abstract: # Background
  .
  Back problems are often recurring or chronic. It is therefore not surprising that chiropractors wish to prevent their return or reduce their impact. This is often attempted with a long-term treatment strategy, commonly called maintenance care. However, some aspects of maintenance care are considered controversial. It is therefore relevant to investigate the scientific evidence forming the basis for its use.
  .
  # Objectives
  .
  A review of the literature was performed in order to obtain answers to the following questions: What is the exact definition of maintenance care, what are its indications for use, and how is it practised? How common is it that chiropractors support the concept of maintenance care, and how well accepted is it by patients? How frequently is maintenance care used, and what factors are associated with its use? Is maintenance care a clinically valid method of approach, and is it cost-effective for the patient?
  .
  # Results
  .
  Thirteen original studies were found, in which maintenance care was investigated. The relative paucity of studies, the obvious bias in many of these, the lack of exhaustive information, and the diversity of findings made it impossible to answer any of the questions.
  .
  # Conclusion
  .
  There is no evidence-based definition of maintenance care and the indications for and nature of its use remains to be clearly stated. It is likely that many chiropractors believe in the usefulness of maintenance care but it seems to be less well accepted by their patients. The prevalence with which maintenance care is used has not been established. Efficacy and cost-effectiveness of maintenance care for various types of conditions are unknown. Therefore, our conclusion is identical to that of a similar review published in 1996, namely that maintenance care is not well researched and that it needs to be investigated from several angles before the method is subjected to a multi-centre trial.
author: Charlotte Leboeuf-Yde; Lise Hestbæk
date: 2008
institute: 1Nordic Institute of Chiropractic and Clinical Biomechanics, part of Clinical Locomotion Science, University of Southern Denmark, Forskerparken 10, DK-5230 Odense M, Denmark
references:
title: Maintenance care in chiropractic – what do we know?

# Background

Chiropractors all over the world are consulted for spinal pain and dysfunction. Because many spinal pain complaints are chronic or recurrent in nature \[1,2\], it is understandable that, once improvement has been achieved, chiropractors attempt to prevent new events or maintain patients at their optimal level. This is usually done by scheduling additional visits over a prolonged period of time but at longer time intervals than during the acute event. Among chiropractors, this approach is named "maintenance care", whereas in public health terms it is described as secondary or tertiary prevention. Secondary prevention is aimed at preventing new events, whereas tertiary prevention means that improved patients with incurable conditions are maintained at the best possible level.

Although it appears perfectly logical to use maintenance care in chronic and recurrent conditions, when informally discussing this phenomenon with chiropractors, we have often detected either a disinclination to discuss, or an ardour of arguments, often resulting in an embarrassing change of subject. In other words, maintenance care appears to be, for some, a politically incorrect topic.

This might be because the indications for treatment in asymptomatic patients depend solely on tests and observations, such as palpation findings, none of which has been shown to be clearly valid \[3\]. When treating an acute problem, however, this lack of valid examination tests is of little or no concern, as the patient's reaction to the treatment will provide feedback on the construct validity of the various treatment procedures. Therefore, there appears to be disagreement among chiropractors as to whether chiropractic treatment is mainly effective in the acute phase or whether it is possible also to prevent the underlying disorder, regardless of whether the patient is symptomatic at the time of examination and treatment.

Jamison has discussed the preventive aspect of maintenance care, when encompassing other than the musculoskeletal conditions. She points out that some chiropractors believe "that subluxations can cause, and spinal adjustments correct, diverse problems ranging from pain to more subtle endocrine, visceral and autonomic dysfunctions" and warns that this scientifically untested theory has considerable ill effects in the scientific and medical communities \[4\]. In general, if chiropractors believe that "spinal health" equals good health, it is understandable that they would try to convince patients to have regular preventive chiropractic treatments. Jamison discusses this in a second paper, where she also mentions the negative repercussions of such practice \[5\]. It could also be that the overzealous use of maintenance care has resulted in problems with various reimbursement systems, as Mitchell warned already in 1980 \[6\]. Some individuals' short-term financial gains could be seen as having negative long-term repercussions for the whole profession.

The concept of maintenance care, therefore, seems to be associated with the very core of disagreement between chiropractors and their styles of practice; those who treat mainly musculoskeletal conditions and those who attempt to treat also other conditions. In addition, it may divide those who believe that their examination method is objective and valid and those who depend (also) on patients' signs and symptoms for their diagnosis and treatment.

Nevertheless, maintenance care seems to be commonly employed, and if it is a useful model of preventive treatment, it should be recognized as such; but if it is ineffective, it should not be part of the chiropractic patient management strategy. Maintenance care therefore, merits being taken seriously and to be subjected to scientific scrutiny.

In 1993, the Mercy Guidelines \[7\] attempted to perform a literature review on this subject but ended up making its recommendations largely on clinical experience "of nearly 100 years". The report suggested that the use of chiropractic adjustments in a regiment of preventive/maintenance care has merit. There are no statements in the guideline in relation to indications, type of treatment, duration and frequency of treatment, nor on effectiveness. It is merely written that maintenance care is "discretionary and elective on the part of the patient" and that when recommended, "it is necessary for the practitioner to clearly identify the type and nature of this care and to give proper patient disclosure".

Aker and Martel, three years later, performed a narrative review and concluded on the basis of the sparse literature that "there is no scientific evidence to support the claim that maintenance care improves health status" and went on to recommend a series of research actions to be taken \[8\]. Our continued monitoring of the literature revealed several additional studies since the time of their publication.

# Objectives

Therefore, a new literature review of this subject appeared timely, with the intent of:

1\. Defining maintenance care and the indications for and nature of its use.

2\. Describing to what degree chiropractors believe in maintenance care and to what degree it is accepted by patients.

3\. Establishing the prevalence with which chiropractors use maintenance care, and factors associated with its use.

4\. Determining its efficacy and cost-effectiveness for various types of conditions.

Because of the few articles that could be traced in relation to the number of questions, only a narrative review could be undertaken.

## Search strategy and inclusion criteria

A librarian-assisted electronic literature search was attempted using the Medline and Mantis databases, with no limitations for language or time period. The search terms were "chiropractic", "maintenance care", and "prevention", both as Mesh-term and free text. However, fewer articles than those already known by the authors appeared, and therefore a manual search was undertaken as well. The index lists of chiropractic journals were examined, as well as reference lists of articles on the topic, which resulted in the 13 articles used in this review. In addition, guidelines and chiropractic textbooks were consulted, but not exhaustively, in order to bring forth some background information on the topic.

The only inclusion criteria for this review were that the texts were research articles published in journals, that they dealt with chiropractors or chiropractic patients, and that they discussed the issue of maintenance care, also described as secondary prevention, tertiary prevention, or regular, long-term chiropractic care. Articles exclusively dealing with primary prevention were not eligible for the review.

# Results

## General description of the studies

We found 13 original articles, in which the issue of maintenance care was investigated. Eleven could be described as surveys and have been briefly described in Table 1 \[Additional file 1\]. Eight were cross-sectional \[4,5,9-14\], one was a file inspection study \[15\], one was a prospective study \[16\], and one was a one-year multi-centre prospective outcome study, in which the prevalence of maintenance care was retrospectively investigated at the one-year follow-up \[17\]. The two non-survey articles consisted of a case-report \[18\] and a preliminary randomized controlled clinical trial, investigating the efficacy of maintenance care in patients with chronic LBP \[19\]. The first of the studies was published in 1976 with data collected in 1973–4 \[15\] and the last 30 years later \[19\].

Five of the surveys were from Australia, three from the USA, two from UK, and one from Norway (Table 1 \[Additional file 1\]). The clinical trial would have been carried out in Canada \[19\], whereas it is unclear in which country the case-report was produced \[18\]. The sample sizes ranged from a case-report of 1 patient \[18\] to 2056 case files \[15\].

Five of the 11 surveys had low response rates: 20% \[5\], 22% \[4\], 35% \[13\], 44% \[11\], 51% \[10\], and in one survey the response rate does not appear to have been reported \[12\]. In the file inspection study, the proportion of participants was higher (71%) than the percentage of practitioners whose files were inspected (35%) \[15\]. Two of the surveys with higher response rates included specific study samples that nevertheless probably did not represent the general chiropractic profession; one of recently graduated Australian chiropractors \[9\] and one in which Australian chiropractors were included on the basis of whether they practised in a chiropractor-dense area or not \[16\].

