- Email: [email protected]
Validity of scientific data — the responsibility of the principal investigator
86
T I B S - M a r c h 1 982
other participants in Reaction [1]. However, prior to the popularization of the superoxide theory, extensive work had been done on the in vitro sensitivity of enzymes and other cell components to hyperbaric oxygen, and the prevailing theory was that enzymes were inactivated in vivo by oxygen. Thus, in Reaction [ 1], a critical enzyme, symbolized by X, would be inactivated ( X + ) , thereby disrupting the overall metabolism of the cell. Haugaard, who reviewed this literature in 19686 , gave two reasons the hypothesis did not gain wider acceptance: 'First, inactivation of enzymes in vitro appeared to be too slow in onset to account for the symptoms that occurred in the intact animal. Second, no conclusive demonstration had been made that any enzyme, in any organ of the intact animal, was inhibited during exposure of the animal to oxygen at an elevated pressure.' However, there are now several examples of enzymes which are rapidly and directly inactivated by dioxygen, and there are clear-cut demonstrations of enzyme inactivation in vivo. Two examples of enzymes inactivated in v i v o , in the presence of superoxide dismutase, are amidophosphoribosyl transferase 2° (purine biosynthesis) and dihydroxyacid dehydratase21 (branched chain amino acid biosynthesis). These and other examples suggest the possibility that understanding oxygen toxicity will require sensitive enzymes be identified, isolated, and characterized in terms of their reactivity towards oxygen in much the same manner one would determine the mechanism of an enzyme. We are left with the question of why cells have superoxide dismutases. The following possibilities should be considered: (a) all superoxide dismutases are involved in protection of cells against oxygen toxicity; (b) superoxide is a metabolite whose concentration must be controlled; (c) superoxide dismutase activity is a trivial property of metalloproteins, inherent to the metal ion, and the real biological functions have not yet been discovered and; (d) combinations of the above. In view of this discussion, possibility (a) seems unlikely. However, possibility (b) cannot be excluded, and certain observations suggest that 02 may play some role in the chemotactic response of macrophages 22. However, it is difficult to imagine why strictly anaerobic organisms which are highly sensitive to oxygen, such as the methanogens, express this type of activity. For some time the author has entertained possibility (c), however, there is no direct evidence in support of alternate functions for these proteins. It is hoped that genetic studies will shed light on this problem. 4" Elsevier Biomedical Press 1982
0376
References
mutases (Michelson, A. M., McCord, J. M. and Fridovich, I., eds), p. 320, AcademicPress 1 McCord, J. M., Keele, Jr., B. B. and Fridovich, I. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 10 Fee, J. A. in Metal Ions in Biological Systems (Sigel, H., ed.), Vol. 13, pp. 259-298, Marcel 1024-1027 Dekker, New York 2 Fee, J. A. in King, T. E., Mason, H. S. and Morrison, M. (eds) Oxidases and Related Sys- 11 Hofmann, H. and Schmutt, O. (1980) Graefes Arch. Opthalmol. 214, 181-185 tems Pergamon Press (in press) Presented at the Third InternationalSymposium on Oxidases and 12 Marshall,L. E., Graham, D. R., Reich, K. A. and Sigman, D. S. (1981)Biochemistry 20, 244-250 Related Redox Systems, July 1--4, 1979, Albany, 13 Archibald,F. S. and Ffidovich, I. (1981)J. BacNew York teriol. 145,442--451 3 Fee, J. A. (1980) in Metal Ion Activation of Dioxygen (Spiro, T., ed.), pp. 209-237, John 14 Stetter, K. O. and Kandler, O. (1973)FEBS Lett 36, 5-8 Wiley and Sons, New York 4 Fee, J. A. (1981) in Oxygen and Oxy-radicals in 15 Hassan, H. M. and Ffidovich, 1. (1977) J. Biol. Chem. 252, 7667-7672 Chemistry and Biochemistry (Rodgers, M. A. J. and Powers, E. L., eds), pp. 205-221, Academic 16 Fee, J. A., Lees, A. C., Bloch, P. L., Gilliland, P. L. and Brown, O. (1980) Biochem. Int. 1, Press, New York 304-311 5 Fee, J. A. (1981) in Oyygen and Life Special PublicationNo. 39, pp. 77-97, The Royal Society 17 Simons, R. S., Jackett, P. S., Carroll, M. E. W. and Lowrie, D. B. (1976) Toxicol. Appl. of Chemistry,BurlingtonHouse, London PharmacoL 37, 271-280 6 Haugaard,N. (1968)Physiol. Rev. 48, 311-373 18 Hassan, H. M. and Fridovich, 1. (1979) Infect 7 Fee, J. A. and Valentine,J. S. (1977) inSuperoxDis. Rev. 1,357-369 ide and Superoxide Dismutases (Michelson, A. M., McCord, J. M. and Fridovich,I., eds), pp. 19 Gersham, R., Gilbert, D. L., Nye, S. W., Dwyer, P. and Fenn, W. O. (1954)Science 119,623--626 19--60AcademicPress, London 8 Bors, W., Saran, M. and Czapski, G. (1980) in 20 ltakura, M. and Holmes, E. W. (1979) J. Biol. Chem. 254, 333-338 Vol. 1 of Biological and Clinical Aspects of Superoxide and Superoxide Dismutases 21 Brown, O. R. and Yein, F. (1978) Biochem. Biophys. Res. Commun. 85, 1219-1224 (Bannister, J. V. and Hill, H. A. O., eds), pp. 22 Petrone, W. F., English, D. K., Wong, K. and 1-3 I, Elsevier/North-HollandBiomedicalPress McCord, J. M. (1980) Proc. Natl. Acad. Sci. 9 Michelson,A. M., McCord, J. M. and Fridovich, U.S.A. 77, 1159-1163 I. (1977) in Superoxide and Superoxide Dis-
