Bologna mice guilty of research misconduct

This is a first-hand, first-person account of a whistleblower. Together with her 3 colleagues, she reported her principal investigator (PI) for data manipulation. The same day the university authorities were informed, 3 of the 4 whistleblowers recanted and lauded their PI’s “scientific rigour”. The remaining whistleblower instead uncovered more evidence of suspected research misconduct and lodged a second report. Eventually, the university investigated, but the internal examiners were busy accusing the whistleblower of incompetence, coercion, document forgery and data theft, while most of her evidence was ignored. Instead, mice were blamed for data manipulations nobody really denied. One rigged experiment was reproduced by the accused PI herself, without any oversight, and the case was closed.

The final report of the misconduct investigation is classified, the university rector prohibited to share it with anyone. Instead, he and the dean wrote letters to the funding charities, reassuring that Elisabetta Ciani was acquitted and requesting for her funding to continue. Incidentally, just before the whistle was blown in 2016, MiaMed, a University of Bologna spin-off (founded by concerned US investor Michael Jasulavic to market Ciani’s “cures”) was sold to the US biotech company Amicus Therapeutics. Its only product: a potential therapy for the cyclin-dependent kinase-like 5 (CDKL5) deficiency, a rare neurological disease affecting infants, patented by Ciani and at least in part, subject to research misconduct allegations.

Ciani and the University of Bologna, together with the investors, were counting on receiving no less than $90 million from that deal. With Amicus lawyers already in touch with whistleblowers, and in absence of any investigative procedure, the oldest university in the world had it easy to whitewash Ciani.

Ciani recently faced 2 retractions, at the no-nonsense Journal of Biological Chemistry (JBC): Trazzi et al 2013 and Ciani et al 2002.

But now, the whistleblower account, complemented with the official university documents.

---

How it all began

In October 2016, myself and three other young researchers employed in the Department of Biomedical and Neuromotor Sciences (DIBINEM) at the University of Bologna (Italy) decided to speak up about the possible misconduct taking place in the laboratory of Prof. Elisabetta Ciani. Ciani has been a group leader and associate professor since 2014, she and two other long-term lab members were involved in several publications flagged by the PubPeer community as problematic (e.g. unacknowledged asymmetric gel splicing) or highly problematic (e.g. image duplication). 

We were four scientists (out of seven group members in that period, including Ciani), who in October 2016 prepared a detailed written report on the inconsistencies and disagreements in experimental results and data analysis in the laboratory, and personally presented it to the PI:

The main scientific focus of the laboratory at that time was a search for underlying mechanisms and new treatment strategies for a rare genetic child disease – a CDKL5 syndrome. The laboratory studied mouse models of the syndrome, conducting both molecular biology and animal behaviour experiments. The research was funded by the CDKL5 children parents’ associations like LouLou Foundation, Telethon Foundation and external grants. 

We found evidence that some experimental results obtained and reported by the team (e.g. as peer reviewed publications) were produced against good laboratory practices and rigorous scientific routine. Our report was made out of the need to express the doubts regarding one to-be-published article as well as already published results obtained in the laboratory in the years 2014-2015, i.e.

1. “Inhibition of GSK3β rescues hippocampal development and learning in a mouse model of CDKL5 disorder” by Fuchs et al., Neurobiol Dis, 2015;
2. “HDAC4: a key factor underlying brain developmental alterations in CDKL5 disorder” by Trazzi et al., Hum Mol Gen, 2016;
3. “Treatment with the GSK3-beta inhibitor Tideglusib improves hippocampal development and memory performance in juvenile, but not adult, Cdkl5 knockout mice” by Fuchs et al., Eur J Neurosci., 2018.

The report was also a manifestation of our helplessness, as three of us had communicated their doubts earlier to the PI and another senior collaborating scientist working at the same department (Renata Bartesaghi, privately Ciani’s aunt) but neither of them took any actions in order to address them. We were also explicitly demanding access to raw data from experiments conducted in the laboratory, which was not a common practice in this team: 

Ciani herself declined several times to reveal raw data from certain experiments, even after she was ordered to do so by the dean of the Faculty and vice-rector of the university. 