In summary, there were only few studies, covering a wide time-period, unevenly distributed across the world, and the study participants were often unlikely to be representative of their target populations.

## Definitions of maintenance care

The definitions of maintenance care that we found were not based on scientific evidence of the clinical validity of maintenance care but perhaps more on opinion and consensus.

Because there is a conviction among some chiropractors that spinal adjustments/manipulations have a preventive effect not only on musculoskeletal problems but also on the general state of health, it is not surprising that one "officially recognized and approved" definition of maintenance care that we found was rather vague. This would allow the chiropractor to apply it according to his/her own scope of practice. The definition is "Appropriate treatment directed toward maintaining optimal body function. This is treatment of the symptomatic patient who has reached pre-clinical status or maximum medical improvement, where condition is resolved or stable" \[20\].

Another definition found in the literature is "...a regimen designed to provide for the patient's continued well-being or for maintaining the optimum state of health while minimizing recurrences of the clinical status" in brief also "continuing care" \[6\]. These definitions resemble that used by Breen, when reporting his study, in 1976: "... treatment, either scheduled or elective, which occurred after optimum recorded benefit was reached, provided there was no evidence of relapse." \[15\]. But on the other hand, according to a recent British study of chiropractors, osteopaths and physiotherapists, at least 10% of each profession reported that they sometimes continue to treat patients with low back pain who show almost no improvement \[14\]. Therefore, it appears that maintenance care is also offered to patients who have *not* improved. Boline and Sawyer \[10\] report on "regular chiropractic care", which we interpreted as meaning maintenance care.

Other authors, who studied this subject, did not define maintenance care at all \[9,13,16,17\] and in one study such non-definition was stated to be purposeful, in order to prevent bias of the investigation process \[11\].

## Indications for maintenance care

There was only sparse information in the literature on the indications for maintenance care and there was no information on specific indications for particular conditions.

The chiropractors in a North-American study by Rupert \[11\] generally agreed that the purposes of maintenance care were to minimize recurrence or exacerbation, maintain or optimize state of health, prevent conditions from developing, provide palliative care for "incurable" conditions, and determine and treat subluxations (all statements with over 80% agreement). Fifty-six percent meant that the purpose of maintenance care was to prevent subluxations, and this was confirmed in a similar study of Australian chiropractors \[13\]. Most of these statements reflect secondary or tertiary preventive approach. Nevertheless, some could also be interpreted as referring to a primary preventive approach.

Interestingly, the concept of "prevention and health promotion" was used in a North American study of maintenance care in relation to chiropractic patients aged at least 65 \[12\]. Boline and Sawyer explored similar concepts, also in a study from North America, in which they investigated attitudes among chiropractors in relation to counselling of patients on a healthy lifestyle, i.e. including elements of primary prevention, as well as regular chiropractic care. \[10\]. This shows that they mean that chiropractors should participate in health promotion and prevention of disease, outside the realm of the purely musculoskeletal.

According to a prospective multicenter study of 115/205 Norwegian chiropractors and 832 patients with persistent low back pain, patients, who after one year reported definite improvement, were treated only a few times and those with poorer outcome had a larger number of consultations over a period of one year. This might indicate that tertiary prevention is offered to and accepted by some of those patients with persistent low back pain, who fail to recover, but that secondary prevention in patients with more satisfactory outcome is less common \[17\]. This information is the only indication of how maintenance care is actually employed in clinical practice.

## The nature of the use of maintenance care

According to the Mercy Guidelines \[7\], spinal adjustments are central in maintenance care. However, the research literature is vague on the contents of a maintenance care consultation and the frequency of treatments required for different types of patients.

Rupert \[11\] asked his North-American study subjects to describe the therapeutic components of maintenance care and concluded that they were adjustments/spinal manipulation, exercises, proper eating habits, patient education, and vitamin supplementation. This finding was confirmed in a similar a study of Australian chiropractors \[13\] and in another study of Rupert et al in North American patients, aged 65 or older \[12\].

Wenban described the outcome in relation to the various outcomes measures used when treating one female patient with a complaint of uncomplicated chronic low back pain \[18\]. His indications for treatment were "tenderness of the patient's vertebral spinous processes, S2 spinous process, and the superior aspect of the posterior superior iliac spines". According to the author, this indicated a "subluxation", and providing that there were no other "more serious indicators" (not further specified), treatment was provided. The treatment consisted of adjustments only, using a combination of diversified and sacro-occipital techniques (a reference is provided to two textbooks\].

Wenban also described the frequency of visits, after a 12 weeks intensive care period, as 2 times per week for 6 weeks, 1 time per week for 2 weeks, and 1 time per 2 weeks for the reminder of the study period of 5.5 months. In addition, Jamison offers some information on this subject. According to the participants in one of her studies: "Maintenance adjustments should be offered on a basis of once a month to once every three to four months" \[4\]. According to one of Rupert's studies, North American patients who agree to receive maintenance care, average 14 visits per year \[11\], and in another study, elderly maintenance care patients (65+ years of age) average 17 visits per year \[12\]. This amounts to a little more than one visit per month.

There were no studies of different types of strategies in relation to different types of patients or conditions.

## Beliefs among chiropractors and acceptance among patients

Boline and Sawyer in a 1987-survery of North American chiropractors noted that 98% of their participants believed that "regular chiropractic care would be important for the 'average' person" \[10\]. Jamison found that 93% of Australian chiropractors considered that at least some patients would require maintenance care (and that 41% thought that all patients would) \[4\]. In another survey, she found that 92% believed that spinal adjustments promote health in asymptomatic patients \[5\].

However, the last two studies dealt primarily with the concept of spinal adjustments and the prevention of endocrine, visceral and autonomic dysfunction, which may have incited chiropractors of specific opinions to participate and, indeed, the very low response rates (22% and 20%, respectively) indicate that this may have been the case.

These beliefs are underpinned by the finding in the study by Rupert \[11\], in which 40% of the chiropractic respondents believe that there is adequate research to support the concept of maintenance care. The Australian respondents were less naïve, with only 22% supporting this statement \[13\].

We found no information on the patients' perspective of maintenance care. However, in one study it was stated that 79% of patients are recommended for maintenance care and that 34% of those "elect to receive these services" \[11\]. It is not clear, whether these estimates are based on an objective count of patient files or on the participating chiropractors' opinions. However, the figure of 34% does not appear unreasonable, given the personal experience of many chiropractors that only some patients are willing and able to continue treatment past the acute event.

Even among patients with persistent low back pain, maintenance care might not be attractive. Of the 832 participants in a large Norwegian multi-centre prospective outcome study, all with persistent low back pain at base line, only 14% were reported by their chiropractors to have received some type of maintenance care during the subsequent year \[17\].

In summary, it is possible that most chiropractors believe in maintenance care but data from unbiased samples are missing. The opinion of patients is unknown, although it might be less positive, as a relatively low percentage of patients seem to accept to continue treatment past the initial treatment program.

## Prevalence of use and factors associated with its use

Although the concept of maintenance care seems to be firmly ensconced in the chiropractic profession, the frequency of its use has not been clearly described.

Breen reported in 1976 that 36% of 2987 case files belonged to patients who received maintenance care \[15\]. These patients were obtained from a sample belonging to 24 British chiropractors, i.e. 35% of the practitioners at that time. They had been selected to give a proportional representation of practitioners in the British hospital regions, and a 20% randomly selected sample of up to 1000 case files was taken from each practice. There were proportionally fewer files from newly established practices. Most of these chiropractors were educated in North America, practising at a time when there were relatively few manipulating practitioners but also at a time when chiropractic was relatively unknown. It was noted that most patients consulted for "rheumatic conditions and in particular low back pain" whereas non-musculoskeletal problems were very rare. This appears to be the first investigative study on this subject, and it probably provides a fair picture of the use of maintenance care in the UK at that time.

Others have reported the proportion of patients who receive maintenance care in the literature. Webb and Leboeuf in 1987 found that 44% of newly graduated Australian chiropractors estimated that at least 34% of their patients were on maintenance care \[9\].

A similar estimate was obtained in another Australian study published two years later \[16\]. In both these two studies, only 6% of the respondents reported that more than 2/3 of their patients received maintenance care. However, although the response rates were better in these, two studies (65% and 82%, respectively); their estimates were based on the practitioners' opinion rather than exact counts of patient files.

