# Failure to Replicate: A Sign of Scientific Misconduct?

^{1}

^{2}

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Analysis of Experiments

#### 2.1. Biochemical and Radiobiological Considerations

#### 2.2. Replication Attempts: Is There a Bystander Effect?

#### 2.2.1. B and C 100% Experiments

_{1}found in Table 1 of the 2001 [2] paper (cf. also figures 3 and 7 in the 1999 paper [1] and figure 1 in the 2001 paper [2]). A’s survivals are markedly different from those of B and C.

**Figure 1.**Graphs of unpublished experiments performed by B and C following the protocols for exposure of 100% (

**A**) and 50% (

**B**) of V79 cells to tritiated thymidine.

_{1}, the theoretical cross-section for survival in mBq/labeled cell, listed in Table 1 of the 2001 paper. The various symbols represent the results of seven experiments by B and C. There are more than 100-fold more survivors in the experiments by B and C than in A’s experiments at the level of 5 mBq/labeled cell. The symbols in 1B show the results of eight 50% V79 experiments of B and C. The dashed line approximates the 50% V79 survival curves depicted in the two papers. The horizontal line is at 0.70 survival, the approximate level at which the survival curves plateau. There are about 70-fold more survivors in B’s and C’s results than in those of A at the level of 20 mBq/labeled cell.

#### 2.2.2. B and C 50% Experiments

#### 2.3. Statistical Analysis of the Raw Data

#### 2.3.1. Terminal Digit Distributions of the Coulter Counts

^{−5}(highly significant).

**Figure 2.**Terminal digit distributions for coulter counts. (

**A**) B’s and C’s Coulter digit distributions, chi-square for fit to uniform, p = 0.462; (

**B**) A’s Coulter digit distributions, chi-square for fit to uniform, p = 4.49 × 10

^{−4}.

**Table 1.**Terminal digit analysis of coulter counts of experiments (Exps) represented in the published figures. The numbers of the various digit distributions were compared to the corresponding uniform distributions to calculate the chi-squared statistic and the corresponding probabilities for nine degrees of freedom.

Figure | Type | # of Exps | Terminal Digit | Total | Chi-square | p | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|

0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |||||||

Controls | --- | 100% | 7 | 17 | 18 | 16 | 18 | 31 | 16 | 22 | 22 | 23 | 20 | 203 | 9.2 | 0.42 |

--- | 50% | 8 | 17 | 22 | 22 | 23 | 20 | 23 | 25 | 33 | 24 | 21 | 230 | 6.8 | 0.66 | |

1999 | 3 and 6 | 50% | 2 | 5 | 4 | 12 | 5 | 2 | 6 | 3 | 7 | 13 | 3 | 60 | 21.0 | 1.3 × 10^{−2} |

3 | 100% | 2 | 6 | 9 | 3 | 4 | 11 | 4 | 7 | 4 | 3 | 6 | 57 | 11.3 | 0.26 | |

7 | 100% | 3 | 10 | 3 | 4 | 1 | 3 | 7 | 4 | 4 | 4 | 2 | 42 | 14.2 | 0.12 | |

2001 | 1 | 100% | 7 | 16 | 22 | 27 | 17 | 8 | 24 | 14 | 12 | 11 | 23 | 174 | 20.7 | 1.4 × 10^{−2} |

2A | 50% | 3 | 4 | 11 | 12 | 4 | 0 | 12 | 1 | 8 | 3 | 17 | 72 | 89.7 | 8.7 × 10^{−6} |

#### 2.3.2. Frequency of the Mean in Triplicate Counts

**Figure 3.**Mid-ratio distributions for colony counts. (

**A**) B’s and C’s colonies, 29 of 88 in the 0.4–0.6 interval, 35.4 ± 5.19 expected (p ~ 0.91); (

**B**) A’s colonies, 83 of 111 in the 0.4–0.6 interval, 34.2 ± 5.1 expected, p ~ 3.73 × 10

^{−18}).