The misconduct notification

We also wanted to know what kind of procedures the raw data was subjected to in order to obtain the final results that had been published already or were about to be published, as the used methods were raising serious concerns. The list of these concerns can be found in Report 1 and includes:

• “recycling” of the controls from one behavioural experiment in other experiments conducted several months or years later. Results obtained for one group of animals were published at least twice, in different journals, serving as a control experiment for different, spatially and temporally independent studies. Such practice is scientifically unacceptable as it undermines the concept of experimental controls while misleading the reader that results of experimental and control groups were obtained within the same study, and not, as it happened in reality, in experiments run several months or years apart pooled on one plot;
• intentional omitting vehicle-treated experimental group but instead, while presenting the results, labelling control untreated animals as vehicle treated ones; 
• “cherry picking” of data points, in extreme case resulting in comparing groups consisting of 2 animals per group; 
• submitting manuscript for publication with changes made without the knowledge of other co-authors;
• manipulating excel files entries containing experimental results without explanation or justification;
• reporting only the results in favour of the given research hypothesis while not mentioning other experiments that were conducted in the laboratory because the results were not “desirable”; 
• conducting comparative experiments on microscopic samples treated differently, e.g. comparison of fluorescent images acquired with 3-dimensional z-stack acquisition (control group) and without the z-stack acquisition (experimental group), which resulted in dramatically different fluorescence signal; 
• manipulating animal genotyping data stored on the common laboratory computer (intentional removing of entries regarding animals’ characteristics, making it impossible to identify particular animals);
• ambiguous reporting of sample sizes in animal experiments to hide the fact that experiments were often conducted on 3 mice per group only; 
• keeping experimental data on private computers (microscopic images, behavioural scores, video recordings, etc.) and not sharing it with other lab members, even when explicitly asked for;
• applying incorrect statistical methods, without appropriate explanation in materials and methods (e.g. comparing means of samples with obviously non-Gaussian distributions or/and unequal variances with parametric tests; treating samples as independent while repeated measures analysis should have been applied, etc.);
• ignoring other scientists from different laboratories who expressed their inability to reproduce experimental results from Ciani’s lab;
• not willing to correct a conclusion-changing mistake in the results description from a behavioural experiment published in a peer-reviewed journal.

As the PI showed little interest, tried to diminish the presented concerns or even discredit the whistleblowers, a few days later I forwarded our report to the dean of the DIBINEM Raffaele Lodi  and the vice-dean Pietro Cortelli. They both proposed to meet. Of the four whistleblowers, only I decided to participate in that meeting. During the meeting it occurred that, surprisingly, the University of Bologna had practically no formalized procedures to deal with allegations regarding potential scientific misconduct or breach of research integrity. 

Whistleblowers switch sides

Due to the unclear procedure, we decided not to sign our report and asked for postponement of action until further notice. Instead, in early November 2016 in a brief email, the dean informed us that our unsigned report has been forwarded to “those in charge of investigating the case”. This turn of events caused a disagreement among the whistleblowers, while none of us knew whether or when the PI would be informed about the ongoing investigation. This uncertainty made the atmosphere in Ciani’s lab unbearable. All my  professional activity was stopped and although I complained to the department’s authorities about it, there was practically no action from the university’s side. 

In the meantime, more proof of the potential scientific misconduct appeared. I myself traced back behavioural experiments dating to the end of 2014 and found more inconsistencies. The new evidence of potential scientific misconduct was added to the updated version of the original report handed to Ciani in October 2016:

Sadly but also not surprisingly, by then none of my co-authors of the initial version of the report wanted to be involved in the case anymore. They instead signed a letter to the dean announcing their dissociation from the report authorship and assuring that

“the work done in Prof. Ciani’s laboratory is of high value and scientific rigour”. 

Interestingly, the dissociation statement was dated 11 November 2016, the day Ciani found out that the report reached the authorities and that an internal investigation was started. That dissociation letter later served as proof that I manipulated my colleagues to defame the PI and that I put their names on the report without their knowledge or consent. Although there was a record of all four persons working on the report (emails and google docs history), no one wanted to acknowledge it. All three whistleblowers who dissociated themselves from the report left the laboratory soon after.