Obviously, the use of maintenance care will affect the clinic income. According to Rupert's participants, 23% of practices' incomes was generated from maintenance care \[11\]. That the use of maintenance care can affect the patient turnover was shown in the Leboeuf et al study from Australia \[16\].

We were unable to find any information on which factors play a role, when a chiropractor decides to offer a maintenance care program to a patient, and no information seems to be available on what considerations patients take into account when deciding to accept such a program.

## Efficacy and cost-effectiveness for various types of conditions

It remains also to study the efficacy and cost-effectiveness of maintenance care for various types of conditions.

To our knowledge, it has only been attempted to test the efficacy of maintenance care in one well-designed pilot study, in which 29 patients with chronic low back pain were randomly allocated to either a non-maintenance care group or a maintenance care group (for a 9-month treatment after one initial month of treatment). At follow-up, there was no difference in pain but the group that received maintenance care had lower disability scores than the control group \[19\]. A full-scale trial is presently underway on patients with neck pain (personal communication – M. Descarreaux).

# Discussion

This literature review reveals that more than 30 years of ad hoc research into maintenance care does not provide much information. In fact, the relative paucity of studies, the obvious biases in many of these, the lack of exhaustive information, and the diversity of findings made it impossible to answer any of our questions.

It is not clear whether chiropractors use maintenance care mainly as a secondary or tertiary preventive measure, and if so how frequently and for which conditions. It is possible that there are considerable differences between countries and perhaps during certain periods. The Australian literature in the 1990s dealt with some fundamental concepts, such as "the healthy spine – freedom from disease" concept, which the UK study 20 years earlier did not concern itself with. In between, Mitchell in 1980 \[6\] transmitted a warning on over-servicing, which is the negative side of maintenance care if used unwisely. None of the studies looked at this very difficult balance.

Several studies indicated that almost all chiropractors believe in the value of maintenance care, but newer studies on unbiased study samples are needed to confirm these findings. In addition, it is possible that differences exist between different groups of chiropractors. Would chiropractors who graduated from a university-based chiropractic institution have the same beliefs as those coming from chiropractic colleges of a more traditional type? Further, the density of chiropractors in a region might have an effect on this aspect.

Patients' opinions and expectations of maintenance care and their satisfaction with maintenance care appear not to have been studied. This is, nevertheless, an important subject. Unless expectations and results match, patients are likely to become unsatisfied.

The treatment program, including contents and time schedule, requires further study. Only one report deals explicitly with the timing of treatments. Wenban \[18\] describes a pre hoc determined and rigid plan for a large number of treatment visits in a young patient with "uncomplicated" low back pain for more than three months. We know, from personal experience that also other, more flexible, treatment plans are used, but how common are these respective approaches?

Despite the bulk of the literature being of poor quality, there seems to be light at the end of the tunnel. It has been shown that it is feasible to conduct a randomized controlled clinical trial of maintenance care, and a full-scale study is presently underway. However, several aspects other than efficacy need to be investigated. For example, it would be relevant to test the cost-effectiveness for patients who choose to be treated regularly vs. those who receive treatment only when symptoms arise. This should be investigated for different types of conditions and using different treatment programs. In other words, the aim should be to be able to say, not only, if maintenance care is a clinically valid treatment approach but, also, for whom it should be used, how and when.

However, before testing the clinical validity of maintenance care, it would be necessary to find out what it is, how it is practised and on which indications, to make sure that such a trial does reflect the situation in real life.

# Conclusion

• There is no evidence-based definition of maintenance care and the indications for and nature of its use remains to be clearly stated.

• It is likely that many chiropractors believe in the usefulness of maintenance care but it seems to be less well accepted by their patients.

• The prevalence with which maintenance care is used has not been established.

• Efficacy and cost-effectiveness of maintenance care for various types of conditions are unknown.

Therefore, the recommendations given by Aker and Martel \[8\] more than a decade ago are still relevant, namely that "before a large-scale, multicentered clinical trial can be pursued, a series of preliminary studies need to be conducted to delineate the parameters of care to be used in the clinical trial, the outcome measures to be used, and the feasibility of conducting such a complicated and resource-intensive study."

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

CY did the literature search. Both authors read the articles and abstracted the data. CY made the first draft of the manuscript. LH revised it critically for intellectual content. Both authors read and approved the final manuscript.

# Supplementary Material

###### Additional file 1

Table 1. Description of 11 surveys in a review on the use of maintenance care among chiropractors. The table provides an overview of the reviewed articles, including author, year of publication, year and country of study, population, response rate, sampling method, data collection and objectives as they relate to the present review.

Click here for file

### Acknowledgements

The manuscript was preared within the scope of the authors' positions at The Nordic Institute of Chiropractic and Clinical Biomechanics. The only role of the Institute was to pay the authors' salary. Otherwise, the Institute had no role in the study design; in the collection, analyis an interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.


date: 2013-03-15
title: Findings of research misconduct.

Findings of Research Misconduct

**Notice Number:**  NOT-OD-13-049**

**

  NOT-OD-13-049**

**

Key Dates

**Release Date:**   March 13, 2013

Issued by

Department of Health and Human Services

Purpose

SUMMARY: Notice is hereby given that the Office of Research Integrity (ORI) has taken final action in the following case:

Adam C. Savine, Washington University in St. Louis: Based on the report from Washington University in St. Louis (WUSTL) and Respondent's admission, ORI found that Mr. Adam C. Savine, former doctoral student, Department of Psychology, WUSTL, engaged in research misconduct in research supported by National Institute of Mental Health (NIMH), National Institutes of Health (NIH), grant R56 MH066078, National Institute on Drug Abuse (NIDA), NIH, grants F31 DA032152 and R21 DA027821, and National Institute on Aging (NIA), NIH, grant T32 AG00030.

ORI found that the Respondent engaged in research misconduct by falsifying data that were included in the following three publications and six conference abstracts:

Publications

1\. Savine, A.C., & Braver, T.S. \`\`Local and global effects of motivation on cognitive control.' Cogn Affect Behav Neurosci. 12(4):692-718, 2012 Dec. (hereafter referred to as Cogn Affect Behav Neurosci. 2012).

2\. Savine, A.C., McDaniel, M.A., Shelton, J.T., Scullin, M.K. \`\`A characterization of individual differences in prospective memory monitoring using the Complex Ongoing Serial Task.' J Exp Psychol Gen. 141(2):337-62, 2012 May (hereafter referred to as J Exp Psychol Gen. 2012).

3\. Savine, A.C., & Braver, T.S. \`\`Motivated cognitive control: Reward incentives modulate preparatory neural activity during task- switching.' J Neurosci. 30(31):10294-305, 2010 Aug 4 (hereafter referred to as J Neurosci. 2010).

Conference Abstracts

1\. Savine, A.C., & Braver, T.S. (November 2010) \`\`The contextual and local effects of motivation on cognitive control.' Psychonomics Society, St. Louis, MO.

2\. Savine, A.C., & Braver, T.S. (November 2010) \`\`A model-based characterization of the individual differences in prospective memory monitoring.' Psychonomics Society, St. Louis, MO.

3\. Savine, A.C., & Braver, T.S. (November 2010) \`\`Motivated cognitive control: Reward incentives modulate preparatory neural activity during task-switching.' Society for Neuroscience, San Diego, CA.

4\. Savine, A.C., & Braver, T.S. (June 2010) \`\`Motivated cognitive control: Reward incentives modulate preparatory neural activity during task-switching.' Motivation and Cognitive Control Conference, Oxford, England.

5\. Savine, A.C., & Braver, T.S. (January 2010) \`\`Neural correlates of the motivation/cognitive control interaction: Activation dynamics and Performance prediction during task-switching.' Genetic and Experiential Influences on Executive Function, Boulder, CO.

6\. Savine, A.C., & Braver, T.S. (June 2009) \`\`Incentive Induced Changes in Neural Patterns During Task-Switching.' Organization for Human Brain Mapping, San Francisco, CA.

As a result of the Respondent's admission, the senior authors will request that the published papers be retracted or corrected.

ORI finds that Respondent falsified data and related text in Cogn Affect Behav Neurosci. 2012, J Exp Psychol Gen. 2012, J Neurosci. 2010, and in six (6) meeting abstracts, by altering the experimental data to improve the statistical results. Specifically, Respondent:

1\. Falsified data in Cogn Affect Behav Neurosci. 2012 to show an unambiguous dissociation between local and global motivational effects. Specifically, Respondent exaggerated (1) the effect of incentive context on response times and error rates in Table 1 and Figures 1 and 3 for experiment 1 and (2) the effect of incentive cue timing on response times and error rates in Table 2 and in Figures 6, 9, and S2 for experiment 2.