Opinion Validity of scientific data- the responsibility of the principal investigator Harvey F. Lodish The complex rules that govern the practice of modern science are rarely set down in writing - at least not in a form which would be read by most practicing biochemists. They are passed - or should be - by example from mentor to Ph.D. student, from lab director to research staff and fellows. To my mind the most important of these concerns the mechanisms by which one ensures that all published data are true and reliable, and can be used as a basis for further investigations by other laboratories. The principal investigator (P.I.) must play the key role in this process. In most American universities and research institutes, this would be the actual P.I., the one who is the recipient of the grant which supports the work. In laboratories with other Harvey F. Lodish is at the Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A.
5067/82/C000 - 0000/$02.75
organizational structures, this would be the person who actually directs the work in question, not necessarily the director of the entire laboratory. In practical terms, this would be the person who submits the paper to the journal, and who (generally) puts his or her name at the end of the list of authors. It is generally unreasonable to expect the Head of a Department, or Director of the Institute, to play an immediate role in the process of validation and verification, given the diverse types of projects and techniques that are underway in even most medium-sized departments or institutes. Many of the recent scandals concerning forged or doctored data stem from the fact that most research groups tend to be rather large, and that the principal investigator or lab chief did not have a direct role in the supervision or conduct of the experiments. Biochemical methodology is evolving very rapidly. It is now very common for a Ph.D.
87
T I B S - M a r c h 1982
student or postdoctoral fellow to he using an instrument, or following a technique, with which the P.I. may have no fin'st-hand experience, and for which the P.I. may not understand all of the principles, pitfalls, and subtleties. In addition, students and postdoctorals are coming under increasing pressure to produce publishable results to attain or maintain a suitable research position. I have seen this situation become especially acute in cases where a fellowship or other sources of funding terminate after a fixed interval. Research groups, and the P.I. in particular, are often under considerable pressure to publish papers before a grant or project is up for review. All these factors underscore the primary role of the P.I. in insuring that all work which emanates from his or her laboratory is absolutely reliable. Relatively few of the problems my colleagues and I face involve outright fraud the deliberate concocting of an entire set of experimental data, although these cases do appear to be widespread in both clinical and basic science. Particularly if the 'results' are important, they will he checked quickly by others in the field. One of the (few) comforting things to emerge from the incident of the protein kinase cascade, for example, was how quickly skepticism was raised by other scientists who were unable to repeat the key results. I am told that most such cases involve extremely intelligent individuals; they know what constitutes 'good' science and can fabricate apparendy 'solid' experimental results. I am not sure one can completely guard against such individuals. However, the farther a senior P.I. is from the day-to-day activities of the laboratory, the more likely it is that a fraud by a subordinate can be perpetrated. To minimize this possibility it is essential that the work of all junior research staff and students be closely supervised. This is a special concern in larger research groups in which the P.I. may designate senior research personnel to direct specific projects. It is also a concern
Here we publish the first in a short series of articles on the practice of science, as outlined in TIBS (1981) Dec. p. i. Among the topics to be covered later in this series are the responsibilities of funding agencies and university administrators, policies on authorship, the imporlance of trust in research, and procedures for judging disputes. TIBS will welcome comments and alternative viewpoints from readers. Those who wish to contribute to this debate should write to the Cambridge office.
