblots produced by the same gel apparatus with similar samples and analyzed with comparable secondary antibodies often exhibit the same shapes and artifacts even if they are from different gels (see image below that illustrates the same artifact in four different blots with different samples!). Not infrequently allegations on PubPeer are mistaken because current software suggests duplications that don’t exist, rendering a paper ‘questioned’ even though it should not be (but giving the commentators another PubPeer entry)!

In addition, some PubPeer accusations wrongly claim image manipulations because of digital image reproduction artifacts. For example, Dr. Bik suggested in a widely retweeted accusation on ‘X’ that an immunoblot we published in 2010 (Supplementary Figure S6b of O.H. Shin et al.; # 37 below)) was intentionally manipulated. This accusation was based on the following analysis:

The areas boxed in different colors were alleged to be intentionally duplicated. Note that the tiny allegedly cloned areas of similar background signals always occur in the same blot, not between blots, partly overlap, do not correspond to gel lanes, and are randomly distributed in the image. Besides the fact that it would make no sense to duplicate such small areas of background – a fraudster could just run a gel with empty lanes – and that such duplications do not improve the data, overlapping duplications like this were nearly impossible to manufacture in 2010 without border effects. Similar artifacts in immunocytochemistry images have caused waves of criticisms. An accusation by Dr. Bik against another lab provides a beautiful example of how such artifacts occur (https://pubpeer.com/publications/C9E4F18C603C449A0CD32876B719A5). A general problem here is that digital reproductions of all kinds of images can create artifactual microduplications especially if the image resolution is changed during reproduction or if the images were processed with older software. These artifacts are not intentional manipulations but byproducts of digital imaging processing procedures.

The Economic and Personal Benefits and Costs of Scientific Fraud Detection Efforts

Fraud constitutes a serious problem in science, especially when it is the basis for a large number of follow-up studies or clinical trials as described in an excellent recent book (Charles Piller “Doctored: Fraud, Arrogance, and Tragedy in the Quest to Cure Alzheimer’s”). Obviously it is essential for scientific papers to present data that are accurate descriptions of experimental results. Moreover, if we as scientists discover errors in a paper that could potentially affect its conclusions, the paper needs to be retracted even without evidence of fraud (see the example of P.Y. Lin at al. (2023) [paper #8 below]). Similarly, if a paper contains potentially manipulated data, even if it is just one figure among many, a retraction is generally considered necessary although the remaining data, often obtained by scientists who had no part in the manipulation, are sound. Intentional reporting of incorrect data is unethical because it wastes scarce resources, potentially aggravates patient suffering, misleads other scientists, and produces a sense of disappointment in all serious scientists.

Having said this, however, it should be noted that there is also a dark side to efforts that aim to uncover scientific fraud. If the alleged mistakes are unintentional errors of no scientific consequence, or worse if the allegations of mistakes are incorrect, due to overeager critics with an incomplete understanding of the science, science integrity efforts can cause anguish and economic waste. Especially the costs to young scientists can be horrendous. Many junior scientists feel personally attacked by accusations of misconduct although their only mistakes are copy-paste errors or other trivial oversights. Since such accusations are published on PubPeer, communicated to journals, and publicized by ‘tweets’ and webposts, accusations of minor errors not only cause private anguish but also public repercussions. Moreover, they incur economic losses since these accusations have to be dealt with in multiple communications, record searches often dating back decades, and sometimes new experiments. In my own lab, such accusations have pushed junior scientists – all female – out of experimental science and led to soul-searching among those who decided to continue in science. Thus, I believe that care should be taken to uncover actual fraud and not to necessarily criticize every possible minor mistake that has no impact on an overall project.

As a scientific community, we need to communicate to the public that we do make mistakes. We have to ask ourselves whether correcting decades-old minor mistakes that nobody would ever notice without A.I. software is warranted. Do we really have to conform to social media pressures from people who are utterly uninterested in the actual science and claim that absolute purity in science is a justifiable goal? Moreover, if a paper contains data manipulations that constitute only a tiny fraction of the project, is it justified to retract the entire paper in the name of scientific purity, or shouldn’t we have a more appropriate mechanism to deal with this problem? We in the scientific community need to abandon the claim of science infallibility: Science does involve mistakes, let’s admit it. At the same time, however, we should also defend the value of imperfect science against attackers whose only goal appears to be to take down scientists without regard to the actual scientific content.

Source of errors in papers from the Südhof lab

As described below, more than 20 students and postdocs in my lab over 20 years committed copy-paste mistakes in papers identified by PubPeer comments and by us. In addition, we discovered major mistakes in two papers, with one paper containing possible image manipulations. The presence of isolated copy-paste errors in papers represents a systemic problem. Nearly all of our errors involve figures containing copious images (usually >20, sometimes >300), most of which depict control conditions that look alike. Once a copy-paste mistake was committed, neither the student/postdoc who assembled the figure nor I could detect it until new computational analysis tools became recently available. The nature of these mistakes – insertion of incorrectly duplicated representative images or duplicated numbers in data files after analysis – means that the mistakes have no effect on the findings or conclusions of a paper. Moreover, none of these mistakes improved the data appearance in a paper. They provided no benefit to the person who made them. A plot of papers published (green circles, see below) and papers with errors (red circles) vs. the year of publication illustrates the temporal occurrence of our lab’s errors compared to the number of papers published.

This plot reveals that errors do not correlate with the number of papers published or with particular postdoctoral fellows/students in the lab but with the advent of the pasting of digital images and of publishing data excel files, both of which are susceptible to copy-paste errors. A preliminary sampling of the literature revealed that similar copy-paste mistakes are widespread and multiple accusations against my lab actually identified copy-paste errors in other labs. The software that enabled detection of these errors in recent years will also prevent their recurrence in future, together with the advances in raw data storage that are now in place.

In addition to the copy-paste errors in our lab that account for the vast majority of questioning, reanalysis of the raw data of one paper (#8 below) revealed that the data were incorrectly analyzed (not identified by PubPeer) and that this mistake was compounded by copy-paste errors, leading us to retract the paper although the raw data are available, there is no evidence of data manipulations, and we continue to believe in the conclusions. Furthermore, we recently discovered with the help of an external image analysis expert that representative images in another paper (#27 below) contain inexplicable abnormalities, which again will warrant a retraction of the paper even though the quantitative analyses and conclusions are correct.

Main PubPeer accusers of the Südhof lab

Originally most PubPeer contributors were apparently idealistic science enthusiasts. More recently, professional “science integrity investigators” who may derive income from their activities appear to dominate. The thrust of criticisms appears to have shifted from examining science content to identifying generally minor mistakes, with the implication that minor mistakes are not just human but signify a big problem. The majority of accusation against our lab on PubPeer are made by four commentators, Dr. Elisabeth Bik, Dr. Maarten van Kampen (pseudonym Orchestus quercus [https://pubpeer.com/publications/F7C42C356B2E7049FDB68A434EF4F8]), ‘Actinopolyspora biskrensis’ (identity unknown), and Dr. Kaveh Bazargan (pseudonym Illex illecebrosus [https://pubpeer.com/publications/5813077CE8B5C29E479FD50C259F77]). The four major accusers of our lab often operate like a tag team creating an echo chamber in which one person (usually Dr. Bik) first makes an accusation and the others then repeat the accusation from various angles.

Dr. Elisabeth Bik is a professional integrity consultant who may provide paid services to universities, journals and other customers. Dr. Bik specializes in image duplications and manipulations but appears to have no formal training in image analysis or computational image processing. Dr. Bik’s comments rarely discuss the actual science, nor do they consider whether alleged data manipulations are relevant for a paper’s findings. Dr. Bik spent 15 years as a long-time postdoctoral research associate at Stanford and several years in industry and science publishing before her current job. Dr. Bik’s PubPeer posts on my lab are mirrored by tweets implying misconduct without explaining the actual content of accusations or their relevance. 

Dr. Maarten van Kampen (pseudonym Orchestus quercus) frequently follows up on Dr. Bik’s comments and echoes them. He is a contributor to the website “ForBetterScience” run by Dr. Leonid Schneider that published ad hominem insults against me personally even though I have never met either him or Dr. Schneider. In repeating Dr. Bik’s accusations, Dr. van Kampen often uses probability arguments but appears to have no training in biomedical research.

Actinopolyspora biskrensis (anonymous) also usually provides secondary reinforcements to Dr. Bik’s accusations without new information. They often attempt to generalize individual mistakes to imply systemic fraud. Like Dr. van Kampen, ‘Actinopolyspora’ is a collaborator with Dr. Leonid Schneider on the website “ForBetterScience” (see https://pubpeer.com/publications/D8EAA6F915EE4008B654738F66ABE6) that published ad hominem insults against me personally. Actinopolyspora’s conflict of interest cannot be assessed owing to the anonymity of the posts.

Dr. Kaveh Bazargan (pseudonym Illex illecebrosus) is a physicist with a PhD in display holography. He founded “River Valley Technologies” (https://rivervalley.io/), a company that offers paid publication and proofing services. Dr. Bazargan’s frequent comments on PubPeer on our work also generally echo Dr. Bik’s accusations, mostly reinforcing her allegations with ‘animations’ that often provide no new information but are amplified on ‘X’ (formerly Twitter). He seems to ask for retractions based on single copy-paste mistakes.

Complete Accounting of Südhof Lab Mistakes and Unfounded Allegations

Given the persistent public scrutiny of our lab's work by the four accusers described above, we here provide full accountability and transparency of our lab's work and errors. We will discuss all PubPeer accusations , describe our uncensored responses, reveal mistakes we identified that have not (yet) been listed on PubPeer, and elaborate our efforts to resolve legitimate concerns.

Südhof lab PubPeer accusations are summarized in reverse order of a paper's first criticism or of our identification of a problem. Accusations are numbered according to PubPeer entries, with gaps in numbers caused by responses or irrelevant comments. We classify accusations based on their resolution as ‘unfounded’, ‘minor error’, and ‘major error'. We include accusations directed against our lab but concerning data from other labs. We realize that as scientists, we are often reluctant to admit mistakes and therefore tried to concede errors wherever these exist, even if trivial. However, PubPeer accusers are also loath to admit mistakes in their allegations and repeatedly try to contend the same point, often with fancy graphics and animations that purport to represent an analysis. This frequently results in an unending discussions on PubPeer in which our responses are censored, prompting us to provide this accounting for the sake of transparency. 