2\. Falsified data in J Exp Psychol Gen. 2012 to show that prospective memory is influenced by three dissociable underlying monitoring patterns (attentional focus, secondary memory retrieval, information thresholding), which are stable within individuals over time and are influenced by personality and cognitive differences. Specifically, Respondent modified the data to support the three category model and to show (1) that individuals fitting into each of the three categories exhibited differential patterns of prospective memory performance and ongoing task performance in Tables 1-3; Figures 5-8, and (2) that certain cognitive and personality differences were predictive of distinct monitoring approaches within the three categories in Figure 9.

3\. Falsified data in J Neurosci. 2010 and mislabeled brain images to show that motivational incentives enhance task-switching performance and are associated with activation of reward-related brain regions, behavioral performance, and trial outcomes. Specifically, Respondent modified the data so that he could show a stronger relationship between brain activity and behavior in Table 2 and Figure 4 and used brain images that fit the data rather than the images that corresponded to the actual Talairach coordinates in Figure 3.

Mr. Savine has entered into a Voluntary Settlement Agreement and has voluntarily agreed for a period of three (3) years, beginning on February 22, 2013:

\(1\) To have his research supervised; Respondent agreed that prior to the submission of an application for U.S. Public Health Service (PHS) support for a research project on which his participation is proposed and prior to his participation in any capacity on PHS-supported research, Respondent shall ensure that a plan for supervision of his duties is submitted to ORI for approval; the supervision plan must be designed to ensure the scientific integrity of his research contribution; he agreed that he shall not participate in any PHS-supported research until such a supervision plan is submitted to and approved by ORI; Respondent agreed to maintain responsibility for compliance with the agreed upon supervision plan;

\(2\) That any institution employing him shall submit, in conjunction with each application for PHS funds, or report, manuscript, or abstract involving PHS-supported research in which Respondent is involved, a certification to ORI that the data provided by Respondent are based on actual experiments or are otherwise legitimately derived and that the data, procedures, and methodology are accurately reported in the application, report, manuscript, or abstract;

\(3\) To exclude himself voluntarily from serving in any advisory capacity to PHS including, but not limited to, service on any PHS advisory committee, board, and/or peer review committee, or as a consultant; and

\(4\) That the senior authors will request that the following papers be retracted or corrected: Cogn Affect Behav Neurosci. 2012, J Exp Psychol Gen. 2012, and J Neurosci. 2010.

Inquiries

Please direct all inquiries to:**

** David E. Wright, Ph.D.**
** Director**
** Office of Research Integrity**
** 1101 Wootton Parkway, Suite 750**
** Rockville, MD 20852


author: John M Barry
date: 2004
institute: 1Distinguished Visiting Scholar, the Center for Bioenvironmental Research of Tulane and Xavier Universities, New Orleans, Louisiana, USA
references:
title: The site of origin of the 1918 influenza pandemic and its public health implications

The 1918–1919 influenza pandemic killed more people than any other outbreak of disease in human history. The lowest estimate of the death toll is 21 million, while recent scholarship estimates from 50 to 100 million dead. World population was then only 28% what is today, and most deaths occurred in a sixteen week period, from mid-September to mid-December of 1918.

It has never been clear, however, where this pandemic began. Since influenza is an endemic disease, not simply an epidemic one, it is impossible to answer this question with absolute certainty. Nonetheless, in seven years of work on a history of the pandemic, this author conducted an extensive survey of contemporary medical and lay literature searching for epidemiological evidence – the only evidence available. That review suggests that the most likely site of origin was Haskell County, Kansas, an isolated and sparsely populated county in the southwest corner of the state, in January 1918 \[1\]. If this hypothesis is correct, it has public policy implications.

But before presenting the evidence for Haskell County it is useful to review other hypotheses of the site of origin. Some medical historians and epidemiologists have theorized that the 1918 pandemic began in Asia, citing a lethal outbreak of pulmonary disease in China as the forerunner of the pandemic. Others have speculated the virus was spread by Chinese or Vietnamese laborers either crossing the United States or working in France.

More recently, British scientist J.S. Oxford has hypothesized that the 1918 pandemic originated in a British Army post in France, where a disease British physicians called "purulent bronchitis" erupted in 1916. Autopsy reports of soldiers killed by this outbreak – today we would classify the cause of death as ARDS – bear a striking resemblance to those killed by influenza in 1918 \[2\].

But these alternative hypotheses have problems. After the 1918–1919 pandemic, many investigators searched for the source of the disease. The American Medical Association sponsored what is generally considered the best of several comprehensive international studies of the pandemic conducted by Dr. Edwin Jordan, editor of ***The Journal of Infectious Disease***. He spent years reviewing evidence from all over the world; the AMA published his work in 1927.

Since several influenza pandemics in preceding centuries were already well-known and had come from the orient, Jordan first considered Asia as the source. But he found no evidence. Influenza did surface in early 1918 in China, but the outbreaks were minor, did not spread, and contemporary Chinese scientists, trained by Rockefeller Institute for Medical Research (now Rockefeller University) investigators, stated they believed these outbreaks were endemic disease unrelated to the pandemic \[3\]. Jordan also looked at the lethal pulmonary disease cited by some historians as influenza, but this was diagnosed by contemporary scientists as pneumonic plague. By 1918 the plague bacillus could be easily and conclusively identified in the laboratory \[3\]. So after tracing all known outbreaks of respiratory disease in China, Jordan concluded that none of them "could be reasonably regarded as the true forerunner" of the pandemic \[3\].

Jordan also considered Oxford's theory that the "purulent bronchitis" in British Army camps in 1916 and 1917 was the source. He rejected it for several reasons. The disease had flared up, true, but had not spread rapidly or widely outside the affected bases; instead, it seemed to disappear \[3\]. As we now know a mutation in an existing influenza virus can account for a virulent flare-up. In the summer of 2002, for example, an influenza epidemic erupted in parts of Madagascar with an extremely high mortality and morbidity; in some towns it sickened an outright majority – in one instance sixty-seven percent – of the population. But the virus causing this epidemic was an H3N2 virus that normally caused mild disease. In fact, the epidemic affected only thirteen of 111 health districts in Madagascar before fading away \[4\]. Something similar may have happened in the British base.

Jordan considered other possible origins of the pandemic in early 1918 in France and India. He concluded that it was highly unlikely that the pandemic began in any of them \[3\].

That left the United States. Jordan looked at a series of spring outbreaks there. The evidence seemed far stronger. One could see influenza jumping from Army camp to camp, then into cities, and traveling with troops to Europe. His conclusion: the United States was the site of origin.

A later equally comprehensive, multi-volume British study of the pandemic agreed with Jordan. It too found no evidence for the influenza's origin in the Orient, it too rejected the 1916 outbreak among British troops, and it too concluded, "The disease was probably carried from the United States to Europe \[5\]."

Australian Nobel laureate MacFarlane Burnet spent most of his scientific career working on influenza and studied the pandemic closely. He too concluded that the evidence was "strongly suggestive" that the disease started in the United States and spread with "the arrival of American troops in France \[6\]."

Before dismissing the conclusions of these contemporary investigators who lived through and studied the pandemic, one must remember how good many of them were. They were very good indeed.

The Rockefeller Institute, whose investigators were intimately involved in the problem, alone included extraordinary people. By 1912 its head Simon Flexner – his brother wrote the "Flexner report" that revolutionized American medical education – used immune serum to bring the mortality rate for meningococcal meningitis down from over 80% to 18%; by contrast, in the 1990s at Massachusetts General Hospital a study found a 25% mortality rate for bacterial meningitis. Peyton Rous won the Nobel Prize in 1966 for work he did at the institute in 1911; he was that far ahead of the scientific consensus. By 1918 Oswald Avery and others at Rockefeller Institute had already produced both an effective curative serum and a vaccine for the most common pneumococcal pneumonias. At least partly because of the pandemic, Avery would spend the rest of his career studying pneumonia. That work led directly to his discovery of the "transforming principle" – his discovery that DNA carries the genetic code.

The observations of investigators of this quality cannot be dismissed lightly. Jordan was of this quality.