in situations where the P.l. puts pressure on subordinates to produce data, and recognizes and rewards subordinates who do produce 'exciting' results without carefully checking them. In all cases, where fraudulent work has been published, the P.I. must bear a large part of the responsibility. A more universal acceptance of this fact may force some P.I.s to pay closer attention to the work that they are supposed to direct. Apparently more common are cases of manipulation of experimental data. Individuals move or eliminate data points to make the data look 'better'. All too often people will publish the 'best experiment', without noting that other repeats of the study generated somewhat different data which might lead to, or support, a different conclusion. Sometimes, an experiment is done only once, and not repeated since the results look so 'good'. Or the results were not checked by an independent technique or approach. Some might argue that these examples fall into the area of sloppy experimental methodology, and should not he treated in the same breath as outright fraud. Are not scientists entitled to a certain measure of self-delusion- if a hypothesis or result looks so good, must it not be right, and should it not be published? However, the effects of publishing unreliable data can be just as bad as fraudulent results. At best, other scientists will waste a few weeks to reproduce the results; at worst, the efforts of an entire field can be misdirected for a considerable period. Science is a collective enterprise which is founded largely on mutual trust, and there is a natural tendency for lab directors to accept the validity of results which are shown to them by members of their research group. However, in carrying out the important responsibility of authenticating the results, the P.I. can - and should impart to his own students and staff important lessons in scientific excellence and honesty. In practice, it is unreasonable for a P.I. to repeat, himself or herself, all of the results which are to be published. However, it is the P.I's responsibility to go over as much of the raw experimental data as possible, as well as the calculated values which are the ones which generally get published. What were the backgrounds which were subtracted? How many plaques or colonies actually were counted? What sorts of averages were made in computing a table or graph? How representative is the micrograph which is selected for publication? In each case, the P.I. must insist that all experiments be repeated, in their entirety, at least once before being published, and more if there is any question concerning the validity of the data. The P.I. should also go over himself/herself the results of each of these
repeats. Ideally, the P.I. should insist that another member of the group repeat each key result, but in most situations this is, unfortunately, not practical. (The notion that all experiments must be repeated is not intuitive for most new graduate students and technicians.) An important lesson which the P.I. should teach his group, especially new students, is to he as critical as one can of one's own results. One should think of all of the possible reasons why a particular conclusion might he wrong, and to insist that all of the proper experimental controls be done. I have found that most beginning students and technical staff do not realize sufficiently the importance of controls - in particular, that controls must he run with each repeat of the experiment. (I am not old enough to have forgotten learning this lesson myself!) Some of this self-criticism can he done in meetings of the whole research group, where all members ought to be involved in these discussions. The attitude of the P.I. toward the quality of the work from his/her group is of great importance. By making it clear that the lab chief is concerned about each experimental control, and each data point, the P.I. can minimize, I feel, most of the problems of fraudulent or sloppy research. One can never eliminate this possibility, but the farther the P.I, becomes from the actual interpretation and criticism of the experimental data from his/her group, the more likely this possibility, becomes. All of the authors on a paper have a responsibility, individually as well as co|lectively, for the validity of all of the data, and all must be encouraged to criticize all of the results presented. Many projects now involve collaborative studies between two or more laboratories. Especially in such cases, no author should allow his or her name to appear on a paper unless he or she is comfortable with all of the data and conclusions presented, and is willing to accept responsibility for the validity of the entire work *. The P.I. hears a special responsibility to ensure that all published papers contain enough detailed information to allow other labs to repeat the experiments. Each lab has its own idiosyncratic way of doing certain procedures, and it is the P.I's duty to make sure that these are emphasized. Importantly, when earlier papers, especially from the same laboratory, are referenced for a pat'0cular experimental protocol, the P.I. should be sure that that is the procedure which is currently being used by the group. Following all of these recommendations * A later article in this serieswill concernpoliciesof authorship.