Total number of cases

• Sudhof papers with minor errors:                                           23
• Sudhof papers with major errors:                                            2
• Total number of unfounded accusations:                            63
• Number of accused Sudhof lab trainees:                             34
• Fraction of errors per image, graph, or table:            <0.002        

Detailed discussion of individual papers

46. Paper: Pak, C.H., Danko, T., Zhang, Y., Aoto, J., Anderson, G., Maxeiner, S., Yi, F., Wernig, M., and Südhof, T.C. (2015) Human Neuropsychiatric Disease Modeling Using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1. Cell Stem Cell 17, 316-328.

PubPeer Weblink:  https://pubpeer.com/publications/4BF247658B9C771C11277691085AE5

#1 (July 2024)

Accusation: Dr. Bik discovered that two panels in Figure S6 are switched.

Resolution: Minor copy-paste mistake during figure assembly

Classification: Minor error

***

45. Paper: Um, J.W., Pramanik, G., Ko, J.S., Song, M.Y., Lee, D., Kim, H., Park, K.S., Südhof, T.C., Tabuchi, K., and Ko, J. (2014) Calsyntenins Function as Synaptogenic Adhesion Molecules in Concert with Neurexins. Cell Reports 6, 1096-1109.

PubPeer Weblink: https://pubpeer.com/publications/10E74576C4033F2A67749B3395FEFD

#1 (July 2024)

Accusation: Dr. Bik identified a possible image duplication in Figure 3A

Resolution: Our lab made only a minor contribution to this paper but the contributing lab who made this mistake acknowledged the duplication.

Classification: Minor error by another lab

***

44. Paper: Boucard, A., Maxeiner, S., and Südhof, T.C. (2014) Latrophilins Function as Heterophilic Cell-Adhesion Molecules by Binding to Teneurins: Regulation by Alternative Splicing. J. Biol. Chem. 289, 387-402.

PubPeer Weblink: https://pubpeer.com/publications/7B45E6171BCB1F3B805C2BDC316DF0

#1-4, #6 & #8 (July 2024)

Accusation: Dr. Bik and associates identify image duplications in panels of Figure 1G; Dr. Bazargan follows it up with ‘animations’, and Actinopolyspora reinforces the accusation with generalized questions.

Resolution: Dr. Bik and Co accurately identified errors in a panel of representative images that were undetectable without A.I. tools.

Classification: Minor error

***

43. Paper: Arancillo, M., Min, S.W., Gerber, S., Münster-Wandowski, A., Wu, Y.J., Herman, M., Trimbuch, T., Rah, J.C., Ahnert-Hiler, G., Riedel, D., Südhof, T.C., and Rosenmund, C. (2013) Titration of Syntaxin 1 in mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability. J. Neurosci. 33, 16698-16714.

PubPeer Weblink: https://pubpeer.com/publications/25105898138827EDD8E4A7332B871D

#1 & #2

Accusation: Dr. Bik claims that the Rab3 and NSF blots in Figure 1G are ‘remarkably similar’ and criticizes the fact that the blots are put together from different gels (she calls this ‘splicing’); Dr. Bazargan (‘Illex’) as always follows this up with ‘animations’.

Resolution: High-magnification images show that the blots are clearly different. Although the bands are very similar as expected for blots run on the same gel apparatuses, the lanes ran differently, with a much larger gap between lanes for the smaller Rab3A protein than the larger NSF protein again as expected (see red circles in image). Assembling illustrative blots from different lanes was perfectly acceptable practice before PubPeer started to criticize it. The blot predates the digital era and was obtained using ECL and film at UT Southwestern.

Classification: Unfounded

***

42. Paper: Sun, W., Liu, Z., Jiang, X., Chen, M.B., Dong, H., Liu, J., Südhof, T.C., and Quake, S. R. (2024) Spatial and single-cell transcriptomics reveal neuron-astrocyte interplay in long-term memory. Nature 627, 374-381.

PubPeer Weblink: https://pubpeer.com/publications/6123C544EAE3A2F1FE1D9CE476E00C

#1 (July 2024)

Accusation: None – simply cites an unreviewed preprint that alleges that our study is ‘unlikely to replicate in future’ because the authors did not agree with some of the statistical analyses in our paper.

Resolution: It is impossible to respond to a reference to an unpublished paper, except to say that this post obviously ‘questions’ yet another paper in the success list of PubPeer to have identified ‘questionable’ papers. However, we applaud the BioRxiv paper authors for initiating an open scientific discussion in which we will engage instead of the anonymous PubPeer allegations that are impossible to discuss fairly.

Classification: Uninterpretable accusation

***

41. Paper: Gallardo, G., Schlüter, O.M., and Südhof, T.C. (2008) A molecular pathway of neurodegeneration linking a-synuclein to ApoE and Ab-peptides. Nature Neurosci. 11, 301-308.

PubPeer Weblink: https://pubpeer.com/publications/02B530E2D617B034C0E76407D16B99

#1 (July 2024)

Accusation: Dr. E. Bik alleges that there is a blot duplication between Figures 2C and 6C.

Resolution: This is another A.I.-driven erroneous identification of a non-existing blot duplication. High magnification images show that the two blots exhibit distinct backgrounds.

Classification: unfounded

#3 (July 2024)

Accusation: Dr. K. Bazargan, commenting as ‘Illex’, echoes Dr. E. Bik’s accusation using an animation that provides no new information.

Resolution: This comment is similar to many others by ‘Illex’ who always seems to follow Dr. Bik’s comments by providing an animation to offers no additional information.

Classification: unfounded

#4 (July 2024)

Accusation: An anonymous commentator alleges that error bars are missing in Figure 4a.

Resolution: The error bars are not missing at all. The SEMs were simply too small to be visible in the graph.

Classification: unfounded

***

40. Paper: Shin, O.-K., Rizo, J., and Südhof, T.C. (2002) Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells. Nature Neurosci. 5, 649-656.

PubPeer Weblink: https://pubpeer.com/publications/9BAEF55F4945697536FB528482F5B7

Date of first mistake allegation: May 2024

#1 to #6 (May 2024)

Accusation: Dr. E. Bik and her colleagues allege that among the 70 different blots shown in this paper, several bands on the same blot, but not between blots, look ‘surprisingly similar’, implying that the images were manipulated. In total, in pairwise comparisons 9 ‘surprising’ similarities were alleged among a total of 556 bands (>300,000 comparisons in total). Strikingly, all allegedly duplicated bands are always adjacent to each other. Thus, the allegations here are not that blots were duplicated but that a fraction of adjacent bands were duplicated in a small subset of bands were duplicated.

Resolution: Since Dr. Bik and her associates somehow convinced Nature Neuroscience that their allegations are worthy an investigation and since PubPeer does not enable a scientific discussion, we respond here in detail.

The blots reported in this paper were produced by ECL amplification of immunoreactive bands that were visualized on film and then scanned or photographed (not sure which since the blots were obtained almost 25 years ago). Photographs of the blots were then pasted into an illustration manually, and the illustration was photographed as a figure for a paper. The original raw data were archived as required by law at UT Southwestern when I left that institution in 2008.

High-magnification images of 3 examples of blots alleged to represent duplications among reveal four interesting features:

First, the bands are indeed very similar – only 9 such cases exist out of >300,000 comparisons. Second, the 9 similar bands are ALWAYS next to each other. Third, the background surrounding the similar bands contains many non-identical features (yellow circles) and is thus clearly different, i.e. not duplicated. Fourth, there is no sign of band ‘splicing’ or of any other manipulations of the images.

The fact that there are no indications of band splicing or other physical or digital manipulations is important given the time when these experiments were performed (approximately 25 years ago) and the non-digital methods used. At that time, no AI-driven image reconstructions were available to seamlessly insert bands of proteins into different backgrounds and all image manipulations would be visible at high magnification. The other feature that is very significant is that the 9 similar bands (out of 556) are always next to each other.

How can we explain the fact that the bands are very similar but the images are overall non-identical and there is no sign of image manipulations? Two plausible explanations are possible. First, that this is an accidental similarity in <2% of the bands of similar samples run on the same gels. Second, the image processing software during scanning of the blots or during publishing may have introduced duplications of internal image components when the software perceived these components to be the same, even if they are not the same. Such phenomena were observed to explain other image manipulations alleged by Dr. Bik. Digitization and compression algorithms, especially older ones, used ‘pattern matching’ and economize by simply duplicating matched patterns. This error was, for example, observed in old Xerox copy machines “The source of the error was a bug in the JBIG2 implementation, which is an image compression standard that makes use of pattern matching to encode identical characters only once. While this provides a high level of compression, it is susceptible to errors in identifying similar characters.” (see https://en.m.wikipedia.org/wiki/Xerox#Character_substitution_bug). This has also been documented for immunocytochemistry images in older papers (see https://pubpeer.com/publications/C9E4F18C603C449A0CD32876B719A5, an example where the responsible scientist was lucky to retain very old images that unfortunately are often not available – otherwise he would have been able to argue against Dr. Bik’s accusations). We are not image analysis algorithm experts and cannot assess what older image processing software may or may not have done to images, nor do we know why only some bands are ‘pattern matched’ whereas others are not.

To summarize, the magnifications show that the images were not intentionally manipulated and the sporadic band duplications in the Shin et al. (2002) paper are most likely a chance even due to image processing artifacts that duplicate matching image elements.

Classification: unfounded

#8 & #9 (July 2024)

Accusation: Dr. E. Bik claims that it is impossible that the isolated band similarities are due to a pattern matching algorithm artefact or a similar computational error in image processing downstream of the actual data production.

Resolution: We are not sure that Dr. E. Bik has the computer science background based on her training to make such a definitive judgement about image processing software in use 25 years ago. Moreover, we are disturbed that we as scientists are thought guilty if we cannot prove our innocence. Dr. Bik doesn’t actually provide an explanation for the small fraction of similar adjacent bands. Instead, she demands that we explain them and if we can’t, we are supposed to be guilty.

Classification: unfounded

***

39. Paper: Hosaka, M., Hammer, R.E., and Südhof, T.C. (1999) A phospho-switch controls the dynamic association of synapsins with synaptic vesicles. Neuron 24, 377-387.

PubPeer Weblink: https://pubpeer.com/publications/3A1A9BCBA901F9A66ED727A7912877

Date of first mistake allegation: June 2024

#1 (June 2024)

Accusation: Dr. E. Bik observes that the immunoblots for Synaptogyrin and Rab3a in Figure 8 look ‘remarkably similar’, implying that they are duplicated, although she concedes that there are differences.