More evidence against Oxford's hypothesis comes from Dr. Jeffrey Taubenberger, well-known for his work extracting samples of the 1918 virus from preserved tissue and sequencing its genome. He initially believed, based on statistical analysis of the rate of mutation of the virus that it existed for two or three years prior to the pandemic. But further work convinced him that the virus emerged only a few months prior to the pandemic (personal communication with the author from J Taubenberger, June 5^th^ 2003).

So if the contemporary observers were correct, if American troops carried the virus to Europe, where in the United States did it begin?

Both contemporary epidemiological studies and lay histories of the pandemic have identified the first known outbreak of epidemic influenza as occurring at Camp Funston, now Ft. Riley, in Kansas. But there was one place where a previously unknown – and remarkable – epidemic of influenza occurred.

Haskell County, Kansas, lay three hundred miles to the west of Funston. There the smell of manure meant civilization. People raised grains, poultry, cattle, and hogs. Sod-houses were so common that even one of the county's few post offices was located in a dug-out sod home. In 1918 the population was just 1,720, spread over 578 square miles. But primitive and raw as life could be there, science had penetrated the county in the form of Dr. Loring Miner. Enamored of ancient Greece – he periodically reread the classics in Greek – he epitomized William Welch's comment that "the results \[of medical education\] were better than the system." His son was also a doctor, trained in fully scientific ways, serving in the Navy in Boston.

In late January and early February 1918 Miner was suddenly faced with an epidemic of influenza, but an influenza unlike any he had ever seen before. Soon dozens of his patients – the strongest, the healthiest, the most robust people in the county – were being struck down as suddenly as if they had been shot. Then one patient progressed to pneumonia. Then another. And they began to die. The local paper ***Santa Fe Monitor***, apparently worried about hurting morale in wartime, initially said little about the deaths but on inside pages in February reported, "Mrs. Eva Van Alstine is sick with pneumonia. Her little son Roy is now able to get up... Ralph Lindeman is still quite sick... Goldie Wolgehagen is working at the Beeman store during her sister Eva's sickness... Homer Moody has been reported quite sick... Mertin, the young son of Ernest Elliot, is sick with pneumonia... Pete Hesser's children are recovering nicely... Ralph McConnell has been quite sick this week (Santa Fe Monitor, February 14^th^, 1918)."

The epidemic got worse. Then, as abruptly as it came, it disappeared. Men and women returned to work. Children returned to school. And the war regained its hold on people's thoughts.

The disease did not, however, slip from Miner's thoughts. Influenza was neither a reportable disease, nor a disease that any state or federal public health agency tracked. Yet Miner considered this incarnation of the disease so dangerous that he warned national public health officials about it. ***Public Health Reports*** (now ***Morbidity and Mortality Weekly Report***), a weekly journal produced by the U.S. Public Health Service to alert health officials to outbreaks of communicable diseases throughout the world, published his warning. In the first six months of 1918, this would be the only reference in that journal to influenza anywhere in the world.

Historians and epidemiologists have previously ignored Haskell most likely because his report was not published until April and it referred to deaths on March 30, after influenza outbreaks elsewhere. In actuality, by then the county was free of influenza. Haskell County, Kansas, is the first recorded instance anywhere in the world of an outbreak of influenza so unusual that a physician warned public health officials. It remains the first recorded instance suggesting that a new virus was adapting, violently, to man.

If the virus did not originate in Haskell, there is no good explanation for how it arrived there. There were no other known outbreaks anywhere in the United States from which someone could have carried the disease to Haskell, and no suggestions of influenza outbreaks in either newspapers or reflected in vital statistics anywhere else in the region. And unlike the 1916 outbreak in France, one can trace with perfect definiteness the route of the virus from Haskell to the outside world.

All Army personnel from the county reported to Funston for training. Friends and family visited them at Funston. Soldiers came home on leave, then returned to Funston. ***The Monitor*** reported in late February, "Most everybody over the country is having lagrippe or pneumonia (Santa Fe Monitor, February 21^st^ 1918)." It also noted, "Dean Nilson surprised his friends by arriving at home from Camp Funston on a five days furlough. Dean looks like soldier life agrees with him." He soon returned to the camp. Ernest Elliot left to visit his brother at Funston as his child fell ill. On February 28, John Bottom left for Funston. "We predict John will make an ideal soldier," said the paper (Santa Fe Monitor February 28^th^, 1918).

These men, and probably others unnamed by the paper, were exposed to influenza and would have arrived in Funston between February 26 and March 2. On March 4 the first soldier at the camp reported ill with influenza at sick call. The camp held an average of 56,222 troops. Within three weeks more than eleven hundred others were sick enough to require hospitalization, and thousands more – the precise number was not recorded – needed treatment at infirmaries scattered around the base.

Whether or not the Haskell virus did spread across the world, the timing of the Funston explosion strongly suggests that the influenza outbreak there did come from Haskell. Meanwhile Funston fed a constant stream of men to other American locations and to Europe, men whose business was killing. They would be more proficient at it than they knew.

Soldiers moved uninterrupted between Funston and the outside world, especially to other Army bases and France. On March 18, Camps Forrest and Greenleaf in Georgia saw their first cases of influenza and by the end of April twenty-four of the thirty-six main Army camps suffered an influenza epidemic \[3\]. Thirty of the fifty largest cities in the country also had an April spike in excess mortality from influenza and pneumonia \[7\]. Although this spring wave was generally mild – the killing second wave struck in the fall – there were still some disturbing findings. A subsequent Army study said, "At this time the fulminating pneumonia, with wet hemorrhagic lungs, fatal in from 24 to 48 hours, was first observed \[8\]." (Pathology reports suggest what we now call ARDS.) The first recorded autopsy in Chicago of an influenza victim was conducted in early April. The pathologist noted, "The lungs were full of hemorrhages." He found this unusual enough to ask the then-editor of ***The Journal of Infectious Diseases*** "to look over it as a new disease" \[3\].

By then, influenza was erupting in France, first at Brest, the single largest port of disembarkation for American troops. By then, as MacFarlane Burnet later said, "It is convenient to follow the story of influenza at this period mainly in regard to the army experiences in America and Europe \[6\]."

The fact that the 1918 pandemic likely began in the United States matters because it tells investigators where to look for a new virus. They must look everywhere.

In recent years the World Health Organization and local public health authorities have intervened several times when new influenza viruses have infected man. These interventions have prevented the viruses from adapting to man and igniting a new pandemic. But only 83 countries in the world – less than half – participate in WHO's surveillance system (WHO's flunet website <http://rhone.b3e.jussieu.fr/flunet/www/docs.html>). While some monitoring occurs even in those countries not formally affiliated with WHO's surveillance system, it is hardly adequate. If the virus did cross into man in a sparsely populated region of Kansas, and not in a densely populated region of Asia, then such an animal-to-man cross-over can happen anywhere. And unless WHO gets more resources and political leaders move aggressively on the diplomatic front, then a new pandemic really is all too inevitable.


abstract: A report on the 2006 Keystone Conference on Signaling Networks, Vancouver, Canada, 30 January-4 February 2006.
author: Nevan J Krogan; Timothy R Hughes
date: 2006
institute: 1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA; 2Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, M5S 3E1, Canada
title: Signals and systems

The boundaries between traditional notions of cellular signaling and more genomic and systematic approaches to biology are becoming increasingly blurred, and the recent Keystone conference on signaling networks reflected an expanded view of signaling. It is becoming increasingly accepted that genes, proteins, cells, and organisms function as components of larger systems, rather than independent activities contributing to a single defined outcome, and many presentations at the conference reflected this. If there was a single theme, it was the heavy reliance on technical approaches in functional genomics, proteomics, and computational biology, such that conceptual and technical discussions often dominated the resulting biology.