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TIBS - March 1982
will help ensure that the published data will prove to be reproducible in the hands of others. In cases where there are conflicts or disagreements between labs concerning the repeatability of a result, the P.I. has a recognized responsibility to offer to exchange reagents, organisms etc. The awareness that the P.I. will be completely open in case of any such disputes encourages junior members of the group to be more precise and careful with their own results. Finally, any question concerning the authenticity of a set of results must also be met quickly by openness and candor on the
part of the P.I. There are, unfortunately, no generally accepted set of rules to be followed in this eventuality, but it would seem that, at a minimum, the P.I. agree to, and even insist upon, a full evaluation by an outside group of scientists. The P.I. should co-operate by making available to this committee all lab notebooks and other raw data, and by insisting that each member of his group co-operate fully with them. The knowledge that any suggestion of fraud will be dealt with completely and fairly may deter some individuals from undertaking such acts.
Students and staff should be taught, by example, that one's scientific reputation is the most valuable thing one possesses, and that once lost - by publication of sloppy or falsified or irreproducible work - it is very difficult to regain.
Acknowledgement I thank many colleagues and friends who read an earlier version of this essay and who made a number of valuable suggestions for improving it.
Reviews Actin-binding proteins Susan W. Craig and Thomas D. Pollard The many different proteins which modify actin polymerization and actin filament structure can be divided into three functional groups. These actin-binding proteins are thought to regulate actin assembly and the formation of the spectacular variety of actin structures found in non-muscle cells. In non-muscle ceils, actin exists in a variety of structural forms. Some are remarkably ordered, such as the cross-linked, core bundles of actin f'daments in microvilli and f'dopodia; others are completely disordered as in lamellipodia, in a ruffling membrane and in the bulk of the cytoplasm where much of the actin is in the form of a randomly cross-linked network of filaments. In addition, as much as half of the cellular actin cannot be easily sedimented even though ionic conditions in the cell and the concentration of actin favor full polymerization. Rapid interconversions of the polymeric states of actin and changes in the viscoelastic properties of the actin network are thought to form part of the structural basis for cell motility and cell shape 1. Since the self-assembly properties of actin described in last month's TIBS 2 account only for the formation of long filaments, additional factors must he responsible for the versatility exhibited by actin in non-muscle cells. The effort to determine how assembly and interconversion of the diverse polymeric forms of actin are regulated has led to the discovery of many Susan W. Craig is at the Department of Physiological Chemistry and Thomas D. Pollard is at theDepartment of Cell Biology and Anatomy, Johns Hopkins UniversitySchool of Medicine, Baltimore, MD 21205, U.S.A. © Elsevier Biomedical Press 1982
actirvbinding proteins (Tables I-II/). With few exceptions, this large group of proteins can be divided into three functional classes (Fig. 1). Aetin crom-liaking proteins The discovery that isolated cytoplasm can undergo gel-sol transformations, depending on pH, Ca 2+ concentration, temperature and ionic strength, similar to those found in motile cells, allowed the use of fractionation and reconstitution methods to identify proteins responsible for gelation (Table I). These studies showed that cytoplasmic gels are formed by filamentous actin and actin cross-linking proteins. MONOMER SEQUESTRATION
Cross-linking proteins have been isolated and assayed by their ability to cause a sharp increase in the apparent viscosity of actin filaments when the amount of crosslinker reaches the critical gelling concentration. In theory*, the cross-linker concentration at the gel point is the numher density of cross-links required to form a continuous three-dimensional network which extends through the volume of solution, i.e., an 'infinite' network. The gel point can he detected experimentally because the 'infinite' network has some rigidity or ability to support a shear stress. The rigidity of a gel increases as the density of cross-links increases, until the progressively closer apposition of filaments results in tight bundling and, finully, precipitation of the network. Characterization of actin cross-linking proteins is at an early stage. Gelation proteins must have more than one binding site for actin, but the sites have not been counted. Avidities of cross-linkers for actin filaments have been measured only for vinculin, fascin, and macrophage actin. binding protein. These avidities (Table I)
CAPPING
CROSSLINKING Short Capped Filaments
Monomer bound to Depolymrizing Protein
Monomers
Long Filament
Network
Bundle
Fig. 1. Regulation of actin assembly by 3 classes of actin-bindingproteins. ClassI cross-linksfilaments into networks or bundles. Class H caps an end of the filament and may sever preformed filaments. Class III inhibits polymerization by binding to actin monomers.