**Table 2.**Analysis of the frequency of the occurrence of the rounded mean in the triplicate samples of colony counts. Column 6 is the number N of triplicate samples that contain the rounded mean of the three counts. Column 7 is the number K that is expected based on our model. The z-scores incorporate the correction for continuity.The numerator in Column 5 is the number of qualifying triplicates (triplicates for which the gap (c-a) is greater than two). The denominator in Column 5 is the total number of triplicates.

Figure | Type | # of Exps | # of Triplicates | N = No. with Mean | K = No. Expected | SD | Z-Score | p (N ≥ K) | |
---|---|---|---|---|---|---|---|---|---|

Controls | --- | 100% | 7 | 65/67 | 14 | 12.1 | 3.2 | 0.44 | 0.32 |

--- | 50% | 8 | 71/71 | 7 | 11.7 | 3.1 | −1.7 | 0.96 | |

1999 | 3&6 | 50% | 2 | 19/19 | 14 | 3.0 | 1.6 | 6.7 | 2.1 × 10^{−8} |

3 | 100% | 2 | 20/20 | 12 | 3.1 | 1.6 | 5.2 | 6.1 × 10^{−6} | |

7 | 100% | 3 | 13/15 | 9 | 3.2 | 1.5 | 3.4 | 9.1 × 10^{−4} | |

2001 | 1 | 100% | 7 | 63/66 | 37 | 11.2 | 3.0 | 8.4 | 2.6 × 10^{−13} |

2A | 50% | 3 | 23/24 | 13 | 3.9 | 1.8 | 4.8 | 1.9 × 10^{−5} |

## 3. Discussion

#### 3.1. Can the Discrepancies Be Related to the Times of the Experiments?

#### 3.2. Can the Discrepancies Be Related to Changes in the Two Protocols?

#### 3.3. 100% Survival Results of B and C Are Compatible with the Experiments of Others

#### 3.4. Statistics Can Be a Powerful Tool to Test Numerical Results

## 4. Conclusions

- (1)
- A review of the literature involving the killing of tissue culture cells that have incorporated tritiated thymidine into their DNA predicts exponential survival as reported in two papers published in the journal Radiation Research [1,2] under conditions that were likely not present in the survival experiments performed by A.
- (2)
- Likewise, the early and recent literature predicts biphasic survival curves that were not seen in the two papers under conditions that were likely to have been present in the experiments performed by A.
- (3)
- The expected biphasic survival was seen in the 15 experiments performed by B and C following the same or similar protocols listed by A. These results of B and C appear to be reliable and indicate that under the experimental conditions that prevailed in all of the studies, there is only a small bystander effect.
- (4)
- Statistical analyses of the numerical data that form the background of survival experiments performed by A and reported in the two papers are not consistent with the expectation of the random distributions of numbers.
- (5)
- Statistical analyses of the numerical data that form the background of survival experiments performed by B and C are consistent with the expectation of random distributions of numbers.
- (6)
- This report emphasizes the importance of familiarity with the published literature in the field in question and of access to the raw data on which scientific reports rely.

