How to avoid inappropriate data processing Ruey-Hwa Chen, IBC

1. How to avoid inappropriate data processingRuey-Hwa Chen, IBC

2. Definition of research misconducts "Research Misconduct" means fabrication, falsification, plagiarism, or other practices that seriously deviate from those that are commonly accepted within the scholarly community for proposing, conducting, publishing or otherwise reporting research. It does not include honest error or honest differences in interpretations or judgments of data. http://www.ncsu.edu/sparcs/policy/references/interim.html

3. Experiments Data acquisition Data analysis Data presentation The most prevalent problem Sloppy experimentation and misrepresentation of data • Data acquisition and analysis (data selection or questionable statistics) • Improperly handling image data (to smarten or prettify images which end up with unintended consequences)

4. Guidelines for image manipulation (J. Cell Biol.) • No specific feature within an image may be enhanced, obscured, moved, removed, or introduced. • Adjustments of brightness, contrast, or color balance are acceptable if they are applied to the whole image and as long as they do not obscure or eliminate any information present in the original. • Nonlinear adjustments (e.g., changes to gamma settings) must be disclosed in the figure legend. • The grouping of images from different parts of the same gel, or from different gels, fields, or exposures must be made explicit by the arrangement of the figure and in the text of the figure legend. (http://www.jcb.org/misc/ifora.shtml#digim)

5. Pixel number change nonlinear adjustments

6. Using a black line to reveal vertically sliced gel

7. Using separate blocks for images derived from different gels

8. Note: Any manipulation that violates these guidelines is a misrepresentation of the original data and is a form of misconduct (JCB 166: 11-15, 2004).

9. Image checking system:Used by JCB, JEM, JGP (Rockefeller University Press) • All digital images in manuscript accepted for publication are scrutinized by our production department for any indication of improper manipulation. • Questions raised by the production department will be referred to the editors, who will request the original data from the authors for comparison to the prepared figures. If the original data cannot be produced, the acceptance of the manuscript may be revolved. • Cases of deliberate misrepresentation of data will result in revocation of acceptance, and will be reported to the corresponding author’s home institution or funding agency. (http://www.jcb.org/misc/ifora.shtml#digim)

10. Be aware of image resolution Make sure the "Resample Image" box in "Image Size" dialog window is notchecked and the "Width", "Height", and "Resolution" boxes should be linked by the graphic chain. (JCB) linked Do not check this box

11. Resampling refers to changing the “pixel dimensions” (and therefore display size) of an image. When you downsample (decrease the number of pixels), information is deleted from the image. When you resample up (increase the number of pixels), new pixels are added. Downsampled (decrease the number of pixels) Original Resampled up (increase the number of pixels)

12. Guidelines for digital images (Nat. Cell Biol.) • Authors should list all image acquisition tools and image software packages used. • Authors should document key image-gathering settings and processing manipulations in the Supplementary information. • Images gathered at different times or from different locations should not be combined into a single image, unless it is stated that the resultant image is a product of time-averaged data or time-lapse sequence. If juxtaposing images is essential, the borders should be clearly demarcated in the figure and described in the legend.

13. Guidelines for digital images (Nat. Cell Biol.) • The use of touch-up tools, such as cloning and healing tools in Photoshop, or any feature that deliberately obscures manipulation, is to be avoided. • Processing (such as changing brightness and contrast) is appropriate only when applied equally across the entire image and when it is applied equally to controls. Contrast should not be adjusted so that data disappear. • When submitting revised final figures upon conditional acceptance, authors may be asked to submit original, unprocessed images. (http://www.nature.com/ncb/about/ed_policies/index.html#images)

14. Guidelines for gels and blots (NCB) • Vertically sliced gels that juxtapose lanes that were not contiguous in the experiment must have a clear separation or a black line delineating the boundary between the gels. • Cropped gels in the paper must retain important bands. • Cropped blots in the body of the paper should retain at least six band widths above and below the band. • High-contrast gels and blots are discouraged. Multiple exposures should be presented in supplementary information if high contrast is unavoidable. Immunoblots should be surrounded by a black lineto indicate the borders of the blot, if the background is faint. • For quantitative comparison, appropriate reagents, controls and imaging methods with linear signal ranges should be used.