0376 - 5067/82/0~30 - 0000/$02.75
T I B S - M a r c h 1 982
other participants in Reaction [1]. However, prior to the popularization of the superoxide theory, extensive work had been done on the in vitro sensitivity of enzymes and other cell components to hyperbaric oxygen, and the prevailing theory was that enzymes were inactivated in vivo by oxygen. Thus, in Reaction [ 1], a critical enzyme, symbolized by X, would be inactivated ( X + ) , thereby disrupting the overall metabolism of the cell. Haugaard, who reviewed this literature in 19686 , gave two reasons the hypothesis did not gain wider acceptance: 'First, inactivation of enzymes in vitro appeared to be too slow in onset to account for the symptoms that occurred in the intact animal. Second, no conclusive demonstration had been made that any enzyme, in any organ of the intact animal, was inhibited during exposure of the animal to oxygen at an elevated pressure.' However, there are now several examples of enzymes which are rapidly and directly inactivated by dioxygen, and there are clear-cut demonstrations of enzyme inactivation in vivo. Two examples of enzymes inactivated in v i v o , in the presence of superoxide dismutase, are amidophosphoribosyl transferase 2° (purine biosynthesis) and dihydroxyacid dehydratase21 (branched chain amino acid biosynthesis). These and other examples suggest the possibility that understanding oxygen toxicity will require sensitive enzymes be identified, isolated, and characterized in terms of their reactivity towards oxygen in much the same manner one would determine the mechanism of an enzyme. We are left with the question of why cells have superoxide dismutases. The following possibilities should be considered: (a) all superoxide dismutases are involved in protection of cells against oxygen toxicity; (b) superoxide is a metabolite whose concentration must be controlled; (c) superoxide dismutase activity is a trivial property of metalloproteins, inherent to the metal ion, and the real biological functions have not yet been discovered and; (d) combinations of the above. In view of this discussion, possibility (a) seems unlikely. However, possibility (b) cannot be excluded, and certain observations suggest that 02 may play some role in the chemotactic response of macrophages 22. However, it is difficult to imagine why strictly anaerobic organisms which are highly sensitive to oxygen, such as the methanogens, express this type of activity. For some time the author has entertained possibility (c), however, there is no direct evidence in support of alternate functions for these proteins. It is hoped that genetic studies will shed light on this problem. 4" Elsevier Biomedical Press 1982
0376
References
mutases (Michelson, A. M., McCord, J. M. and Fridovich, I., eds), p. 320, AcademicPress 1 McCord, J. M., Keele, Jr., B. B. and Fridovich, I. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 10 Fee, J. A. in Metal Ions in Biological Systems (Sigel, H., ed.), Vol. 13, pp. 259-298, Marcel 1024-1027 Dekker, New York 2 Fee, J. A. in King, T. E., Mason, H. S. and Morrison, M. (eds) Oxidases and Related Sys- 11 Hofmann, H. and Schmutt, O. (1980) Graefes Arch. Opthalmol. 214, 181-185 tems Pergamon Press (in press) Presented at the Third InternationalSymposium on Oxidases and 12 Marshall,L. E., Graham, D. R., Reich, K. A. and Sigman, D. S. (1981)Biochemistry 20, 244-250 Related Redox Systems, July 1--4, 1979, Albany, 13 Archibald,F. S. and Ffidovich, I. (1981)J. BacNew York teriol. 145,442--451 3 Fee, J. A. (1980) in Metal Ion Activation of Dioxygen (Spiro, T., ed.), pp. 209-237, John 14 Stetter, K. O. and Kandler, O. (1973)FEBS Lett 36, 5-8 Wiley and Sons, New York 4 Fee, J. A. (1981) in Oxygen and Oxy-radicals in 15 Hassan, H. M. and Ffidovich, 1. (1977) J. Biol. Chem. 252, 7667-7672 Chemistry and Biochemistry (Rodgers, M. A. J. and Powers, E. L., eds), pp. 205-221, Academic 16 Fee, J. A., Lees, A. C., Bloch, P. L., Gilliland, P. L. and Brown, O. (1980) Biochem. Int. 1, Press, New York 304-311 5 Fee, J. A. (1981) in Oyygen and Life Special PublicationNo. 39, pp. 77-97, The Royal Society 17 Simons, R. S., Jackett, P. S., Carroll, M. E. W. and Lowrie, D. B. (1976) Toxicol. Appl. of Chemistry,BurlingtonHouse, London PharmacoL 37, 271-280 6 Haugaard,N. (1968)Physiol. Rev. 48, 311-373 18 Hassan, H. M. and Fridovich, 1. (1979) Infect 7 Fee, J. A. and Valentine,J. S. (1977) inSuperoxDis. Rev. 1,357-369 ide and Superoxide Dismutases (Michelson, A. M., McCord, J. M. and Fridovich,I., eds), pp. 19 Gersham, R., Gilbert, D. L., Nye, S. W., Dwyer, P. and Fenn, W. O. (1954)Science 119,623--626 19--60AcademicPress, London 8 Bors, W., Saran, M. and Czapski, G. (1980) in 20 ltakura, M. and Holmes, E. W. (1979) J. Biol. Chem. 254, 333-338 Vol. 1 of Biological and Clinical Aspects of Superoxide and Superoxide Dismutases 21 Brown, O. R. and Yein, F. (1978) Biochem. Biophys. Res. Commun. 85, 1219-1224 (Bannister, J. V. and Hill, H. A. O., eds), pp. 22 Petrone, W. F., English, D. K., Wong, K. and 1-3 I, Elsevier/North-HollandBiomedicalPress McCord, J. M. (1980) Proc. Natl. Acad. Sci. 9 Michelson,A. M., McCord, J. M. and Fridovich, U.S.A. 77, 1159-1163 I. (1977) in Superoxide and Superoxide Dis-