## Acknowledgments

## Author Contributions

## Conflicts of Interest

## References

- Bishayee, A.; Rao, D.V.; Howell, R.W. Evidence for pronounced bystander effects caused by nonuniform distributions of radioactivity using a novel three-dimensional tissue culture model. Radiat. Res.
**1999**, 152, 88–97. [Google Scholar] [CrossRef] - Bishayee, A.; Hill, H.Z.; Stein, D.; Rao, D.V.; Howell, R.W. Free radical-initiated and gap junction-mediated bystander effect due to nonuniform distribution of incorporated radioactivity in a three-dimensional tissue culture model. Radiat. Res.
**2001**, 155, 335–344. [Google Scholar] [CrossRef] - Tobey, R.A.; Anderson, E.C.; Petersen, D.F. The effect of thymidine on the duration of G1 in Chinese hamster cells. J. Cell Biol.
**1967**, 35, 53–59. [Google Scholar] [CrossRef] - Puck, T.T. Studies of the Life Cycle of Mammalian Cells. Cold Spring Harb. Symp. Quant. Biol.
**1964**, 29, 167–176. [Google Scholar] [CrossRef] - Bjursell, G.; Reichard, P. Effects of thymidine on deoxyribonucleoside triphosphate pools and deoxyribonucleic acid synthesis in Chinese hamster ovary cells. J. Biol. Chem.
**1973**, 248, 3904–3909. [Google Scholar] - Fox, R.M.; Tripp, E.H.; Tattersall, M.H. Mechanism of deoxycytidine rescue of thymidine toxicity in human T-leukemic lymphocytes. Cancer Res.
**1980**, 40, 1718–1721. [Google Scholar] - Hiramoto, K.; Narahara, K.; Kimoto, H. Synchronization culture of amniotic fluid cells using excess thymidine block followed by deoxycytidine release and its application to high-resolution banding analysis of chromosomes. Jpn. J. Hum. Genet.
**1990**, 35, 195–206. [Google Scholar] [CrossRef] - Morris, N.R.; Fischer, G.A. Studies concerning inhibition of the synthesis of deoxycytidine by phosphorylated derivatives of thymidine. Biochim. Biophys. Acta
**1960**, 42, 183–184. [Google Scholar] [CrossRef] - Wheater, R.F.; Roberts, S.H. An improved lymphocyte culture technique: Deoxycytidine release of a thymidine block and use of a constant humidity chamber for slide making. J. Med. Genet.
**1987**, 24, 113–114. [Google Scholar] [CrossRef] - Ehmann, U.K.; Williams, J.R.; Nagle, W.A.; Brown, J.A.; Belli, J.A.; Lett, J.T. Perturbations in cell cycle progression from radioactive DNA precursors. Nature
**1975**, 258, 633–636. [Google Scholar] [CrossRef] - Hoy, C.A.; Lewis, E.D.; Schimke, R.T. Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols. Mol. Cell Biol.
**1990**, 10, 1584–1592. [Google Scholar] - Hu, V.W.; Black, G.E.; Torres-Duarte, A.; Abramson, F.P. 3H-thymidine is a defective tool with which to measure rates of DNA synthesis. FASEB J.
**2002**, 16, 1456–1457. [Google Scholar] - Pollack, A.; Bagwell, C.B.; Irvin, G.L., 3rd. Radiation from tritiated thymidine perturbs the cell cycle progression of stimulated lymphocytes. Science
**1979**, 203, 1025–1027. [Google Scholar] - Bedford, J.S.; Mitchell, J.B.; Griggs, H.G.; Bender, M.A. Cell killing by gamma rays and beta particles from tritiated water and incorporated tritiated thymidine. Radiat. Res.
**1975**, 63, 531–543. [Google Scholar] [CrossRef] - Burki, H.J.; Koch, C.; Wolff, S. Molecular suicide studies of 125I and 3H disintegration in the DNA of Chinese hamster cells. Curr. Top. Radiat. Res. Q
**1978**, 12, 408–425. [Google Scholar] - Burki, H.J.; Okada, S. Killing of cultured mammalian cells by radioactive decay of tritiated thymidine at −196 °C. Radiat. Res.
**1970**, 41, 409–424. [Google Scholar] [CrossRef] - Burki, H.J.; Roots, R.; Feinendegen, L.E.; Bond, V.P. Inactivation of mammalian cells after disintegration of 3H or 125I in cell DNA at −196 °C. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
**1973**, 24, 363–375. [Google Scholar] [CrossRef] - Chan, P.C.; Lisco, E.; Lisco, H.; Adelstein, S.J. The radiotoxicity of iodine-125 in mammalian cells II. A comparative study on cell survival and cytogenetic responses to 125IUdR, 131TUdR, and 3HTdR. Radiat. Res.
**1976**, 67, 332–343. [Google Scholar] [CrossRef] - Marin, G.; Bender, M.A. Survival Kinetics of Hela S-3 Cells after Incorporation of 3h-Thymidine or 3h-Uridine. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
**1963**, 7, 221–233. [Google Scholar] [CrossRef] - Drew, R.M.; Painter, R.B. Action of tritiated thymidine on the clonal growth of mammalian cells. Radiat. Res.
**1959**, 11, 535–544. [Google Scholar] [CrossRef] - Drew, R.M.; Painter, R.B. Further studies on the clonal growth of HeLa S3 cells treated with tritiated thymidine. Radiat. Res.
**1962**, 16, 303–311. [Google Scholar] [CrossRef] - Keprtova, J.; Minarova, E. The effect of 3H-thymidine on the proliferation of in vitro cultured mammalian cells. Gen. Physiol. Biophys.
**1985**, 4, 81–92. [Google Scholar] - Persaud, R.; Zhou, H.; Baker, S.E.; Hei, T.K.; Hall, E.J. Assessment of low linear energy transfer radiation-induced bystander mutagenesis in a three-dimensional culture model. Cancer Res.
**2005**, 65, 9876–9882. [Google Scholar] [CrossRef] - Panter, H.C. Cell inactivation by tritium decays at 37 and -196 degrees C: Some comparisons with X rays. Radiat. Res.
**1981**, 87, 79–89. [Google Scholar] [CrossRef] - Buyse, M.; George, S.L.; Evans, S.; Geller, N.L.; Ranstam, J.; Scherrer, B.; Lesaffre, E.; Murray, G.; Edler, L.; Hutton, J.; et al. The role of biostatistics in the prevention, detection and treatment of fraud in clinical trials. Stat. Med.
**1999**, 18, 3435–3451. [Google Scholar] [CrossRef] - Carlisle, J.B. The analysis of 168 randomised controlled trials to test data integrity. Anaesthesia
**2012**, 67, 521–537. [Google Scholar] [CrossRef] - Hudes, M.L.; McCann, J.C.; Ames, B.N. Unusual clustering of coefficients of variation in published articles from a medical biochemistry department in India. FASEB J.
**2009**, 23, 689–703. [Google Scholar] [CrossRef] - Al-Marzouki, S.; Evans, S.; Marshall, T.; Roberts, I. Are these data real? Statistical methods for the detection of data fabrication in clinical trials. BMJ
**2005**, 331, 267–270. [Google Scholar] - Baggerly, K.A.; Coombes, K.R. Deriving chemosensitivity from cell lines: Forensic bioinformatics and reproducible research in high-throughput biology. Ann. Appl. Stat.
**2009**, 3, 1309–1334. [Google Scholar] [CrossRef] - Simonsohn, U. The lesson from two cases of fabricated data detected by statistics alone. Available online: http://ssrn.com/abstract=2114571 or http://dx.doi.org/10.2139/ssrn.2114571 (accessed on 21 November 2012).
- Forensic Tools. Office of Research Integrity: Rockville MD, USA. Available online: http://ori.hhs.gov/forensic-tools (accessed on 26 August 2014).

© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

## Share and Cite

**MDPI and ACS Style**

Hill, H.Z.; Pitt, J.H.
Failure to Replicate: A Sign of Scientific Misconduct? *Publications* **2014**, *2*, 71-82.
https://doi.org/10.3390/publications2030071

**AMA Style**

Hill HZ, Pitt JH.
Failure to Replicate: A Sign of Scientific Misconduct? *Publications*. 2014; 2(3):71-82.
https://doi.org/10.3390/publications2030071

**Chicago/Turabian Style**

Hill, Helene Z., and Joel H. Pitt.
2014. "Failure to Replicate: A Sign of Scientific Misconduct?" *Publications* 2, no. 3: 71-82.
https://doi.org/10.3390/publications2030071