Resolution: This is a typical A.I.-driven erroneous identification of a non-existing blot duplication. The two blots are expected to be 'remarkably similar' given that Synaptogyrin and Rab3a are both synaptic vesicle proteins which biologically behave the same way in this particular experiment, and that the immunoblots are for proteins of similar but different molecular weights (~35 kDa vs. ~25 kDa) that were analyzed on the same gel and probed on the same blot which was cut into stripes. As a result, the bands exhibit the same relative changes and display the same blotting  artifacts. Note also that this blot derives from the predigital area when copy-paste mistakes were very unusual.

Classification: unfounded

#2

Accusation: It is improper for us to thank a lab member for an initial technical contribution to the project in the Acknowledgement section of the paper.

Resolution: We do not believe that thanking a lab member in an Acknowledgement section is inappropriate.

Classification: unfounded

#4, #6 #8, #10, #12 & #14 (July 2024)

Accusation: Dr. K. Bazargan (commenting as ‘Illex Illecebrosus’) and ‘Actinopolyspora biskrensis’ continue to repeatedly allege that the two blots are the same and that there is a suspicious duplication without any new information.

Resolution: To avoid the real possibility that a journal, Stanford, or NIH takes these fraud allegations seriously, I will describe in detail these experiments that were performed more than 25 years ago. The blots reported in this paper were produced by ECL amplification of immunoreactive bands that were visualized on film and then scanned or photographed. The scanned or photographed images were then manually pasted into an illustration, photographed, and processed digitally for publication. The original raw data were archived as required by law at UT Southwestern when I left that institution in 2008. A high-magnification image of a section of the blot is shown below:

The bands are indeed very similar as would be expected from the experiment (see explanation above). However, there are significant differences that show that the blots are NOT identical (see areas marked by yellow circles that exhibit differences in background). These subtle differences in addition to the fact that the bands exhibit distinct thicknesses as would be expected again from slightly different antibody affinities show that the blots are different.

Classification: unfounded

#16 (July 2024)

Accusation: The ‘moderator’ (who may well be identical to the major accusers of our lab) states “Dr Südhof is obviously unwilling and/or unable to address the issues. However unsatisfactory that conclusion may be, we may limit fruitless, repetitive discussion.”, basically claiming that our responses do not address the issues.

Resolution: We feel this comment exposes the nature of PubPeer: Even the ‘moderator’ is partisan. No wonder that our comments get censored

Classification: unfounded

***

38. Paper: Pang, Z.P., Sun, J., Rizo, J., Maximov, A., and Südhof, T.C. (2006) Genetic Analysis of Synaptotagmin 2 in Spontaneous and Ca2+-Triggered Neurotransmitter Release. EMBO J. 25, 2039-2050.

PubPeer Weblink: Not yet publicized by PubPeer but posted on the first author’s website

Date of first mistake identification/allegation: May 2024

#1

Mistake identified: Supplementary Figure S1 contains a single blot image duplication involving a control condition.

Resolution: The journal has been contacted and the correct blot has been submitted.

Classification: Minor error identified by lab

***

37. Paper: Shin, O.-H., Lu, J., Rhee, J.-S., Tomchick, D.R., Pang, Z.P., Wojcik, S., Camacho-Perez, M., Brose, N., Machius, M., Rizo, J., Rosenmund, C., and Südhof, T.C. (2010) Munc13 C2B-domain – an activity-dependent Ca2+-regulator of synaptic exocytosis. Nature Struct. Mol. Biol. 17, 280-288.

PubPeer Weblink: https://pubpeer.com/publications/CF9BF3F6AF7EACB2FB3C0581A8AB76

Date of mistake identification/allegation: May 2024

#1

Mistake identified: Dr. E. Bik claims that the Suppl. Figure 6b immunoblot stripes (reproduced digitally at low resolution by the journal from a non-digital original blot) contains tiny areas of microduplications in the background pattern (not the actual signal).

These areas are tiny, within a blot, randomly distributed, and only digitally identifiable. She implies that these blots are suspicious and could be manipulated, and claims to have detected them by eye. Subsequent comments by Drs. Bazargan and van Kaampen, Actinopolyspora and others echo Dr. Bik’s accusation repeatedly without new information. Moreover, the accusers also allege that it is improper to ask why someone would want to commit a fraud consisting of tiny background duplications that do not conform to lanes or bands (see image) and that we as scientist have a duty to explain these allegedly duplicated areas and that they as accusers have a right to allege that these are manipulated without further evidence or apparent benefit to the manipulator.

Resolution: This is an unusual accusation that targets bits and pieces of two blot stripes from the pre-digital era in a supplementary figure panel containing 7 such stripes. We dispute the accusers claims at several levels. First, we note that if these micro-areas were truly intentionally duplicated and not created by an image-processing artifact with a non-digitally acquired blot that are reproduced at low resolution, then these manipulations would serve no purpose since they are in an area of the blot that contains no signal. Second, the allegedly intentionally duplicated micro-areas do not conform to the lanes of the blot or of the protein signal. Third, the two sets of allegedly duplicated micro-areas are only within a blot stripe but not between blot stripes. The last finding is diagnostic: clearly the scrambling of bits of image pieces was intrinsic to the image. The accusers could argue that maybe there were lots of smudges on the gel that were supposed to be covered up, with each smudge covered by a different ‘cloned’ micro-area. However, there is no evidence of smudges in any of the other blots. Moreover, the covering would have to be done without a trace at a time at which seamless image manipulations were difficult and would use different pieces within a blot without sharing between blots – a scenario that is highly unlikely. Finally, we feel that it is legitimate to ask about motivation: who benefits? Even though Dr. Bik maintains that she won’t speculate about motivations in her accusations and tries to exclude consideration of motivation, her own accusations do imply a motivation  as evidenced by her use of the word ‘suspicious’. The most likely explanation for this finding, like for many of the ‘mistakes’ identified by Dr. Bik’s A.I.-powered software, is that these random microduplications are simply a reproduction artifact of a digitized image.

Classification: unfounded

***

36. Paper: Maximov, A., Shin, O.-H., and Südhof, T.C. (2007) Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release. J. Cell Biol. 176, 113-124.

PubPeer Weblink: https://pubpeer.com/publications/1623AD50C70F6D92C1BD05B69EEED3

Date of mistake identification/allegation: May 2024

#1 & #2 (May 2024)

Mistake identified: A control electrophysiology trace may be duplicated. As usual, Dr. Bik’s accusation is followed by Dr. Bazargan’s  ‘animation’ that repeats and amplifies the accusation without adding information.

Resolution: We will contact UT Southwestern which retained all original data by law to determine if this is a duplication or simply a similar trace.

Classification: Likely minor error

#4 (August  2024)

Mistake identified: Dr. Bik demands to see the original traces

Resolution: As mentioned above, the experiments were performed 20 years ago at UT Southwestern, which retained all original data as required by law. These data are property of UT Southwestern and  no longer under our control.

Classification: Irrelevant comment

***

35. Paper: Gokce, O., and Südhof, T.C. (2013) Membrane-Tethered Monomeric Neurexin LNS-Domain Triggers Synapse Formation. J. Neurosci. 33, 14617-14628. PMCID: PMC3761060

PubPeer Weblink: https://pubpeer.com/publications/DB2D4970F91BF62729727D5DE39975

Date of mistake identification/allegation: We identified and corrected this mistake in the journal but Dr. Bik afterwards repeated our own identification of the mistake without new information

#1 (May 2024)

Mistake identified: Dr. Bik repeats the identification of the copy-paste mistakes in the images, possibly to be able to ‘question’ another paper from our lab

Resolution: No resolution necessary. A correction has been published

Classification: Minor mistake uncovered by the lab

***

34. Paper: Lee, K., Kim, Y., Lee, S.-J., Qiang, Y., Lee, D., Woo Lee, H., Kim, H., Je, H.S., Südhof, T.C., and Ko, J. (2013) MDGAs selectively interact with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development. Proc. Natl. Acad. Sci. U.S.A. 110, 336-341.

PubPeer Weblink: Not yet publicized by PubPeer but PNAS published a Correction

#1 (May 2024)

Mistake identified: Image duplication in Suppl. Figure 2B

Resolution: A correction has been published by the journal

Classification: Minor mistake by another lab and uncovered by the lab

***

33. Paper: Wöhr, M., Fong WM, Janas JA, Mall M, Thome C, Vangipuram M, Meng L, Südhof, T.C., and Wernig, M. (2022) Myt1l haploinsufficiency leads to obesity and multifaceted behavioral alterations in mice. Mol. Autism, 13, 19. PMCID: PMC9087967

PubPeer Weblink: https://pubpeer.com/publications/6AEE2ED764E4EA67E352B62B92D882

#1 (April 2024)

Accusation: Image duplication in Supplementary/Additional File 2

Resolution: The accusation is likely correct but the data are not from our lab

Classification: Likely minor mistake from another lab

***

32. Paper: Ko, J., Soler-Llavina, G.J., Fuccillo, M.V., Malenka, R.C., and Südhof, T.C. (2011) Neuroligins/LRRTMs prevent activity- and Ca²⁺/calmodulin-dependent synapse elimination in cultured neurons. J. Cell Biol. 194, 323-334.

PubPeer Weblink: https://pubpeer.com/publications/6A24D71A02F213024FBA3452BD89DA

#1  (April 2024)

Mistake identified: Supplementary Figure 3 contains a duplicated image

Resolution: We self-reported the error

Classification: Minor mistake identified by us

***

31. Paper: Ko, J., Fuccillo, M., Malenka, R.C., and Südhof, T.C. (2009) LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation. Neuron 64, 791-798.

PubPeer Weblink: https://pubpeer.com/publications/13DFE3B5D880F57C51C7ACB03CFC6C

#1 (April 2024)

Mistake identified: Supplementary Figure 3 contains a duplicated image

Resolution: We self-reported the error

Classification: Minor mistake identified by us

#2 (April 2024)

Accusation: Supplementary Figure 3 contains a second duplicated image

Resolution: We missed this mistake when we self-reported the first error but confirm that the second mistake exists in the same figure

Classification: Minor mistake

#4 (July 2024)

Accusation: ‘animation’ by Dr. Bazargan of accusation #2 three months after it was already resolved

Resolution: No need for additional resolutions

Classification: Irrelevant comment

***

30. Paper: Shimojo, M., Madara, J., Pankow, S., Liu, X., Yates, J. 3rd, Südhof, T.C., and Maximov, A. (2019) Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity. Genes and Dev. 33, 365-376.

PubPeer Weblink: https://pubpeer.com/publications/2999ACA0C61FDB9B3F71E366688A37

#1 & #3 (April 2024)

Accusation: Dr. Bik, echoed by Dr. Bazargan, claim that in Figure 1D some immunoblot sections containing no signal were duplicated.