15. Authors are encouraged to present full scans of gel/Western blot data in Supplementary Information. NCB 12: 583, 2010 Supplementary information

16. Guidelines for microscopy images (NCB) • Cells from multiple fields should not be juxtaposed in a single field; instead multiple supporting fields of cells should be shown as supplementary information. • Adjustments should be applied to the entire image. Threshold manipulation, expansion or contraction of single ranges and the altering of high signals should be avoided. • Pseudo-coloring and nonlinear adjustment (e.g., gamma changes) are only allowed if unavoidable and must be disclosed. • Adjustments of individual color channels are sometimes necessary on “merged” images, but this should be noted in the figure legend.

17. Examples of improper image manipulations Rossner and Yamada What’s in a picture? (JCB: 166 11-15, 2004)

18. (JCB 166: 11-15, 2004)

19. X O Adjustment the intensity of a single band. Improper adjustment of contrast (JCB 166: 11-15, 2004)

20. (JCB 166: 11-15, 2004)

21. Manipulated images (left)- cut individual band and paste it to a new image -revealed by contrast adjustment (right) NCB 5: 320-329, 2003/Corrigendum in NCB 6: 373, 2004/Retraction in NCB 9:480 2007

22. (JCB 166: 11-15, 2004) Enhancing a specific feature – the immunogold particles. • Acceptable ways to highlight a feature such as immunogold particle: • Add arrows • Pseudocoloring particles without altering the brightness of individual pixels (e.g., colorize function of Photoshop) : should be disclosed in the figure legend.

23. Misrepresentation of a microscope field-combine images from separate microscope fields into a single field. (JCB 166: 11-15, 2004)

24. JCB 176: 131-132, 2007 Detecting image manipulation in the Hwang et al. stem cell paper. The image in the top row purports to show negative staining for a particular cell surface marker in four different cell lines. Adjustment of tonal range in Photoshop clearly shows that the two middle images are identical (lower panel).

25. Data acquisition Garbage in = Garbage out Sample selection Unbiased, representative (patients, cells from a population) • Define the question. => What information are needed? • Human tissue samples (well defined clinical manifestation; pathology; pedigree) • Tumor vs. surrounding (normal) tissues • Question => required information => collection of relevant data

26. Data selection It is easy to see the results you expect, and ignore the rest. Don’t fool yourself ! The unexpected, unfit data may be meaningful. Selecting or discarding certain data should have explicit rationale. Best to describe the rationale clearly. Double-blind test

27. Reproducibility A reliable result should be reproducible (by you and by others). Single case: A single event (patient, comet), but multiple observations. A single observation: not reliable. Extraordinary claims demand extraordinary proof.

28. Expected result 2.3kb (expected size) DNA sequence => M71-H Unexpected results. 1 kb (localized to chr. 13) Not associated with any other OR OR regulated by another trans element? Presenting ALL information could be meaningful ! Inverse PCR from an Olfactory Receptor (OR) gene, to look for association with an H enhancer element Lomvardas et al. (2006) Cell 126: 403-13. Not specific to olfactory tissue. Common to nose and spleen? Ubiquitous regulation? Chrosomsomal association?

29. Define colocalization as 25% overlap of pixels from the H and OR signals on sections from a Z series The inability to detect colocalization of H with OR genes in all cells is likely due to our inability to retain these interchromosomal interactions stoichiometrically throughout the fixation and denaturation procedures. Describe and discuss the criteria for data acquisition and analysis Colocalization of the H Enhancer with OR Promoters (identified by Anti-M50) DNA FISH M50/M71/OMP: DIG; FITC-conjugated anti-DIG antibody (green). H: biotin; rhodamine-conjugated neutravidin (red) Anti-M50/M71 (blue) Lomvardas et al. (2006) Cell 126: 403-13.