In the meantime, things were heating up as the mutual accusations and distrust spread. After I sent another email to the university authorities asking for assistance, protection and enabling me to continue to work, I was finally invited for a second meeting with the deans, at the dean’s clinic office. The meeting was described as informal.

Time was passing, at the end of November 2016 I wrote a registered letter to the dean’s office asking about the investigation and my protection as whistleblower. This letter was ignored, as all my attempts to obtain information about whistleblower’s rights and legal assistance. No one ever answered, even when it was forwarded to the Committee for equal opportunities, enhancement of the well-being of workers and against discrimination in the workplace (CUG) and anti harassment advisor. After several weeks and reminders, they finally stated that it was outside their competence, since the whistleblower was a postdoc. 

In the meantime, in December 2016 I had two other meetings that I had asked for – with the two vice-rectors of the university; Antonino Rotolo, Vice Rector for Research, and Elena Trombini, Vice Rector for Student Affairs. Apparently, they had been informed about the case already in November, but officially no progress in the case was made. Again, no minutes were taken despite my requests.  As a consequence, I was not able to enforce the agreements I made with the university as there was no hard proof that any such agreements were ever made. As I complained several times about psychological distress, the university finally offered 5 meetings with the university’s psychologist, free of charge. 

Internal investigation

Finally, in the middle of January 2017, an internal commission to investigate the case was formed, and official hearings took place. Specific concerns regarding the proceeding of the commission were as follows (all based on a memory recording of the meeting, there were no minutes or protocols from the hearing):

• Composition: commission’s members were Vincenzo Scarlato, Giovanni Romeo and Claudio Franceschi, all at least partially affiliated with the University of Bologna. 2 out of 3 of the commission members belonged to the same board structure within the university (university clinic “Sant’Orsola”).
• Insufficient expertise of the commission members in rodent behavioural studies. For instance, they had to explained the basics of the standard and commonly used memory test (Morris Water Maze).
• Commission members were often referring to a counter-report that Ciani had produced together with the other two accused lab members, Claudia Fuchs and Stefania Trazzi (both now appointed as assistant professors). I was never allowed to see that counter-report.
• Inappropriate questions asked by the commission, e.g.: 
  • The commission interrogated me about my scientific background, publication record, previous work experience, types of working contracts in the past and their length. They also asked me to send my CV to the commission members.
  • I was questioned about the scientific project I had carried on between October 2015 and March 2016, which was irrelevant to the case. Questions were very detailed such as “what type of vector was used for cloning” and had nothing to do with any allegation raised in the report. The commission expressed interest on my “background and expertise and role in the group” and insinuated several times about me “that her experience is limited”. This was also the line of defence of Ciani who tried to discredit the whistleblower’s professional expertise (although she never previously complained about our collaboration, she started to after the allegations against her were raised).
• Intimidating comments by commission members, not relevant to the merit of the allegations, e.g.:
  • “One of the counterarguments regarding your report is that you had access to something that you should not have had access to. […] Do you think that you had unauthorized access to data that you should not have accessed?”. This referred to my discovery of data irregularities, presented in my second report, when Ciani tasked me with re-analysis of old raw data files in 2015, up until the day she learned of the internal investigation (11 November 2016). Then, she officially accused me of stealing data from a common computer and banned me from the behavioural facility. 
  • Commission members were insisting that I manipulated my colleagues and put their names on the report without their consent (although a record of a common work on this document existed). The commission mentioned the dissociation statement the co-authors signed. They were insisting that I answer “Why are there four names here, no signature, and that actually 3 out of 4 retracted their names” and “How can you explain, do you have any idea why the other 3 names retracted”. Next the commission claimed that I wrote the report all by myself: “Can you for sure state that it was not written by you”. The commission was also not aware that all four co-authors of the report delivered it personally to the PI and that three of them retracted their names the same day Ciani learned about the report being forwarded to the dean.
  • Disregard for insufficient sample sizes (experimental groups composed of 2 animals per group) and the fact that some statistically insignificant results were falsely marked as significant. Although the study containing such errors was ready to be submitted for a publication (approved by co-authors) and presented on a conference, a commission member commented that it should be treated as “preliminary data and should be repeated”, that “conference is just discussion with colleagues” and that ”the final version of the paper until it is not submitted can be revised.” Although the final version of the paper was ready to be submitted while no further experiments were planned (Ciani in her email: “Let’s read and submit it”), a commission member commented: “this doesn’t mean anything” and “maybe it was superficiality? Let’s see what happens if you repeat the experiment”.