Resolution: The figure and data were not from the Südhof lab, but the senior author responded on PubPeer to explain that this allegation is unfounded

Classification: Unfounded

#2 (April 2024)

Accusation: Dr. Bik claims that since I am the only author from my current lab on this paper published in 2019, I must be an honorary author

Resolution: The accusation overlooks that my affiliation was listed as UT Southwestern, as was that of the senior author Dr. Maximov, and that Dr. Maximov initiated the project in my lab at UT Southwestern and took the project and the reagents he generated with him in 2007 to his own lab, where he completed the project to be published in 2019.

Classification: Unfounded

#4 (April 2024)

Accusation: Dr. Bik alleges that the blots shown in Supplementary Figure 1 are overexposed

Resolution: This is a question of taste. These blots were from the pre-digital age when we often preferred long exposures with ECL visualizations to identify possible minor signals.

Classification: Unfounded

***

29. Paper: Sando, R., Jiang, X., and Südhof, T.C. (2019) Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins. Science 363, pii: eaav7969.

PubPeer Weblink: https://pubpeer.com/publications/3782092ABD5E5AC83CBB9F899C0D59

#1, #2, #4 & #6 (April 2024)

Accusation: Dr. Bik and her associates used sophisticated computational tools to identify image duplications in Figure 3B, animated as usual by Dr. Bazargan.

Resolution: The allegation is correct and the accidental image duplications that were undetectable without specialized software has been corrected.

Classification: Minor mistake

***

28. Paper: Eichel, K., Uenaka, T., Belapurkar, V., Lu, R., Cheng, S., Pak, J.S., Taylor, C.A., Südhof T.C., Malenka, R., Wernig, M., Özkan, E., Perrais, D., and Shen, K. (2022) Endocytosis in the axon initial segment maintains neuronal polarity. Nature 609, 128-135.

PubPeer Weblink: https://pubpeer.com/publications/5423F032DB2E73F1ACD449E4B5BA96

#1 & #2 (April 2024)

Accusation: Dr. Bik identified a blot image duplication in Extended Data Figure 5a

Resolution: The allegations is likely correct but the data are not from a our lab

Classification: Minor mistake in a collaborator’s paper

***

27. Paper: Chen, L.Y., Jiang, M., Zhang, B., Gokce, O., and Südhof, T.C. (2017) Conditional Deletion of All Neurexins Defines Diversity of Essential Synaptic Organizer Functions for Neurexins. Neuron 94, 611-625.

PubPeer Weblink: A minor copy-paste mistake was initially posted by the lab and later amplified by others https://pubpeer.com/publications/F7C42C356B2E7049FDB68A434EF4F8. However, Dr. Matthew Schrag sugsequently discovered more serious image problems

#1 (April 2024)

Mistake identified: The paper’s first author, Dr. L.Y. Chen, reported that she inadvertently and incorrectly copy-pasted an image in Figure 2D into Figure S3A, creating an image duplication

Resolution: We self-reported this mistake on PubPeer as soon as we discovered it; a correction has been filed.

Classification: Minor mistake identified by us.

#2 (April 2024)

Accusation: ‘Aneuris’ repeats Dr. Chen’s report

Resolution: No response needed

Classification: Unfounded

#3-18 (April 2024)

Accusation: ‘Aneuris’, Dr. Bazargan, and Dr. Van Kampen allege that Figure S4B contains cloned micro-areas but Dr. M. Schrag notes that such microareas are often image processing artifacts

Resolution: We note that ‘cloned micro-areas are often image processing artifacts. See another recent accusation by Dr. Bik that reveals how common such artifacts are that are not image manipulations (see https://pubpeer.com/publications/C9E4F18C603C449A0CD32876B719A5). The comments did not identify the real problem that Dr. Schrag advised us of later in a very civil interaction outside of PubPeer, namely that figure S4B contains a partial image duplication in the green channel.

Classification: The PubPeer accusations by themselves are unfounded but the figure contains problems not identified by PubPeer.

#25-52 (May 2024)

Accusation: In extensive repetitive posts, Dr. Maarten Van Kampen (posting as ‘Orchestes quercus’) illustrates by multiple annotated images and supported by assisting comments claims that Figure S4B contains tiny subsections that appear to be duplicated within two images and are thus ‘cloned’, i.e. intentionally manipulated within that image. Moreover, the comments return to Figures 2D and S3A that the first author in post #1 reported to be a mistake.

Resolution: Same as above.

Classification:  Pending but possible image manipulation of the cerebellum images.

#58-67 (July 2024)

Accusation: Dr. E. Bik and Bazargan add their voices to amplify the previous comments.

Resolution: We continue to believe that the majority of the criticisms  Micro-duplications are typical image processing artifacts that may have occurred during image stitching or article production. An intentional image manipulation involving micro-duplications makes no sense because it the effect is to make the image worse, not better. However, subsequent image analyses that were performed by an external expert discovered manipulations in Figure 1D that were not micro-duplications but involved the insertion of the same blue background vGluT2 control stain in both the test and control condition. This manipulation could have been intentional but its purpose is puzzling. The discovery by the independent image analysis expert of a possible image manipulation has been referred to the journal and Stanford

Classification:  Pending.

Postscriptum: Based on our analysis with an invaluable contribution of an independent analysis performed by Dr. Matthew Schrag, we at present conclude that this paper has three problems, two minor and one major. First, an inadvertent image duplication error (post #1); second, duplications of micro-areas in cerebellar figures that are likely image processing artifacts (posts #3-67), and third inexplicable duplications of larger image areas in only one channel in Figure 1D and S4B that cannot be explained by image processing artifacts. The third problem is potentially serious and at present cannot be explained by the first author who is responsible for these figures.

***

26. Paper: Seigneur, E., Polepalli, J., and Südhof, T.C. (2018) Cbln2 and Cbln4 are expressed in distinct medial habenula-interpeduncular projections and contribute to different behavioral outputs. Proc. Natl. Acad. Sci. U.S.A. 115, E10235-E10244.

PubPeer Weblink: https://pubpeer.com/publications/25B0A673C668139C77CEAC19A99B11

#1 (April 2024)

Accusation: We discovered and reported two image duplications in Figure S4

Resolution: Posted by us to preempt social media shaming

Classification: Minor mistake identified by us.

#2 (July  2024)

Accusation: Our attempt to preempt a social media accusation because of this copy-paste mistake failed: Dr. Illex repeats the accusation 3 months after it was reported

Resolution: None necessary

Classification: Irrelevant comment

***

25. Paper: Mall, M., Kareta, M.S., Chanda, S., Ahlenius, H., Perotti, N., Zhou, B., Grieder, S.D., Ge., X., Drake, S., Ang, D.E., Walker, B.M., Vierbuchen, T., Fuentes, D.R., Brennecke, P., Nitta, K.R., Jolma, A., Steinmetz, L.M., Taipale, J., Südhof, T.C., and Wernig, M. (2017) A proneuronal transcription factor repressing many non-neuronal fates. Nature 544, 245-249.

PubPeer Weblink: https://pubpeer.com/publications/D4D96DB690DAC5A3D912EE64E92AC3

#1 & #2 (April 2024)

Accusation: Extended Data Figure 5 contains two image duplications.

Resolution: The allegation concerns data that are not from the Südhof lab but are correct. The postdoc from the Wernig lab involved has initiated a Correction with the journal.

Classification: Minor mistake by other lab

***

24. Paper: Bacaj, T., Ahmad, M., Jurado, S., Malenka, R.C., and Südhof, T.C. (2015) Synaptic Function of Rab11Fip5: Selective Requirement for Hippocampal Long-Term Depression. J. Neurosci. 35, 7460-7474.

PubPeer Weblink: https://pubpeer.com/publications/52D37ED6C4D16682694521FA04B2BA

#1, 2, 4 & 6 (April 2024)

Accusation: Drs. Bik, van Kampen, and Bazargan allege that In Figure 3D, the immunoblotting bands for Cpx and Sph look ‘unexpectedly similar’, implying they are duplicated. As usual, Dr. Bik makes the initial claim and Drs. Van Kampen and Bazargan then follow up.

Resolution: The allegation is a common image analysis mistake. Immunoblotting bands are ‘expectedly similar’ when samples run on the same apparatus and analyzed by Coomassie or blotted with similarly clean secondary antibodies. We demonstrate in our response on the site that this is a frequent occurrence but now make sure in our papers that bands look as dissimilar as possible to avoid future allegations.

Classification: Unfounded

***

23. Paper: Golf, S,R,, Trotter, J,H,, Nakahara, G., and Südhof, T.C. (2023) Astrocytic Neuroligins Are Not Required for Synapse Formation or a Normal Astrocyte Cytoarchitecture. bioRxiv 10:2023.04.10.536254. doi: 10.1101/2023.04.10.536254. Preprint.

PubPeer Weblink: https://pubpeer.com/publications/98784D9AF9B1E8B5B1818E516B5001  

#1 (March 2024)

Accusation: Dr. Bik alleges that one control panel in the 70 blots of Figure 3 is duplicated.

Resolution: There are two problems with this accusation. A. This is not a published paper, but a preprint posted to elicit comments on BioRxiv. Dr. Bik instead chose to ‘comment’ on PubPeer which is typical of her agenda. B. Blot duplications are often mis-identified because similar samples, when run on the same gel apparatuses at the same relative positions in the gel produce the same blotting artifacts. This doesn’t mean they are duplicated, but is expected.

Classification: Unfounded

#3 & #4 (April 2024)

Accusation: Dr. Bik repeats the allegation that one control panel in the 70 blots of Figure 3 is duplicated and Dr. Bazargan ‘animates’ the allegation.

Resolution: Same response as for the first allegation

Classification: Unfounded

#6 & #7 (April 2024)

Accusation: Drs. Bik and Bazargan demand to see the unpublished raw data.

Resolution: We consider the demand for unpublished raw data inappropriate

Classification: Unfounded

***

22. Paper: Seigneur, E., and Südhof, T. C. (2018) Genetic ablation of all cerebellins reveals synapse organizer functions in multiple regions throughout the brain. J. Neurosci. 38, 4774-4790.

PubPeer Weblink: https://pubpeer.com/publications/D8EAA6F915EE4008B654738F66ABE6

#1 (March 2024)

Accusation: Dr. Bik’s artificial intelligence software identified two duplicated blots in Figure 2 that contains 72 individual blots and 36 graphs.

Resolution: The allegation is correct. We copy-pasted the wrong blots! Original data are now posted on PubPeer.

Classification: Minor mistake

#2 & #3 (March 2024)

Accusation: Dr. Bik’s software discovered two image duplications in Figure 3 that contains 67 individual images and would not be detectable without artificial intelligence examination.