# RNA interference screening

The impact of technology on the study of signaling networks was most evident in the widespread application of RNA interference (RNAi) screens. Screens to study signaling networks in multicellular organisms using RNAi technology are being performed with success, although several hurdles clearly remain. One of these is the apparently high rate of false positives. In his keynote lecture, Norbert Perrimon (Harvard Medical School, Boston, USA) predicted an uncomfortably high rate of false positives due to off-target effects, at least in *Drosophila*. Consistent with this, Phil Beachy (Johns Hopkins University School of Medicine, Baltimore, USA) described an ongoing RNAi screen in *Drosophila* looking for proteins involved in the Wingless signaling pathway. Following efforts to study a previously uncharacterized gene identified by this screen, it was noted that there were 16 bases in the interfering RNA that were identical to *armadillo*, a known gene in the pathway, suggesting that it is an unanticipated off-target that could completely explain the phenotype conferred by the interfering RNA. Perrimon proposed that incorporating other types of data, such as protein-protein interaction information, can help decipher which hits are physiologically relevant. Rene Bernards (Netherlands Cancer Institute, Amsterdam, The Netherlands) commented that reporting lists of unconfirmed hits from genome-wide RNAi screens in humans might be detrimental to future work by creating distractions from more productive lines of research, and that scientists (and journals) perhaps need to be more mindful of this.

Despite these limitations, work presented on *Caenorhabditis elegans* showed unquestionably that useful biological information could be extracted from RNAi screens. Gary Ruvkun (Massachusetts General Hospital, Boston, USA) and colleagues have been involved in a variety of screens looking for factors involved in lifespan, molting, defects in fat storage, and the RNAi pathway itself. Ruvkun described the complete list of hits from the molting screen as a "rogues' gallery" of phenotypes and molecular functions. One could argue that this might be evidence not so much of a high false-positive rate as of the dependence of a system on many components and processes; a corollary of this is that perturbation of an individual gene can have a myriad of physiological effects. Andrew Fraser (Wellcome Trust Sanger Institute, Cambridge, UK) presented work that supports this view. His group has been performing synthetic interaction screens in *C. elegans* in which previously characterized worm mutants defective in the epidermal growth factor (EGF)-Ras-Raf-MAP kinase pathway are treated with libraries encoding double-stranded RNAs, in order to identify synthetic genetic interactions. A significant outcome of this work was that RNAi of genes in certain functional categories results in synthetic effects with a high proportion of all mutants tested, suggesting that some cellular functions buffer or canalize many physiological traits, rather than perform a single physiological function.

David Sabatini (Massachusetts Institute of Technology, Cambridge, USA) presented a thorough overview of technical aspects of the development and use of genome-scale lentiviral RNAi libraries for human and mouse. A consortium of groups in the public and private sectors is contributing to the creation of the libraries, which are sold by both Sigma (St. Louis, USA) and Open Biosystems (Huntsville, USA) and are available as glycerol stocks, plasmids and viruses. Sabatini commented that while this effort is still incomplete, some functional categories (for example, transcription factors) are fairly comprehensively covered. The efficacy of the system has been validated by targeting tyrosine kinases, and Sabatini reported that 90% of these genes have at least one target that results in a significant knockdown of expression. Sabatini also discussed issues of high-content screening, such as image archiving (a screen can generate 1 Tb of images), image analysis, and defining a 'hit'. He described Cell Profiler <http://jura.wi.mit.edu/cellprofiler/>, a free, open-source software package for analysis of thousands of cell images, which among other statistics can output cell counts, DNA content and mitotic index.

Sabatini gave his view on how to define true positives: as a rule of thumb, he suggested requiring two independent hairpins that work (the consortium library contains several for each gene), dose dependence, and complementation if possible. Finally, he noted that as antibodies are not available for the majority of human proteins, it is currently difficult to ascertain whether protein levels are impacted by RNAi. On a similar theme, Sumit Chanda (Genomic Institute of the Novartis Research Foundation, San Diego, USA) pointed out that 50% of work published in humans corresponds to less than 10% of the genome, with few (or no) publications for most known and predicted genes, which provides a justification for comprehensive screening efforts.

# Chemical screening

Chemicals have long been used to perturb specific biological activities in experiments, and they have a particular appeal because of their potential application as drugs. Justin Lamb (Broad Institute, Cambridge, USA) described an ongoing effort to construct a transcriptional connectivity map for biomedical discovery, which relates microarray gene-expression profiles that are due to drug effects, disease, and gene perturbations. An initial goal is to find off-target uses for existing drugs; Lamb's group is currently analyzing 1,500 drugs approved by the US Food and Drug Administration (FDA), 500 non-drug bioactive compounds used as research tools, and the effects of the knockdown of 500 potential drug targets using lentivirally delivered short hairpin RNAs (shRNAs). A distinguishing feature of the connectivity map is the use of the Kalmogorov-Smirnov statistic to score a relationship between two experiments, even in the absence of an overall correlation. Lamb presented evidence from these analyses to indicate that connections can be drawn between drugs and diseases even if the experiments are performed in different cell types, or possibly even different organisms, which is significant because large-scale RNAi screens are typically performed in well behaved cultured cells.

An alternative assay for chemical activities was presented by Soren Jensby Nielsen (BioImage, Soeborg, Denmark), who described screening compounds for their impact on protein translocation. He reported the effects of around 50 compounds on the localization of a dozen different green fluorescent protein (GFP) fusion proteins. A surprising number of effects were observed, and although effects appear to be protein-specific (that is, they do not correlate with growth inhibition or other general effects), Nielsen stated that this screening approach has not yet identified any primary targets, indicating that the observed mislocalizations are probably downstream effects of perturbing the primary target. Nevertheless, this is an intriguing approach to screening, especially as it requires only a relatively basic setup compared with high-content assays.

# Protein-interaction networks

Giulio Superti-Furga (CeMM Center for Molecular Medicine, Vienna, Austria) and one of us (N.K.) both described efforts to purify protein complexes for the entire proteome of *Saccharomyces cerevisiae*. The two groups used a similar strategy involving the immunoprecipitation of affinity-tagged proteins followed by mass spectrometry. Although proteomic studies have been previously published in budding yeast, the current studies attempt to tag and purify approximately 10 times as many unique proteins than any previous study. Both studies have just been published and comparison of the datasets with each other and with others previously generated should allow the compilation of the most comprehensive protein-interaction map ever generated for an organism.

A next goal will be to define similar physical interaction maps in higher organisms. Marc Vidal (Dana-Farber Cancer Institute, Boston, USA) presented work on uncovering interacting proteins in human, focusing on two-hybrid analyses that are then confirmed by pulldowns. Like Perrimon, Vidal was enthusiastic about weighting the significance of interactions on the basis of multiple independent data types, most notably phenotypic profiles.

# Pathways and networks

The term signaling pathway implies a cause-and-effect relationship among a series of steps. Two talks concerned the development of computational methods for the inference of cause and effect, or for determining the importance of the nodes in a network, from quantitative genetic and phenotypic analysis. Eric Schadt (Rosetta Inpharmatics, Seattle, USA) pointed out that in genetic crosses information can only flow from genotype to phenotype. In contrast, gene-expression patterns can be either a cause or an effect of phenotypes. He gave a conceptual overview of the statistical approaches his group is using to distinguish whether gene-expression phenotypes are upstream or downstream of other phenotypes in large mouse crosses. In summary, one can ask which network structure is most consistent with the observed correlations, with quantitative trait loci used as an anchor. Alon Kaufman (Hebrew University, Jerusalem, Israel) presented algorithms for calculating the contribution of genes to a function that has been quantified via set of multiple mutants in factors that influence that function. These algorithms, known as the 'multi-perturbation shapely value analysis' and the 'functional influence network' algorithms, can recreate some features of known networks and should aid in predicting aspects of unknown pathways.

Analysis of the consequences of multiple perturbations has historically been a primary approach to dissecting genetic and signaling networks. On the other hand, these computational analyses require a collection of quantitative genetic data. Two talks described methods for generating such data on a large scale in budding yeast. Jef Boeke (Johns Hopkins University, Baltimore, USA) described a comprehensive genetic analysis of approximately 70 genes involved in DNA integrity. Analysis of the genetic patterns enables the establishment of 'congruence groups', functionally related clusters that share the same pattern of synthetic sick or lethal (SSL) interactions, but may not participate in synthetic sick or lethal relationships among themselves. One of us (N.K.) described the generation of an epistatic miniarray profile (a quantitative analysis of double-mutant growth phenotypes among a logically-selected subset of genes) of approximately 850 genes involved in most aspects of chromosome function. This analysis detected not only synthetic sick or lethal interactions, but also cases in which double deletions grow better than would be expected from the growth of each single mutant when crossed to most other mutants on the array. The genes involved in these latter interactions often displayed similar sets of genetic profiles and correlated well with physical interactions. Will RNAi enable the generation of such networks in higher organisms? Perhaps to some degree; however, in the question-and-answer period, Fraser suggested that his group had encountered a high false-negative rate in RNAi x RNAi 'crosses' in *C. elegans*, relative to RNAi x germline mutation, presumably because most RNAi knockdowns are incomplete.