I tried to get the attention of the Italian National Research Council (CNR) and exchanged several emails with the head of the Research Ethics and Bioethics Committee, however with little effect. The head of the Committee, although initially vividly interested and sympathetic, stopped replying to the emails from one day to another.

The final reports

In March 2017 the commission issued a “Final report” (above, which later on was referred to as “preliminary”). I was not allowed to see the document for the next 2 months as the official request I had to make to the rector to access it was opposed by a nameless “interested party”. 

Eventually, the report became classified. The university sent a formal prohibition of disseminating, even translated the prohibition into English (it was the only document that the university took trouble to translate, all the remaining official communication was in Italian):

“I would like to remind you that, in view of the merely preparatory nature of the work leading to the Report, all the data contained in the document may be used only for the objectives of your request of 22 March 2017, as prescribed by the Law no. 196/2003, and may not be disclosed to third parties who are not qualified or permitted to use these data.“

The commission’s report was highly critical with regard to both scientific matters and laboratory management by the PI. Although the commission hardly referred to any of the allegations raised by the laboratory members, it identified “scientifically unacceptable practices” such as using very small sample sizes and running behavioural experiments without necessary controls (control groups were evaluated separately, sometimes several months apart from the experimental ones). All those led the commission to question the experimental murine model as a whole. Moreover, the report underlined the lack of transparency and proper communication between the PI and other laboratory members and publications’ co-authors.  

Although the commission’s report was classified, within the next weeks several bodies requested access to this document (including funders of the dubious research), at least some requests were rejected by the rector of the university. Surprisingly, the rector himself was disseminating a letter stating that the commission’s report “regards a single experiment from a single research line (…) that is being repeated”. 

This statement was simply not true, as it was not in line with the report that mentioned “behavioural experiments”(in plural, without specifying them). The rector did not refer to any of the allegations originally delivered to the university by the whistleblowers. He was falsely reassuring external bodies and hiding the scale of the problem. Instead, he was very active in contacting different institutions and reassuring them that the ongoing case was nothing serious, by twisting and selectively representing the statements of the investigative commission.

Consequences?

The only notable consequence that Ciani’s laboratory faced as a result was a temporary suspension of one of the research funding lines. The decision was made by Telethon – a non-profit organisation supported by the Italian Ministry of Education, Universities and Research – independently, since (to our best knowledge) the university did not inform any of the funding bodies about the ongoing investigation or the commissions’ reports. The funding however resumed after the second report by the investigation commission was issued in September 2017.

That second report again highlighted the lack of transparency and information flow between the PI and the group members as well as poor laboratory management. It also gave recommendations to the PI to be implemented in her future work. The investigators again questioned the research line based on the murine model employed in the laboratory. 

In the March 2017 report, a behavioural experiment published in the journal Human Molecular Genetics (HMG)  was scheduled to be repeated. That was because the control group of animals, serving as a reference point for the entire study, was run several months before the rest of all the other experimental groups. The experiment was ordered to be repeated by… Ciani herself. Not surprisingly the “new” result confirmed the validity of the one questioned. In its final September 2017 report, the commission stated that Ciani provided sufficient proof that the experiment published in HMG was reproduced and scientifically valid. 

---

In the second part, you can read how the journal Human Molecular Genetics and its editor Dame Kay Davies dismissed all whistleblower concerns and refused to act in any way on the evidence of research misconduct.

---