Opinion Validity of scientific data- the responsibility of the principal investigator Harvey F. Lodish The complex rules that govern the practice of modern science are rarely set down in writing - at least not in a form which would be read by most practicing biochemists. They are passed - or should be - by example from mentor to Ph.D. student, from lab director to research staff and fellows. To my mind the most important of these concerns the mechanisms by which one ensures that all published data are true and reliable, and can be used as a basis for further investigations by other laboratories. The principal investigator (P.I.) must play the key role in this process. In most American universities and research institutes, this would be the actual P.I., the one who is the recipient of the grant which supports the work. In laboratories with other Harvey F. Lodish is at the Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A.
5067/82/C000 - 0000/$02.75
organizational structures, this would be the person who actually directs the work in question, not necessarily the director of the entire laboratory. In practical terms, this would be the person who submits the paper to the journal, and who (generally) puts his or her name at the end of the list of authors. It is generally unreasonable to expect the Head of a Department, or Director of the Institute, to play an immediate role in the process of validation and verification, given the diverse types of projects and techniques that are underway in even most medium-sized departments or institutes. Many of the recent scandals concerning forged or doctored data stem from the fact that most research groups tend to be rather large, and that the principal investigator or lab chief did not have a direct role in the supervision or conduct of the experiments. Biochemical methodology is evolving very rapidly. It is now very common for a Ph.D.
87
T I B S - M a r c h 1982
student or postdoctoral fellow to he using an instrument, or following a technique, with which the P.I. may have no fin'st-hand experience, and for which the P.I. may not understand all of the principles, pitfalls, and subtleties. In addition, students and postdoctorals are coming under increasing pressure to produce publishable results to attain or maintain a suitable research position. I have seen this situation become especially acute in cases where a fellowship or other sources of funding terminate after a fixed interval. Research groups, and the P.I. in particular, are often under considerable pressure to publish papers before a grant or project is up for review. All these factors underscore the primary role of the P.I. in insuring that all work which emanates from his or her laboratory is absolutely reliable. Relatively few of the problems my colleagues and I face involve outright fraud the deliberate concocting of an entire set of experimental data, although these cases do appear to be widespread in both clinical and basic science. Particularly if the 'results' are important, they will he checked quickly by others in the field. One of the (few) comforting things to emerge from the incident of the protein kinase cascade, for example, was how quickly skepticism was raised by other scientists who were unable to repeat the key results. I am told that most such cases involve extremely intelligent individuals; they know what constitutes 'good' science and can fabricate apparendy 'solid' experimental results. I am not sure one can completely guard against such individuals. However, the farther a senior P.I. is from the day-to-day activities of the laboratory, the more likely it is that a fraud by a subordinate can be perpetrated. To minimize this possibility it is essential that the work of all junior research staff and students be closely supervised. This is a special concern in larger research groups in which the P.I. may designate senior research personnel to direct specific projects. It is also a concern
Here we publish the first in a short series of articles on the practice of science, as outlined in TIBS (1981) Dec. p. i. Among the topics to be covered later in this series are the responsibilities of funding agencies and university administrators, policies on authorship, the imporlance of trust in research, and procedures for judging disputes. TIBS will welcome comments and alternative viewpoints from readers. Those who wish to contribute to this debate should write to the Cambridge office.