Biological networks tend to have distinctive mathematical properties, most notably a scale-free degree distribution, the cause of which has been debated extensively. Ravi Iyengar (Mount Sinai Medical Center, New York, USA) and Albert-Laszlo Barabasi (University of Notre Dame, South Bend, USA) both presented arguments and modeling results supporting the notion that the origin of scale-free network structures is likely to be a consequence of selection pressure following gene duplication. Briefly, the idea is that newly duplicated genes have increased chances of survival if they develop a function that is tied to that of a gene that is already functionally coupled to many other genes, creating a 'rich get richer' scenario. The results of simulations of this paradigm appear to match experimentally derived network structures.

In contrast to all the network technology, a biological highlight was the description by Cori Bargmann (Rockefeller University, New York, USA) of her lab's demonstration that *C. elegans* can learn to smell which bacteria it should and should not eat, using a network of only a few hundred neurons. Overall, the conference successfully integrated a broad range of biological topics, including novel technologies and computational strategies that biologists can apply to obtain meaningful systems-level biological information on signaling networks.


abstract: We assess five years of usage of the major genome-wide collections of mutants from *Saccharomyces cerevisiae*.
author: Bart Scherens; Andre Goffeau
date: 2004
institute: 1Institut de Recherches Microbiologiques J.M. Wiame, Campus CERIA, Av. E. Gryson 1, 1070 Bruxelles, Belgium; 2Institut des Sciences de la Vie, Université Catholique de Louvain, Croix du Sud 2-20, 1348 Louvain-la-Neuve, Belgium
references:
title: The uses of genome-wide yeast mutant collections

In April 1996, the completely annotated genome sequence of the yeast *Saccharomyces cerevisiae* was made publicly available \[1,2\], the first eukaryotic genome sequence to be completed. Eight years later, thanks to the united efforts of the large yeast research community and to the unique genetic and physiological properties of yeast, this humble servant of mankind provides by far the best annotated eukaryotic genome \[3\]. The completeness of the yeast genome sequence has allowed the development of many novel tools for analyzing all molecular components of the cell and their interactions. These tools include three high-throughput collections of mutants that were first produced in 1999 and that have been analyzed in the five years since then. Here, we review the uses of these collections and their contribution to the identification of the components of basic physiological and developmental pathways of *S. cerevisiae*.

# The yeast deletion mutant collection

A set of over 20,000 knockout strains was created by a consortium of European and North American laboratories \[4,5\]. The collection currently contains homozygous and heterozygous diploid strains corresponding to deletions of each of 5,916 genes (including 1,159 essential genes) and one haploid strain of each mating type for every non-essential gene (4,757 genes). Each knockout strain is marked by two unique 20-nucleotide 'bar codes', allowing quantitative and qualitative identification by DNA microarray hybridization of each strain in the pools used to assess the strains under different growth conditions (see Figure 1). The original article \[4\] describing this collection has been cited more then 560 times in the five years since its publication, according to the ISI Web of Science \[6\]. The complete collection of strains can be obtained at low cost from Euroscarf \[7\], ATCC \[8\] and Invitrogen \[9\].

The deletion collection has been used in dozens of novel exhaustive screens for phenotypes that occur under a variety of physiological conditions; these include growth in minimal medium, in high salt and low salt, in galactose or sorbitol, at pH 8, after heat or cold shock, under stress by hydrogen peroxide (all in \[10\]); growth on non-fermentable carbon substrates \[11\], in saline conditions \[12\] or after treatment by ionizing radiation or DNA-damaging agents \[13-17\]; and the collection has also been screened for defects in meiosis, sporulation and germination \[18,19\]. This approach has uncovered numerous new putative components of well-known pathways; for instance, the number of genes known to have sporulation or germination phenotypes when deleted has been doubled by these analyses \[18\].

More sophisticated screens, for example for suppressors of the accumulation of mutations \[20\], have been developed more recently, as well as screens involving transformation of the deletion strains in order to identify genes needed for non-homologous DNA end-joining \[21\]. Novel protocols requiring individual transformations of each mutant have allowed the identification of host factors that influence the fate of the Ty family of long-terminal-repeat retrotransposable elements \[22\] and of genes involved in the unfolded protein response induced by heterologous introduction of mutant human Huntingtin protein or fragments of α-synuclein, both of which form disease-associated aggregates \[23\]. Similarly, proteins that interfere with the assembly of endoplasmic-reticulum structures termed karmellae, which are induced by elevated levels of HMG-CoA reductase under specific growth and genetic conditions, have been identified using the collection \[24\]. Several morphological screens have been developed, for example for defects in the selection of the bipolar bud site \[25\], in cell-size distribution \[26,27\], in cell morphology \[28\] and in meiotic chromosomal segregation \[29\]. Another approach is the screening of individual colonies. For instance, mis-sorting and secretion of vacuolar carboxypeptidase Y were detected by colony immunoblotting \[30\]. A second example of colony screening is the transformation of each single-deletion strain to express viral replicase proteins and an RNA replication template in which the capsid gene was replaced by a luciferase reporter gene, which was used to monitor viral expression in yeast colonies \[31\]. Finally, to identify the genes affecting glycogen storage, the deletion mutant colonies were blotted and stained by iodine vapor; the intensity of coloration allowed assessment of glycogen accumulation \[32\].

## Use of the yeast deletion collection in screens for synthetic lethal mutants and to study drug targets

Synthetic lethality is the phenomenon that occurs when two mutations that are each viable are combined and the double mutant is lethal. A method has been developed for the systematic construction of double mutants, which is called synthetic genetic array analysis (SGA) \[33,34\]. Haploid strains with mutations in non-essential genes were crossed to an array of the whole haploid deletion collection; the resulting diploid cells were made to sporulate and the lethal combinations identified, indicating the existence of essential interactions between gene products. In a first SGA screen using eight query genes, 291 interactions among 204 genes were identified \[33\]. Three years later \[34\], the search was expanded to 132 query genes, and 4,000 interactions were identified among 1,000 genes with roles in cytoskeletal organization, cell-wall biosynthesis, microtubule-based chromosome segregation and DNA metabolism.

A more recent development of this approach is the use of DNA-DNA hybridization protocols to assess lethality, termed 'synthetic lethality analysis by microarray' (SLAM). In this method, a pool of haploid deletion strains is transformed with a cassette that replaces the gene of interest with either a deletion construct or the wild-type form. Transformants are pooled and genomic DNA is isolated; the barcodes are amplified by PCR and labeled with either Cy3 (green) or Cy5 (red) fluorescent dyes; and hybridization to an array containing all the deletion tags allows identification of the synthetic-lethal combinations (which are missing). The SLAM method has been validated by identifying members of the DNA helicase interaction network \[35\]. Synthetic genetic arrays have also been used for high-resolution genetic mapping of suppressor mutations \[36\]; this method, termed SGA mapping (SGAM), is in principle also applicable to the analysis of multigenic traits.

The yeast deletion collection has also been used to identify members of the pathways modified by more than 25 different chemical ligands. This approach has identified the L-carnitine transporter Agp2p as a novel transporter of bleomycin in yeast, implicating membrane transport as a key determinant of resistance to this widely used anticancer agent \[37\]. In another study, the transcription factor Rpn4p was shown to compensate for proteasome inhibition by PS-341, a drug that is being studied as a treatment for cancer \[38\]. Other ligands tested include the phosphatidyl kinase inhibitor wortmannin \[39\]; anticancer agents \[10,37,40,41\]; antifungal agents including nystatin and calcofluor \[10,41\]; antibiotics including hygromycin B \[42\] and rapamycin \[43\]; statins (a class of drugs that lower cholesterol), the smooth muscle relaxant alverine citrate, the local anaesthetic dyclonine, yeast K1 killer toxin \[44\], 6-azauracil (a drug that depresses cellular nucleoside triphosphate levels \[45\]) and even the detergent sodium dodecyl sulfate \[44\].