Resolution: Again the allegation is correct. Once we copy-pasted the wrong images we would have been unable to detect it. Original data are now posted on PubPeer.

Classification: Minor mistake

#8 (April 2024)

Accusation: Dr. Bik demands a journal correction.

Resolution: A journal correction has been made.

Classification: Irrelevant

#9 & #11, 12, 16 & 19 (April 2024)

Accusation: The anonymous ‘Actinopolyspora’ demands access to unpublished data and claims that the fact that 25 postdocs in the Sudhof lab over 15 years committed copy-paste errors suggests that there is systemic fraud in the lab. Moreover, both this anonymous commenter and Dr. Bik claim that they have no conflict of interest.

Resolution: No resolution possible although the demands for unlimited data and the claim of a lack of a conflict of interest are increasingly common on PubPeer. Note that the raw data have already been posted previously (https://purl.stanford.edu/cc564dr1376).

Classification: Irrelevant

#23 (July 2024)

Accusation: Dr. Bazargan ‘animates’ one of Dr. Bik’s accusations.

Resolution: No resolution necessary.

Classification: Pointless repetition of a resolved accusation

***

21. Paper: Wang, J., Miao, Y., Wicklein, R., Sun, Z., Wang, J., Jude, K.M., Fernandes, R.A., Merrill, S.A., Wernig, M., Garcia, K.C., and Südhof, T.C. (2021) RTN4/NoGo-Receptor Binding to BAI Adhesion-GPCRs Regulates Neuronal Development. Cell 184, 5869-5885. PMCID: PMC8620742

PubPeer Weblink: https://pubpeer.com/publications/5813077CE8B5C29E479FD50C259F77

#1 (March 2024)

Accusation: Dr. Bik identified an image duplication in a supplementary figure containing 36 panels of illustrative images in a paper with 373 images and more than 130 graphs

Resolution: Dr. Bik’s scrutiny correctly identified a duplication that occurred during figure assembly and could only be detected by artificial intelligence because the images are very similar

Classification: Minor mistake

#6, 8, 10, 12, 14 & 15  (July 2024)

Accusation: Dr. Bazargan reiterates and ‘animates’ Dr. Bik’s identification of a duplicated image, ‘Actinopolyspora’ confirms it again, and another anonymous commenter also reinforces the fact that may be 1 error among 500 display items of this paper.

Resolution: Same as above

Classification: No need for a response except to note that the enthusiasm in repeating allegations is interesting – PubPeer is a huge echo chamber

***

20. Papers: Jiang, X., Sando, R., and Südhof, T.C. (2021) Multiple signaling pathways are essential for synapse formation induced by synaptic adhesion molecule. Proc. Natl. Acad. Sci. U.S.A. 118, e2000173118; Li, J., Xie, Y., Cornelius, S., Jiang, X., Sando, R., Kordon, S., Pan, M., Leon, K., Südhof, T.C., Zhao, M., and Araç, D. (2020) Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism. Nature Comm. 11, 2140.

PubPeer Weblink: https://pubpeer.com/publications/027E93962D3C5DB86482283739C67D#10

#9 (March 2024)

Accusation: Dr. Bik noticed that a control image in the two papers cited above is the same even though the stated conditions appear different, suggesting fraud

Resolution: Dr. Bik’s artificial intelligence-powered scrutiny of all of our papers correctly identified the same control image for the same type of experiment in two different studies performed at the same time. Moreover, in the Nature Communications paper the control condition is labeled as 'Ctrl' whereas in the PNAS paper it is labeled as 'NPR-mut', which may seem to indicate that different conditions are attributed to the images in the two papers. However, the Methods section clearly states that the 'Ctrl' of the Nature Communications paper is the 'NPR-Mut' that was used in the PNAS paper and thus the conditions are the same. It would have been better if we had indicated that the same control condition was used for the same experiment in two different projects. Five years ago we lived in a less prosecutorial and censorious environment and the lead author did not think of stating this explicitly, which was an oversight.

Classification: Unfounded

***

19. Paper: Burré, J., Sharma, M., and Südhof, T.C. (2012). Systematic Mutagenesis of a-Synuclein Reveals Distinct Sequence Requirements for Physiological and Pathological Activities. J. Neurosci. 32, 15227-15242.

PubPeer Weblink: https://pubpeer.com/publications/0FECC6D2E9498F9876CFCC24D2E03E#8

#1-#3 (December 2023 & March 2024)

Accusations: Dr. Bik & Co noticed that the blots shown in Figure 5, 6 and 7 are assembled from multiple individual blots and accuses the lab of ‘splicing’ blots together inappropriately.

Resolution: These blots assemble analyses of 26 samples that cannot be examined on a single gel. Thus the samples were run in parallel on multiple gel electrophoresis apparatuses and blots. There are no standard apparatuses that could fit so many samples. No attempt was made to hide this fact. A decade ago, before the current atmosphere of prosecution, it was assumed and accepted that composite blots like the one cited by the accusers would be assembled from multiple individual blots, each of which is clearly recognizable as a separate blot. Nowadays this is considered deception even though there is no attempt to hide it.

Classification: Unfounded

#4 & #6 (March 2024)

Accusations: Presumably based on artificial intelligence searches, Dr. Bik identified image duplications in two sets of panels in Figure 7D.

Resolution: The accusation is correct. During copy-pasting of representative images, two images were erroneously duplicated and differentially cropped. There are no implications for the actual science.

Classification: Minor mistake

#5 (March 2024)

Accusations: Dr. Bik’s alleges a duplication an individual panel in Figure 9A.

Resolution: Again, Dr. Bik’s A.I. search correctly discovered a duplication among the 208 panels of Figure 9A. This mistake which likely happened during copy-pasting of representative images and was undetectable before A.I. tools became available.

Classification: Minor mistake

#9 (March 2024)

Accusations: Dr. Bik repeats the accusation that the combination of different blots in Figures 5 and 6 is inappropriate. She concedes that in general it is unavoidable with large numbers of samples but that in this particular case it is ‘suspicious’ because different blots were combined at different positions.

Resolution: We simply disagree. At the time of this study -15 years ago- people found this perfectly acceptable.

Classification: Unfounded

#16 & #18 (April 2024)

Accusations: ‘Actinopolyspora biskrensis’ alleges that there is a pattern of fraud in my lab because we retracted another paper in which we (not PubPeer) identified an inappropriate analysis of data. The comment implies that Dr. Burre’s isolated errors are also fraud and that more than 25 members of my lab each committed fraud by manipulated a panel each in one of their papers.

Resolution: It is unfortunate that PubPeer has become a forum for people who appear to have an agenda of taking down scientists by implausible suspicions and insinuations, often hiding behind pseudonyms. The occurrence of isolated errors in numerous papers from my lab that could not have been identified prior to the advent of AI-driven software and likely similarly exist in hundreds of other papers is not an indication that each of the postdocs who are responsible committed an isolated act of fraud in their paper.

Classification: Inappropriate comment.

#19 (July 2024)

Accusations: Dr. K. Bazargan commenting as ‘Illex’ provides an animation of Dr. Bik’s comment #4 that was resolved previously (see above), possibly to amplify the accusation even though it was long dealt with

Resolution: No need for further responses.

Classification: Unfounded

***

18. Paper: Lin, P.Y., Chen, L.Y., Jiang, M., Trotter, J.H., Seigneur, E., and Südhof, T.C. (2023) Neurexin-2: An Inhibitory Neurexin That Restricts Excitatory Synapse Formation in the Hippocampus. Sci. Advances 9, eadd8856.

PubPeer Weblink: https://pubpeer.com/publications/C22E0805CB0B55CB7388F488611145

#1 (March 2024)

Accusation: One set of representative images in Figure 4B shows enlarged views of two synapses that cannot be found next to each other in the low-magnification view shown in the paper, and therefore these images represent image manipulations. In order to illustrate this accusation, the accuser drew new boxes into the published figure that are not in the paper.

Resolution: As explained in the figure legend, these are representative synapse images taken from the same experiment but are not adjacent in low-magnification images shown. The low-magnification image is show specifically to illustrate adjacent synapses whereas the high-magnification images illustrate particular features of synapses. All raw data were submitted to a public database (https://purl.stanford.edu/nb252dn4150)

Classification: Unfounded

#4, #6 & #7

Accusation: These accusations by Dr. Bik & Co make the same point as #1: Because we did not explicitly state in the figure legend that the representative images shown were not adjacent to each other in the section, this  is scientifically wrong.

Resolution: Journal restrictions on legend sizes make it impossible to explain every detail but there was clearly no intent of hiding the fact that the representative images are just that, representative images, that were selected from a larger set of images. Otherwise we would have indicated this using customary boxes.

Classification: Unfounded

***

17. Paper: Wang, S., DeLeon, C., Sun, W., Quake, S.R., Roth, B.L., and Südhof, T.C. (2024) Alternative Splicing of Latrophilin-3 Controls Synapse Formation. Nature 626, 128-135.

PubPeer Weblink: https://pubpeer.com/publications/A04E94FAF81B5D7EC9E6B1668085EA

#1a (February 2024)

Accusation: The p values in Figure 5 must be wrong because they are the same for Exon 31 (E31) and Exon 32 (E32) conditions.

Resolution: The P values in Fig5 (and also throughout the paper) are identical for E31 and E32 in the same comparison groups because the splicing of E31 and E32 are mutually exclusive. Therefore, each PSI datapoint in E31 plot always has a corresponding datapoint with the value of 100-PSI in E32 plot, and for the same comparison group (e.g. KCl 0hr vs 6hr), the p value for E31 must be equal to E32.

Classification: Unfounded

#1b  (February 2024)

Accusation: The p values shown in Figure 5 are different from those of the Supplemental Tables and therefor one of them must be wrong.

Resolution: Figure 5 used a t-test as specified in the legend. The supplemental tables use Tukey’s test to calculate pairwise p values after correcting family-wise error as specified again in the legend. In the “Statistics and reproducibility” section we explicitly stated: “Most statistical tests were performed using two-sided t-tests, as indicated. To control for family-wise error during multiple comparisons, two-sided Tukey’s tests were used in parallel and the adjusted P values are summarized in Supplementary Tables 1 and 2, and do not change the conclusions drawn from t-tests in this work.”

Classification: Unfounded

#1c  (February 2024)

Accusation: Figures 5E and 5F are duplications because they are the same graph rotated 180 degrees.

Resolution: The distribution of Figure 5e and 5f are expected to be precise mirror-images of each other because E31 and E32 are mutually exclusive.