in situations where the P.l. puts pressure on subordinates to produce data, and recognizes and rewards subordinates who do produce 'exciting' results without carefully checking them. In all cases, where fraudulent work has been published, the P.I. must bear a large part of the responsibility. A more universal acceptance of this fact may force some P.I.s to pay closer attention to the work that they are supposed to direct. Apparently more common are cases of manipulation of experimental data. Individuals move or eliminate data points to make the data look 'better'. All too often people will publish the 'best experiment', without noting that other repeats of the study generated somewhat different data which might lead to, or support, a different conclusion. Sometimes, an experiment is done only once, and not repeated since the results look so 'good'. Or the results were not checked by an independent technique or approach. Some might argue that these examples fall into the area of sloppy experimental methodology, and should not he treated in the same breath as outright fraud. Are not scientists entitled to a certain measure of self-delusion- if a hypothesis or result looks so good, must it not be right, and should it not be published? However, the effects of publishing unreliable data can be just as bad as fraudulent results. At best, other scientists will waste a few weeks to reproduce the results; at worst, the efforts of an entire field can be misdirected for a considerable period. Science is a collective enterprise which is founded largely on mutual trust, and there is a natural tendency for lab directors to accept the validity of results which are shown to them by members of their research group. However, in carrying out the important responsibility of authenticating the results, the P.I. can - and should impart to his own students and staff important lessons in scientific excellence and honesty. In practice, it is unreasonable for a P.I. to repeat, himself or herself, all of the results which are to be published. However, it is the P.I's responsibility to go over as much of the raw experimental data as possible, as well as the calculated values which are the ones which generally get published. What were the backgrounds which were subtracted? How many plaques or colonies actually were counted? What sorts of averages were made in computing a table or graph? How representative is the micrograph which is selected for publication? In each case, the P.I. must insist that all experiments be repeated, in their entirety, at least once before being published, and more if there is any question concerning the validity of the data. The P.I. should also go over himself/herself the results of each of these
repeats. Ideally, the P.I. should insist that another member of the group repeat each key result, but in most situations this is, unfortunately, not practical. (The notion that all experiments must be repeated is not intuitive for most new graduate students and technicians.) An important lesson which the P.I. should teach his group, especially new students, is to he as critical as one can of one's own results. One should think of all of the possible reasons why a particular conclusion might he wrong, and to insist that all of the proper experimental controls be done. I have found that most beginning students and technical staff do not realize sufficiently the importance of controls - in particular, that controls must he run with each repeat of the experiment. (I am not old enough to have forgotten learning this lesson myself!) Some of this self-criticism can he done in meetings of the whole research group, where all members ought to be involved in these discussions. The attitude of the P.I. toward the quality of the work from his/her group is of great importance. By making it clear that the lab chief is concerned about each experimental control, and each data point, the P.I. can minimize, I feel, most of the problems of fraudulent or sloppy research. One can never eliminate this possibility, but the farther the P.I, becomes from the actual interpretation and criticism of the experimental data from his/her group, the more likely this possibility, becomes. All of the authors on a paper have a responsibility, individually as well as co|lectively, for the validity of all of the data, and all must be encouraged to criticize all of the results presented. Many projects now involve collaborative studies between two or more laboratories. Especially in such cases, no author should allow his or her name to appear on a paper unless he or she is comfortable with all of the data and conclusions presented, and is willing to accept responsibility for the validity of the entire work *. The P.I. hears a special responsibility to ensure that all published papers contain enough detailed information to allow other labs to repeat the experiments. Each lab has its own idiosyncratic way of doing certain procedures, and it is the P.I's duty to make sure that these are emphasized. Importantly, when earlier papers, especially from the same laboratory, are referenced for a pat'0cular experimental protocol, the P.I. should be sure that that is the procedure which is currently being used by the group. Following all of these recommendations * A later article in this serieswill concernpoliciesof authorship.