30. Are these images representative (typical)? Describe the phenotypic range and distribution Yao & Sun (2005) EMBO J. 24: 2602-12. Sample size

31. Keep clear lab notes Belongs to the lab. • Organize your thoughts • Historical records • Helps you to remember • Allows other people to replicate your experiment • Defense against fraud • Content: • Date (bound note book, not loose leaf; numbered pages) • Title of the experiment • Brief statement of purpose • Description of the experiment • Summary (interpretation) of the results Record everything as soon as you can. Ref: At the Bench: A Laboratory Navigator. Chapter 5, Laboratory Notebooks. CSHL Press

32. Always preserve raw data Microscopy Society of America on ethical digital image processing (as published in Microscopy Today Nov/Dec 2003, p61): Ethical digital imaging requires that the original uncompressed image file be stored on archival media (e.g., CD-R) without any image manipulation or processing operation. All parameters of the production and acquisition of this file, as well as any subsequent processing steps, must be documented and reported to ensure reproducibility. Generally, acceptable (non-reportable) imaging operations include gamma correction, histogram stretching, and brightness and contrast adjustments. All other operations (such as Unsharp-masking, Gaussian blur, etc.) must be directly identified by the author as part of the experimental methodology. However, for diffraction data or any other image data that is used for subsequent quantification, all imaging operations must be reported.

33. Responsibility of PI • Check all primary data? (may be difficult) • Pay attention to details. Do not look only at final assembled figures. • Establish lab culture, attitude for proper ethics

34. Responsibility of the scientist You may be pushed to obtain certain results. In many cases of fraud, the perpetrator has blamed the PI, saying that the PI expected a particular result, and the researcher felt compelled to produce it. It is true that a PI may want a result. But your data are your responsibility, and it is up to you to be sure the data are recorded honestly and accurately. Ref: At the Bench: A Laboratory Navigator. Chapter 5, Laboratory Notebooks. CSHL Press

35. Responsibility of the coauthors • Authorship should be justified • Each coauthor should write down his/her specific contribution. Neural Develop 3(1):26

36. Who can be listed as an author? • Authorship criteria defined by International Committee of Medical Journal Editors: Authors are ones who have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data, who have drafted an article or revised it critically for important intellectual content, or who have made final approval of the revision to be published.

37. Responsibilities of coauthorship • Anyone who allows his or her name to appear among the authors of a paper assumes major responsibilities. • Coauthors should be able and willing to defend the paper in public, and must be confident about the integrity of the data. • Coauthorship should never be conferred or accepted as an honor or simply as a reward for providing resources or sponsoring a junior investigator who has done all the work. ~ by Arnold Relman, editor of the New England Journal of Medicine

38. Plagiarism • The action to pass off someone else’s work as your own • Self-plagiarism: ranging from duplicate publication to “salami-slicing”, where authors add small amounts of new data to a previous paper (e.g., The previous paper analyzed 15 patient samples and the new one adds 15 more).

39. General rules to avoid plagiarism • The article cannot contain a large chunk of material (e.g., a whole paragraph) that has been published previously by others– including Introduction, Materials and Methods... etc. • If reuse of substantial part of your own work is necessary, clear citation of the previous publication is required. Warning: a number of publishers have implanted plagiarism screen tool (e.g. Crosscheck) to screen published and submitted content. http://www.crossref.org/crosscheck.html

40. Avoid duplicate publication—even for control data Proc. Natl. Acad. Sci. USA 107: 7616, 2006 Fig. 3E Gao et al.

41. Dealing with fraud • Prevention is better than treatment. • Catch it within the lab. • Whistle-blowing and dealing with whistle-blowing. • Do not treat it lightly. A single case may destroy your career. • Credibility is key in science. • Investigation; internal and external • http://www.ncsu.edu/sparcs/policy/references/interim.html • Damage control. • Admission to wrong-doing. • Honesty is the best principle.

45. Additional Info Sources: Responsible Conducts in Research UC, San Diego http://ethics.ucsd.edu/resources/resources-topics.html Online Research Ethics Course http://ori.hhs.gov/education/products/montana_round1/research_ethics.html Office of Research Integrity http://ori.dhhs.gov/

46. Acknowledgements • Dr. Henry Sun (IMB, Academia Sinica) • Dr. Hong-Chen Chen (National Chung Hsing University)