Classification: Unfounded

#3  (April 2024)

Accusation: Self-reported mis-labeling of a Figure in the paper committed by Nature staff as an illustration that published papers also contain errors introduced during processing

Resolution: No need for corrections except if Nature is convinced by PubPeer tweets that it should correct its own mistake

Classification: Unfounded

***

16. Paper: Woerman AL, Stöhr J, Aoyagi A, Rampersaud R, Krejciova Z, Watts JC, Ohyama T, Patel S, Widjaja K, Oehler A, Sanders DW, Diamond MI, Seeley WW, Middleton LT, Gentleman SM, Mordes DA, Südhof TC, Giles K, Prusiner SB. (2015) Propagation of Prions Causing Synucleinopathies in Cultured Cells. Proc. Natl. Acad. Sci. USA 112, E4949-4958.

PubPeer Weblink: https://pubpeer.com/publications/F80D8161AB29FEF18967FAF9A0D228#2

#1, 3-5 (December 2023)

Accusation: Based on data reconstructions, the statistical significance of panel A of Figure 6 is p = 0.0679 instead of p<0.05 as stated in the paper. Again, the accusation is supported by an echo chamber of commentators.

Resolution: The incriminated data are not from the Südhof lab but small differences in reconstructed data points could easily be responsible for the tiny difference in calculated p values, especially since figures are generally constructed from data points by programs and subsequent shuffling of panels introduces inaccuracies in the representations. Given that the alleged p value difference is small, the conclusion that the stated p value is wrong seems to be unjustified, although saying so was criticized by PubPeer as inappropriate.

Classification: Unfounded

***

15. Paper: Jiang, X., Sando, R., and Südhof, T.C. (2021) Multiple signaling pathways are essential for synapse formation induced by synaptic adhesion molecule. Proc. Natl. Acad. Sci. U.S.A. 118, e2000173118. PMCID: PMC7826368

PubPeer Weblink: https://pubpeer.com/publications/027E93962D3C5DB86482283739C67D

#1 (November 2023)

Accusation: The stated statistical significance of the right graph in Figure 5C must be wrong because the error bars overlap

Resolution: Prism software indicates that the two conditions are significantly different, consistent with the fact that overlap of confidence intervals is not a reliable indicator of statistical significance (Schenker, Nathaniel, and Jane F. Gentleman. 2001. “On Judging the Significance of Differences by Examining the Overlap Between Confidence Intervals.” The American Statistician 55 (3): 182–86. http://www.jstor.org/stable/2685796.)

Classification: Unfounded

#3-6 (November 2023)

Accusation: Using the error bars as a guide to reconstruct the statistical significance between the two conditions, the two conditions cannot be significantly different

Resolution: We went back to the original data and confirmed that the error bars are SDs, not SEMs, and that the two conditions are statistically significantly different. An error in the figure legend was identified.

Classification: Minor mistake in figure legend identified

#8, 9, & 11 (March 2024)

Accusation: The same control was used in two different papers

Resolution: Correct – the experiments were carried out at the same time with the same controls

Classification: Unfounded

***

14. Paper: Ho, A., Morishita, W., Hammer, R.E., Malenka, R.C., and Südhof, T.C. (2003) A role for Mints in transmitter release: Mint 1 knockout mice exhibit impaired GABAergic synaptic transmission. Proc. Natl. Acad. Sci. U.S.A. 100, 1409-1414.

PubPeer Weblink: https://pubpeer.com/publications/866AA1811F89014742E1EAEB2BD25D

#1 (October 2023)

Accusation: The day 60 day data point in Figure 3C cannot be statistically significantly different because of the error bars almost overlap.

Resolution: In the incriminated graph only the day 60 day data point is statistically significantly different. This is not a biologically significant result as discussed in the paper, but the existing statistical significance was nevertheless reported as mandated by publishing rules. Also see #1 under Paper 15 above.

Classification: Unfounded

#2 (October 2023)

Accusation: The fact that neurexin protein levels are lower in heterozygous than in homozygous KO mice in Table 1 is suspicious.

Resolution: Protein measurements are notoriously noisy. As a result, small changes in protein levels are not interpretable, especially if they were statistically not significantly different as in this case.

Classification: Unfounded

***

13. Paper: Biederer, T., Cao, X., Südhof, T.C., and Liu, X. (2002) Regulation of APP-dependent transcription complexes by Mints/X11s: Differential functions of Mint isoforms. J. Neurosci. 22, 7340-7351.

PubPeer Weblink: https://pubpeer.com/publications/34D6A8F36DFC9F19D753DCAF6B96FE    

#1 (October 2023)

Accusation: The lack of error bars on panel D of Figure 3 raises concerns about the validity of the data

Resolution: Error bars are not visible in Figure 3D because the error bars are too small

Classification: Unfounded

***

12. Paper: Fernandez-Chacon, R., Shin, O.-H., Königstorfer, A., Matos, M.F., Meyer, A.C., Garcia, J., Gerber, S.H., Rizo, J., Südhof, T.C., and Rosenmund, C. (2002) Structure/function analysis of Ca²⁺-binding to the C₂A-domain of synaptotagmin 1. J. Neurosci. 22, 8438-8446.

Weblink: https://pubpeer.com/publications/1FD9572D003130580F6C598C39D9C6

Date of accusation: September 2023

Accusation: Unknown since comment was removed by ‘moderator’

Resolution: No criticism recorded

Classification: Unfounded

***

11. Paper: Trotter, J.H., Hao, J., Maxeiner, S., Tsetsenis, T., Liu, Z., Zhuang, X., and Südhof ,T.C. (2019) Synaptic Neurexin-1 Assembles into Dynamically Regulated Active Zone Nanoclusters. J. Cell Biology 218, 2677-2698. PMCID: PMC6683742

PubPeer Weblink: https://pubpeer.com/publications/71F24BE796880C3A39AC29501382AB

#1 (July 2023)

Accusation: Control blots in Figure 7C are duplicated

Resolution: Original blots document that the control blots were likely not duplicated even though the blots show similar blotting artifacts as documented, although given the similarity of the blots one might easily conclude that they might be identical. A common error of PubPeer posts is that blots with similar artifacts are alleged to be duplicated, which neglects the fact that samples analyzed on the same blotting apparatus with the same antibodies exhibit similar artifacts even though they are different.

Classification: Unfounded

Postscript: Upon reviewing the primary data, we discovered a blot mixup in the images shown that we corrected with an Erratum but that was not detected on PubPeer

Classification: Minor error identified by the lab

#4 (October 2023)

Accusation: One bar in the Figure 9C graph is not labeled as significantly different but looks like it should have been labeled as significantly different

Resolution: PRISM software analysis of the data suggests that it is not significantly different

Classification: Unfounded

#6 (March 2024)

Accusation: Dr. Bik acknowledges that we published a correction not of any mistake PubPeer identified, but of a mistake we identified

Resolution: No resolution needed – the comment was just meant to amplify the fact that we found and acknowledged a mistake

Classification: Unfounded

#8 (March 2024)

Accusation: Dr. Bik alleges that there is a mistake in Figure S6E because two representative images overlap. As often, her comments are then echoed and amplified by Dr. Bazargan and Actinopolyspora

Resolution: The overlap exists but is perfectly legitimate since the panels illustrate sample images from the same experiment

Classification: Unfounded

***

10. Paper: Burré, J., Sharma, M., Tsetsenis, T., Buchman, V., Etherton, M., and Südhof, T.C. (2010) a-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro. Science 329, 1664-1668.

PubPeer Weblink: https://pubpeer.com/publications/2452F555579F6D021205B875814D82

#1 (May 2023)

Accusation: The blots in Figure 2B, 4B, and 4C are duplicated

Resolution: Original blots demonstrate that the blots are not duplicated. This is the same common error in PubPeer accusations as in paper 11, which alleges blot duplications based on similar artifacts but neglects the fact that similar samples run on the same blotting apparatuses exhibit similar artifacts

Classification: Unfounded

#3 (May 2023)

Accusation: Demands higher resolution images of the blots that we presented in PubPeer

Resolution: We feel the original blots we presented on PubPeer are sufficient to demonstrate that the accusation that the blots were duplicated is mistaken.

Classification: Unfounded

#5 & #6  (March 2024)

Accusations: Dr. Bik reinforced/repeated #1 and #3 accusations.

Resolution: The first author of the paper now posted the original blots of the paper to document that there is no conclusive evidence that the control blots were duplicated and that these blots exist. Moreover, it should be noted that this is enormous exercise in determining whether or not the accusers or our lab are right refers to control blots that are clearly without signal in the original blots.

Classification: Unfounded

***

9. Paper: Patzke, C., Brockmann, M.M., Dai, J., Gan, K.J., Grauel, M.K., Fenske, P., Liu, Y., Acuna, C., Rosenmund, C., and Südhof, T.C. (2019) Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses. Cell 179, 498-513. PMCID: PMC7159982

PubPeer Weblink: https://pubpeer.com/publications/4AEAAAE084C8DFE9E26107D350B0B5

#1, 2, 4 & 5 (March 2023)

Accusation: The source data files for Figures 6E, 6F, 6K, S6F, S6G, S6Q, S6R, and S7C contain instances of data duplication.

Resolution: The accusation is largely correct. During assembly of the source data files, copy-paste errors of several blocks of numbers were committed for which a Correction has been filed. In addition, isolated number identities occurred that are not errors but intrinsic to the scientific method used.

Classification: Minor mistake identified by PubPeer

***

8. Paper: Lin, P.Y., Chen, L.Y., Zhou, P., Lee, S.H., Trotter, J.H., and Südhof, T.C. (2023) Neurexin-2 restricts synapse numbers and restrains the presynaptic release probability by an alternative splicing-dependent mechanism. PNAS 120, e2300363120

PubPeer Weblink: https://pubpeer.com/publications/DAF32F6DB6C166337E5381F769AE52

Note: This paper elicited a large number of comments because, as we confirmed in our reanalysis of the raw data deposited here (https://purl.stanford.edu/cp231wr9194), some of the source data were not properly analyzed, copy-paste errors were committed during assembly of the data, and the drafting of some figures was incorrect because the copy-paste errors were transmitted into the figures. Moreover, as always many comments were censored by the ‘Moderator’ who usually disallows author comments but generally does not censor accuser comments. Finally, a key author of the study was medically challenged during data assembly and the intense time of PubPeer criticism and provided incorrect answers on PubPeer.  