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TIBS - March 1982
will help ensure that the published data will prove to be reproducible in the hands of others. In cases where there are conflicts or disagreements between labs concerning the repeatability of a result, the P.I. has a recognized responsibility to offer to exchange reagents, organisms etc. The awareness that the P.I. will be completely open in case of any such disputes encourages junior members of the group to be more precise and careful with their own results. Finally, any question concerning the authenticity of a set of results must also be met quickly by openness and candor on the
part of the P.I. There are, unfortunately, no generally accepted set of rules to be followed in this eventuality, but it would seem that, at a minimum, the P.I. agree to, and even insist upon, a full evaluation by an outside group of scientists. The P.I. should co-operate by making available to this committee all lab notebooks and other raw data, and by insisting that each member of his group co-operate fully with them. The knowledge that any suggestion of fraud will be dealt with completely and fairly may deter some individuals from undertaking such acts.
Students and staff should be taught, by example, that one's scientific reputation is the most valuable thing one possesses, and that once lost - by publication of sloppy or falsified or irreproducible work - it is very difficult to regain.
Acknowledgement I thank many colleagues and friends who read an earlier version of this essay and who made a number of valuable suggestions for improving it.
Reviews Actin-binding proteins Susan W. Craig and Thomas D. Pollard The many different proteins which modify actin polymerization and actin filament structure can be divided into three functional groups. These actin-binding proteins are thought to regulate actin assembly and the formation of the spectacular variety of actin structures found in non-muscle cells. In non-muscle ceils, actin exists in a variety of structural forms. Some are remarkably ordered, such as the cross-linked, core bundles of actin f'daments in microvilli and f'dopodia; others are completely disordered as in lamellipodia, in a ruffling membrane and in the bulk of the cytoplasm where much of the actin is in the form of a randomly cross-linked network of filaments. In addition, as much as half of the cellular actin cannot be easily sedimented even though ionic conditions in the cell and the concentration of actin favor full polymerization. Rapid interconversions of the polymeric states of actin and changes in the viscoelastic properties of the actin network are thought to form part of the structural basis for cell motility and cell shape 1. Since the self-assembly properties of actin described in last month's TIBS 2 account only for the formation of long filaments, additional factors must he responsible for the versatility exhibited by actin in non-muscle cells. The effort to determine how assembly and interconversion of the diverse polymeric forms of actin are regulated has led to the discovery of many Susan W. Craig is at the Department of Physiological Chemistry and Thomas D. Pollard is at theDepartment of Cell Biology and Anatomy, Johns Hopkins UniversitySchool of Medicine, Baltimore, MD 21205, U.S.A. © Elsevier Biomedical Press 1982
actirvbinding proteins (Tables I-II/). With few exceptions, this large group of proteins can be divided into three functional classes (Fig. 1). Aetin crom-liaking proteins The discovery that isolated cytoplasm can undergo gel-sol transformations, depending on pH, Ca 2+ concentration, temperature and ionic strength, similar to those found in motile cells, allowed the use of fractionation and reconstitution methods to identify proteins responsible for gelation (Table I). These studies showed that cytoplasmic gels are formed by filamentous actin and actin cross-linking proteins. MONOMER SEQUESTRATION
Cross-linking proteins have been isolated and assayed by their ability to cause a sharp increase in the apparent viscosity of actin filaments when the amount of crosslinker reaches the critical gelling concentration. In theory*, the cross-linker concentration at the gel point is the numher density of cross-links required to form a continuous three-dimensional network which extends through the volume of solution, i.e., an 'infinite' network. The gel point can he detected experimentally because the 'infinite' network has some rigidity or ability to support a shear stress. The rigidity of a gel increases as the density of cross-links increases, until the progressively closer apposition of filaments results in tight bundling and, finully, precipitation of the network. Characterization of actin cross-linking proteins is at an early stage. Gelation proteins must have more than one binding site for actin, but the sites have not been counted. Avidities of cross-linkers for actin filaments have been measured only for vinculin, fascin, and macrophage actin. binding protein. These avidities (Table I)
CAPPING
CROSSLINKING Short Capped Filaments
Monomer bound to Depolymrizing Protein
Monomers
Long Filament
Network
Bundle
Fig. 1. Regulation of actin assembly by 3 classes of actin-bindingproteins. ClassI cross-linksfilaments into networks or bundles. Class H caps an end of the filament and may sever preformed filaments. Class III inhibits polymerization by binding to actin monomers.
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