#1 - #4, #7, #11, and #13-#16 (March & April 2023)

Accusation: The source data files for Figures 2-6 contain extensive data duplications

Resolution: The accusation is correct. The data duplications initially appeared to be a minor mistake due to extensive copy-paste errors, but reanalysis of the data (see below) showed that the errors were transmitted into the figures. Moreover, the paper’s 1^st author then posted a ‘corrected’ set of replacement data that were not previously seen by the lab and did not appear to be from the Südhof lab, suggesting they were not based on a scientific analysis. This was the trigger that led to a reanalysis of the raw data by the lab. The reanalysis showed that the data duplications in the source data files were copy-paste errors made during assembly of the summary file resulting from an original analysis of the raw data that was not correctly performed, and that the copy-paste errors were erroneously included in the figures.

Classification: Data duplications are a minor error, but the original raw data analysis and figures include major errors that were not detected by PubPeer comments but uncovered in our re-analysis – PubPeer only identified the copy-paste errors and the implausible nature of the replacement data posted by the 1^st author.

#18 - #21, #23-#26, #28, #30, #31, #33-#35, #37, #38, #40, #42, #44, and #46 (April-August 2023)

Accusation: The unpublished replacement data posted by the 1^st author contain irregularities suggesting possible falsification

Resolution: The accusation is correct. The replacement data were not reviewed or endorsed by the Südhof lab prior to being posted by the 1^st author and their origin is unclear.

Classification: Post does not refer to a Südhof lab publication

#64-#67 and #69 (October 2023)

Accusation: The posted raw data may or may not contain technical issues

Resolution: The discussion here was largely based on an incomplete understanding of electrophysiological methods as one of the comments points out. Electrophysiological results are inherently noisy.

Classification: Unfounded

#68 (October 2023)

Accusation: The review of the paper was flawed because both reviewers are alumni of the Südhof lab

Resolution: The accusation is incorrect since Prof. Josh Huang was never associated with, or collaborated with, the Südhof lab, although Prof. Katsuhiko Tabuchi was in the Südhof lab 20 years ago but has since established a successful large independent research program as a full professor in Japan.

Classification: Unfounded

Postscriptum: The Südhof lab posted as response #70 (October 2023) the following statement:

“My lab has surveyed the many criticisms of this paper and reanalyzed part of the raw data. We observed multiple errors. The ones we know of, both discovered by us and raised by others, I’m listing here. Our preliminary conclusions are:

a. The published source data and the raw data associated with the paper are often inconsistent.

b. There are extensive raw data falling into two categories: raw data that were labeled as used for the paper and raw data that were not used from analogous experiments without a clear difference in quality or rationale.

c. The limited reanalysis of anonymized data confirms the conclusions of the paper, although we have analyzed only a small part of the data and the conclusions are not always statistically significant for all parameters. The absolute values of the mean parameters differ as would be expected when raw data are analyzed independently. Needless to say that does not confirm the paper as we also need to relate the data to the original lab notebook.

d. As regards comment #68, although Prof. Katsuhiko Tabuchi was a fellow in my lab 25 years ago, there has been no connection to my lab since then, and Prof. Josh Huang would indeed be surprised to learn that he was supposed to have been a fellow in my lab.

Overall, we concur that there are major inconsistencies with the source data of this paper compared to the existing raw data, which we are puzzled by. Note that the raw data, unlike blots or images, cannot be manipulated and contain identifiable metadata. Our current preliminary conclusion thus is that, consistent with some of the comments, the source data of this paper contain major flaws that cannot be explained by copy-paste mistakes as I had mistakenly surmised earlier but must have another origin.”

An ’Editorial Expression of Concern’ was published by PNAS that was immediately re-published as comment #71 on PubPeer and in accusatory blogs on ForBetterScience and Spectrum websites.

#72 and #73 (October 2023)

Accusation: The mean value in Figure 1B does not fit the datapoints associated with it, suggesting that the mean value is wrong

Resolution: The accusation is correct that the mean control value does not fit the datapoints but incorrect in concluding that the mean value is wrong. Replotting of the raw data in the source file clearly shows that the datapoints were accidentally shifted during construction of the figure, probably because in the adobe illustrator software independent image objects are often linked and movement of an object can inadvertently cause movement of other linked objects.

Classification: Minor mistake identified by PubPeer

Update. We retracted the P.Y. Lin et al. paper after our independent analysis of the raw data in 2024 (https://www.pnas.org/doi/10.1073/pnas.2403021121) with the following statement: “We wish to retract the paper because re-analysis of the original raw data for Figs. 2, 4, and 6 (https://purl.stanford.edu/cp231wr9194) revealed that, although our analyses of the original data are supportive of the conclusions of the paper, unresolvable differences exist between these raw data and the published data source file that cannot be corrected by a simple erratum. In addition, the data source file contained copy-paste errors, and Fig. 1 included shifted data points that occurred during figure drafting. We thank Dr. Daniel Matus of Stanford University for his independent analysis of the primary raw data."

Further analyses revealed two mistakes associated with the publication of this paper, a partly incorrect analysis and the incorporation of copy-paste mistakes from data files into figures.

***

7. Paper: Dai, J., Liakath-Ali, K., Golf, S., and Südhof, T.C. (2022) Distinct Neurexin-Cerebellin Complexes Control AMPA- and NMDA-Receptor Responses in a Circuit-Dependent Manner. E-Life 11, e78649.

PubPeer Weblink: https://pubpeer.com/publications/68D8490A4754CE00F936214C3931F2

#1 & #4 (March 2023)

Accusation: The source data file of the paper contains duplicated values for several cells

Resolution: The accusation is correct. The duplications are copy-paste errors that were corrected with an Erratum in the journal.

Classification: Minor mistake identified by PubPeer

#5, #7 & #8 (November 2023 & July 2024)

Accusation: An anonymous commentator confirms an Erratum was published, another commentator demands all raw data, and finally Dr. Bik 9 months later re-confirms that an Erratum has been published.

Resolution: The confirmation is correct but we are puzzled why it was posted except if it was meant to amplify a questioning signal.

Classification: Irrelevant comment

***

6. Paper: Zhang, X., Lin, P.Y., Liakath-Ali, K, and Südhof, T.C. (2022) Teneurins Assemble into Presynaptic Nanoclusters that Promote Synapse Formation via Postsynaptic Non-Teneurin Ligands. Nature Comm. 13, 2297.

PubPeer Weblink: https://pubpeer.com/publications/EC9A138F8BFAAE1F2FB803106703AB

#1 (March 2023)

Accusation: Two columns in the source data file for Figure 8 are duplicated

Resolution: The accusation is correct. The columns were accidentally duplicated in a copy-paste error during assembly of the file for sharing after figures were drafted. The error was corrected with an erratum. All raw data were submitted to a public database (https://doi.org/10.25740/kq306bq2466)

Classification: Minor mistake identified by PubPeer

#5 & #6 (July 2024)

Accusation: Dr. Bik, followed as usual by Dr. Bazargan’s ‘animation’, finds that two of the 413 fluorescence images of the paper may exhibit a partial overlap.

Resolution: We could not confirm the alleged overlap despite the animation but the complete raw data are publicly available for reanalysis (see https://doi.org/10.25740/kq306bq2466)

Classification: Unfounded

***

5. Paper: Dai, J., Patzke, C., Liakath-Ali, K., Seigneur, E., and Südhof, T.C. (2021) GluD1, A signal transduction machine disguised as an ionotropic receptor. Nature 595, 261-265.

PubPeer Weblink: https://pubpeer.com/publications/0B87E141DC8DFFF4826A9250A94BAD

#1 (February 2023)

Accusation: The PPR data in the source file are missing

Resolution: The corresponding data were mislabeled as belonging to ‘1f’ instead of ‘1k’ in the source data file due to a typing error. This mistake is being addressed in a ‘Correction’ in the journal.

Classification: Unfounded

#2 & #4 (February & March 2023)

Accusation: The source file contains duplicated values for two rows of numbers

Resolution: Correct. We introduced two copy-paste mistakes during transfer of data from experimental logs to the source file, as occurs when the ‘copy’ key is not pressed sufficiently. This mistake is being addressed in a ‘Correction’ in the journal.

Classification: Minor mistake identified by PubPeer

#3, #9, & #11 (March & June 2023)

Accusation: Some of the full-sized blots in Suppl. Figure 1b and 1c do not correspond to the cropped or quantified data in the Extended Data figure.

Resolution: Correct. Reassessment of original blots identified two related mistakes. In Suppl. Figure 1b one blot was mislabeled and not all blots were included. In Suppl. Figure 1c a different blot of the same experiment with identical results was shown. Again, these mistakes are being addressed in a ‘Correction’ in the journal.

Classification: Minor mistake identified by PubPeer

#12-#16 & #18 (July & November 2023)

Accusation: The data on the effect of a mutation on a function should not have been tested by t-tests

Resolution: T-tests are the standard test for manipulations that contains only a single independent variable and only compare that variable to the control, but not among samples. The graph reports a comparison of multiple single tests to the same shared controls, and could be plotted as multiple graphs consisting of a control and test condition. We agree that the question of the appropriate statistical test can be contentious especially if one focuses less on the biological experiment and more on the graph format but feel that the t-test is appropriate here.

Classification: unfounded

***

4. Paper: Sclip A, and Südhof, T.C. (2020) LAR receptor phospho-tyrosine phosphatases regulate NMDA-receptor responses. E-Life 9, pii: e53406. PMCID: PMC6984820

PubPeer Weblink: https://pubpeer.com/publications/613AFEC1A22C0725BB6D5A9E5CFE76

#1 (February 2023) & #4 (April 2023)

Accusation: Alleged duplication of blots and falsification of data because (i) quantifications of blots were made on the basis of different ‘n’s’ for different proteins but (ii) only a single Tuji control blot is shown for the quantifications.

Resolution: Ad (i), the ‘n’s’ (number of replicates) differ between experiments because each protein quantification is performed separately with a different antibody using the same samples. Different numbers of repeat experiments are performed for various proteins because the noise levels differ between proteins, with the noise level in turn being determined by a protein’s abundance and the quality of an antibody. Each antibody is different, and each immunoblotting quantification is a separate experiment compared to the same controls run on the same gels for multiple antigens. Ad (ii), a single sample control blot is shown for each set of experiments because illustrating samples of the Tuji control blots separately for each protein seems superfluous and adds no information. The same control blot is shown for panels B and C because these experiments were run on the same gels to illustrate that fact.

Classification: Unfounded

#7 & #9 (July 2024)

Accusation: Drs. Bik and Bazargan (in yet another ‘animation’) restate a year later the accusation that the control blot in panels B and C was duplicated, even though we explained already above that they are purposely the same. Dr. Bik states “The lengthy explanations given above [our earlier response in PubPeer, when we still tried to discuss data with PubPeer accusers, which is clearly impossible] seem to not address the concern raised above in #1, which is that two Tuji blots representing different samples (presynaptic vs active zone) look unexpectedly similar. I understand that each protein-of-interest might have been normalized using its own Tuji re-probe, and that protein quantifications are difficult, but that still does not explain the abovementioned similarity. Can the authors please address the actual concern? Also, these figures are not "for illustration purposes only" - they are the data.”

Resolution: Although our explanation was lengthy, we apologize that we apparently didn’t make it understandable possibly because it wasn’t lengthy enough. First, the samples analyzed are NOT different, they are the same. Second, these samples are run on the same gels and analyzed on the same blots and thus share Tuji controls. The two Tuji blots are thus ‘expectedly similar’ because they are the same. Third, we show the same Tuji blot for pre- and postsynaptic proteins because these were analyzed on the same gels. This is real data but illustrative data – we do not show every blot we ever did. All this was described in earlier responses. We realize that Drs. Bik and Bazargan focus on illustrative representative data, not on the actual science and how science is done, and we appreciate the many questions they raise. We also realize that in order to not attract Drs. Bik’s and Bazargan’s accusations, we should choose different Tuji examples even though they are run on the same gels. At this point, scientists in our labs are becoming paranoid that something may look ‘unexpectedly similar’ and cause PubPeer comments that can destroy careers without having any impact on the science.

Classification: Unfounded

#12 (July 2024)

Accusation: An anonymous commenter understood that the samples are not different, for which we are grateful

Resolution: We appreciate that another commenter reads our responses

Classification: helpful comment

***

3. Paper: Dai, J., Aoto, J., and Südhof, T.C. (2019) Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA- and AMPA-Receptor Responses. Neuron 102, 993-1008. PMCID: PMC6554035

PubPeer Weblink: https://pubpeer.com/publications/568B4CF8B40A979424B7F343F3B061

#1 (February 2023)

Accusation: The y-axis labels of Figure 6B and 6C are incorrect

Resolution: Correct - the y-axes were mislabeled

Classification: Minor mistake identified by PubPeer

#3-#11, #22, #23  (February & March 2023)

Accusation: The source data for Figure 2D, 4A, 5E, 6A, 6B, 8, S2, S4, and S8 contain duplications and possible calculation errors

Resolution: Correct - the source data contain multiple isolated errors produced by copy-paste mistakes that also lead to errors in downstream calculations but have no discernable effect on figures or conclusions.

Classification: Minor mistake identified by PubPeer

#25-#28 and #30   (March 2023)

Accusation: The statistical tests used for experiments examining 3 experimental groups – derived from littermate SS4+ and SS4- mice vs. unrelated WT samples – are generally incorrect

Resolution: The accusers express their view that the three experimental groups should be treated as equivalent in pairwise comparisons whereas biologically they are not equivalent. The 3 experimental groups comprise a genetically distinct WT sample expressing both SS4+ and SS4- variants of neurexins and two genetically identical samples that express either only SS4+ or SS4- variants of neurexins. Thus, the statistics are more complex than a simple 2-way ANOVA with a post-hoc correction since the comparison of the two genetically identical samples between each other is inherently different from their comparison with the WT sample. Given this experimental configuration, we believe our statistical approach may be the most appropriate, but it is possible that a non-traditional test that accounts for the experimental non-equivalency might be even better.

Classification: Unfounded

#33-#37  (March 2024)

Accusation: Dr. Bik states that the Correction we published on this paper to rectify the labeling mistakes and copy-paste errors in our paper represent a ‘Mega Correction’ and another accuser criticized that we did not address the accuser’s concerns about statistics.

Resolution: We feel that our correction of multiple copy-paste errors in supplementary tables and of mislabeled graphs in the supplement is not a ‘Mega Correction’, although we deeply regret these errors and mistakes. These mistakes had no consequences for the study, either its data or its conclusions. We started to publish primary data before artificial intelligence-powered software was available to detect mistakes, such as copy-pasted numbers or images, that we in the pre-artificial intelligence era overlooked, which clearly should not have happened. The impact of our 'Mega Corrections' is purely procedural and has no consequences for the actual science.

Classification: Difficult to classify since we already stated that we believe that this is a minor correction and that we do not agree with the statistical criticism

***

2. Paper: Sclip, A., Bacaj, T., Giam, L., and Südhof, T.C. (2016) Extended synaptotagmin (ESyt) triple knock-out mice are viable and fertile without obvious endoplasmic reticulum dysfunction. PlosONE 11, e0158295.

PubPeer Weblink: https://pubpeer.com/publications/FBC43D21E0E903A65AF81CD8D1CAF1

#1 (August 2022)

Accusation: Alleged duplication of blots in Figure 3 and S1.

Resolution: It is correct that the same tubulin and actin blots were used as loading controls for experiments that were performed at the same time. This is not a duplication of blots but the use of the same controls for an experiment that was performed at the same time, and we should have noted this in the legends. An Erratum to document this detail is being prepared.

Classification: Minor mistake identified by PubPeer

#3 (December 2022)

Accusation: A correction was made in the published paper without notification

Resolution: This is incorrect – no correction was made

Classification: Unfounded

#5 (December 2022)

Accusation: Claims that the data are no longer in the PlosONE website

Resolution: A screenshot from the PlosONE website shows that the data are clearly still there.

Classification: Unfounded

#7 (June 2023)

Accusation: The Munc18 and Syt1 blots are identical, i.e. represent duplications

Resolution: Original blots of the experiments as now shown on PubPeer demonstrate that the Munc18 and Syt1 blots were derived from the same experiments run on the same gels but probed with species-specific distinct antibodies. Since these proteins have similar sizes, the shape of their bands is nearly identical, creating an appearance of duplication. However, based on direct communication of the accusers with PlosONE ‘Ethics’, the PlosONE ‘Ethics’ administrators demand that we amend the blots to show samples from the same original publicly deposited blots that make the bands look different, which will be published as a ‘Correction’. The ‘Correction’ doesn’t actually correct anything but journals are very sensitive to PubPeer criticism and their Ethics departments are tasked with eliciting Corrections.

Classification: Unfounded

#10 (August 2023)

Accusation: Maintains that he/she/they cannot see the blots properly

Resolution: Original blots were publicly deposited (https://purl.stanford.edu/vq040hz0549)

Classification: Unfounded

#12 (March 2024)

Accusation: Dr. Bik states “the blot provided in #2 appears to contain munc18 bands in green and syt1 bands in red, just under the munc18 bands, while the published panels show both bands separately (no double bands) and both in red. So it appears you either did not provide the originals, or the published panels have been color-converted with double bands removed.”

Resolution: We have already published a ‘Correction’ that explains better the various blots with inclusion of full-length original blots and boxed areas, and all original blots have been posted on PubPeer and are also publicly available at the published URL as high-resolution images. However, the accusations that "So it appears you either did not provide the originals, or the published panels have been color-converted with double bands removed" are incorrect. In the incriminated blot, two different species-specific antibody signals were monitored on the same blot (for Munc18 and synaptotagmin that have almost identical molecular weights) in different optical channels. Thus, no double band has been removed, nor are colors converted because every color represents an arbitrary assignment of a digital signal. We chose to show the Munc18 and synaptotagmin blots in the same false color in our paper because we thought that this would make it easier to compare the blots, but this has clearly confused people who are not familiar with this type of experiments.

Classification: Unfounded

#13 (July 2024)

Accusation: Dr. Bik quotes the Correction

Resolution: No resolution necessary since this comment is just meant to amplify the number of comments on PubPeer.

Classification: Unfounded

***

1. Paper: Yi, F., Danko, T., Botelho SC, Patzke C, Pak C, Wernig, M., and Südhof, T.C. (2016) Autism-associated SHANK3 haploinsufficiency causes Ih channelopathy in human neurons. Science 352, aaf2669.

PubPeer Weblink: https://pubpeer.com/publications/CBAA10B0FBF31CC41E9B7D56C8B0C3

#1 (April 2018)

Accusation: Since the resting membrane potential of the human neurons derived from stem cells in the experiments is at approximately -40 mV, the neurons must be leaky and sick and it is therefore not possible to draw any conclusions from these neurons

Resolution: All human neurons produced from stem cells are immature and exhibit a decreased resting potential, even though they are not leaky or sick. They exhibit robust active and passive membrane properties and form fully functional synapses

Classification: Unfounded

#3 (April 2018)

Accusation: How can different clones in different sets of experiments produce very similar readouts given that evoked EPSC amplitudes are dependent on intensity of stimulation

Resolution: The electrophysiological approaches used here were validated previously in a number of papers and shown to be highly reproducible across experiments (e.g., see Zhang, Y., Pak, C.H., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Xu, W., Yang, N., Patzke, C., Chen, L., Wernig, M., and Südhof, T.C. (2013) Rapid Single-Step Induction of Functional Neurons from Human Pluripotent Stem Cells. Neuron 78, 785-798. PMCID: PMC3751803; Pak, C., Danko, T., Mirabella, V.R., Wang, J., Liu, Y., Vangipuram, M., Grieder, S., Zhang, X., Ward, T., Huang, Y.W.A., Jin, K., Dexheimer, P., Bardes, E., Mittelpunkt, A., Ma, J., McMachlan, M., Moore, J.C., Qu, P., Purmann, C., Dage, J.L., Swanson, B.J., Urban A.E., Aronow, B.J., Pang, Z.P., Levinson, D.F., Wernig, M., and Südhof, T.C. (2021) Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-Derived NRXN1-Mutant Neurons. Proc. Natl. Acad. Sci. U.S.A. 118, e2025598118. PMCID: PMC8179243)

Classification: Unfounded

#4 (April 2018)

Accusation: The electrophysiological experiments are unreliable (e.g., “Anyone knows that hyperpolarizing MP from -40 to -70 takes heroic current magnitude” and “Not all cells should be active at rest, even not ~60 %”)

Resolution: The electrophysiological approaches used here are consistent with a large number of previous studies by multiple independent experimenters not only the Südhof lab but also in others, and the data shown in the paper are internally fully consistent

Classification: Unfounded

#5 (April 2018)

Accusation: Mean amplitudes and intervals do not correlate with the 'representative traces'

Resolution: Electrophysiological measurements vary between cells and representative traces are only an illustration of the results, they do not depict an average of traces

Classification: Unfounded

#10 & #11 (July 2024)

Accusation: Desires to know the current used to measure the resting membrane potential

Resolution: We unfortunately do not have the resources to research all questions posed by the audience if there is no pressing rationale

Classification: Unfounded