## Major scientific contributions arising from the use of the yeast deletion collection

In a total of 33 publications that have used the deletion collection, in which over 100 different conditions were explored, more then 5,000 genes were attributed a phenotype (see Table 1 for an overview). Many of these genes were not previously known to contribute to the pathways scrutinized, even though in many cases - such as in studies of sporulation, the cell cycle, radiation damage and growth in defined media - there had previously been apparently exhaustive classical mutagenesis or DNA microarray screens. Altogether, these analyses have shown 71 genes previously thought to be non-essential to have a nonviable phenotype \[46\]; and the systematic deletion approach has been claimed to be five-fold more sensitive then a microarray gene-expression analysis for finding genes involved in pathways of interest \[11\].

Some new phenotypes have emerged from these analyses, although many known phenotypes have not yet been explored \[47\]. As an example of recent novel information provided by the systematic-deletion approach, over 100 new genes resulting in a defective bipolar budding pattern \[25\] have been identified. Other examples are the identification of a new putative bleomycin transporter \[37\]; the *NEJ1* gene involved in non-homologous DNA end-joining and claimed to be one of the guardians of the cancer cells \[21\]; and 52 genes, the deletion of which causes lethality in yeast transfected with a fragment of heterologous human Huntingtin \[23\]. The latter set, which includes genes involved in lipid metabolism, vesicle transport and of unknown function, may help to define the still poorly defined pathway termed the unfolded protein response \[23\].

## Uncertainties in the results obtained from systematic deletion mutants

The yeast deletion collection allows powerful novel screens and has revealed many novel components of complex metabolic and developmental pathways. The data produced should be examined with great care, however, as there are several problems with the use of the collection (for further details, see two reviews by Elisabeth Winzeler \[46,48\]). A first issue is that the collection is incomplete, as a few hundred genes were missed in the first annotation of the genome that was used as the basis of the deletions (see updates of yeast genome data in *Saccharomyces* genome database (SGD) \[49\]). Moreover, relative amounts of some deleted strains may have been distributed unequally during the initial creation of the pools and further amplified \[4\]. Secondly, there can be problems with mutations or variations elsewhere in the genome that affect the phenotype. For instance, because the transformation procedure that was used to create the deletion library is a mutagenic event, mutations in sites some distance away from the intended deletion might be present in some strains \[50\]. Also, up to 8% of the deleted strains are known to retain a wild-type copy of the targeted gene, presumably because of aneuploidy or some duplication event \[50\]. Even in diploid strains, mutations that apparently have no phenotype may in fact modify the phenotype of other mutations in subtle ways (a phenomenon termed haploinsufficiency) \[51\].

When deletions are moved into a different genetic background, their phenotypes may change; for instance, up to 18% of synthetic-lethal interactions were not confirmed in different backgrounds \[33,46\]. Another source of discrepancy might be genetic variation among the individual isolates of the strains that are amplified under given selective conditions. For instance, the phenotype for a given mutant strain can be masked by extracellular complementation by a protein product of another strain in the pool.

Other factors are related to the use of the barcodes. Their presence may modify the phenotype or, conversely, the stress created by the knockout procedure may mutagenize the barcode. About 17% of the 4,600 homozygous strains in the collection have a hybridization signal similar to that of the background. Moreover, trivial technical factors may affect the hybridization data, for example when the wrong piece of DNA is deposited on the microarray or when the tag modifies the secondary structures and thus causes poor hybridization. Also, some published datasets have too few repetitions of experiments to be statistically valid.

These and other problems concerning the specificity and sensitivity of the screen used must be taken into account when interpreting data obtained using the yeast deletion collection. As a gross estimate, over one third of the data may consist of false positives or false negatives. As a golden rule, therefore, the data from a primary screen of the collection should always be confirmed by independent approaches.

# Collections of strains mutated using transposons

Transposons are mobile genetic elements that can be used to disrupt genes in a non-targeted fashion. Insertional mutagenesis using transposons can disrupt non-coding as well as coding regions and can lead to partial loss or gain of function; it is thus complementary to deletion of genes \[52\]. The 'mini-Mu' transposon modified for insertional mutagenesis in yeast was pioneered by Daignan-Fornier and Bolotin-Fukuhara \[53\]. Ross-Macdonald *et al*. \[52\] used a version of the same transposon including a reporter gene (*lacZ*), an epitope tag (encoding hemagglutinin) and selectable markers for yeast and bacteria to perform random transposon mutagenesis. The approach uses a minitransposon that was introduced into the yeast genome by a two step method: a yeast genomic DNA library was mutagenized in *Escherichia coli* using the minitransposon; the library plasmids were then cut to excise the yeast genomic DNA with the transposon insertion, which was transformed into diploid yeast, replacing one chromosomal copy by homologous recombination \[52\]. This transposon-mutagenesis system can be used for the analysis of gene expression, detection of subcellular localization and identification of phenotypes caused by the insertion, and the transposon can be deleted using the Cre-LoxP recombinase system, removing the selectable marker. The insertion point in the genome can be determined for each mutant by sequencing outwards from the transposon sequence into the surrounding yeast sequences. The article describing the first such screen \[52\] has been cited 209 times in the five years since its publication \[6\]. The collection of transposon-inserted strains created in this work \[52\] is available free of charge from Michael Snyder's laboratory \[54\].

A database called 'transposon-insertion phenotypes, localization and expression in *Saccharomyces cerevisiae*' (TRIPLES) \[55,56\] contains the results obtained from exploiting the mutants obtained by the transposon-insertion approach. A total of 240,768 yeast insertional mutants were screened for transposon-encoded β-galactosidase activity, and a collection of 28,428 yeast strains was generated that produce β-galactosidase under conditions of vegetative growth and/or sporulation. The insertion point of 23,191 of these strains has been sequenced, identifying 3,750 different yeast genes, and the subcellular localization of 2,744 different yeast proteins has been documented (A. Kumar, personal communication).

The transposon-insertion collection \[52,54\] has been used to identify genes involved in sporulation and vegetative growth. Analysis of the collection has also identified 137 new genes that were not originally annotated by the yeast genome sequencing consortium \[57\]. Ferreira *et al*. \[58\] have used the collection - which was produced in the S288C strain used to sequence the yeast genome \[52\] - to mutagenize the W303-1A strain (which is known to be more responsive to gene inactivation than the S288C). Ferreira *et al*. \[58\] identified genes required for growth in high-salt medium (61 insertions in 31 genes) and survival of hypo-osmotic shock and growth at 15°C (31 insertion in 10 genes). A similar approach was also used by Takahashi *et al*. \[59\] to screen in other strains for ethanol tolerance and modification of the cell wall, and by Suzuki *et al*. \[60\] to screen for the effects of insertions on the response to nitrogen starvation, on invasive or filamentous growth and on sporulation.

Other approaches for insertional mutagenesis have also been applied to yeast. Firstly, instead of bacterial mini-Mu transposons, T-DNA from *Agrobacterium tumefaciens* has been used to mutagenize yeast on a genome-wide scale \[61\]. A second approach is the use of non-homologous insertion of DNA cassettes not derived from mobile elements. This process, which exploits the cellular machinery that normally repairs DNA double-strand breaks by non-homologous end joining, is increased by gamma irradiation \[62\]. Thirdly, Blanc and Adams \[63\] used mutations resulting from insertion of the Ty1 transposon to identify yeast mutations that generate evolutionarily significant phenotypes by causing small but positive increments of fitness. Finally, an application of transposon mutagenesis called direct allele replacement technology (DART) allows rapid transfer of any insertion allele into any strain \[64\]. A transposon library consisting of a collection of plasmids containing yeast genomic DNA with transposon insertions is sequenced to identify the exact insertion point in the yeast genomic DNA \[64\]. After excision from the plasmid, the yeast genomic DNA containing the transposon is used to transform a yeast strain of choice by homologous recombination. The procedure was validated by identification of 29 insertions into 17 genes involved in apical growth \[64\].

Insertional mutagenesis using transposons has several potential advantages over targeted deletion: for instance, insertion occurs in the non-coding as well as in the coding segments, so regulatory regions and other non-genie regions can be disrupted. Moreover, depending on the site of insertion, transposon mutagenesis may lead to partial loss of function or gain of function and hence to the identification of novel functions that would not be found from studies of complete knockouts of genes \[52\]. Conditional alleles may be generated, as well as mutants in promoter or terminal regions. Also, apart from phenotype analysis of the mutation, the level of expression of the targeted gene can be measured *in vivo* and the subcellular localization of its product can be determined. A disadvantage, however, is that transposon insertions are not random, and this method may therefore never cover all the genes in the genome. Also, several of the problems that were mentioned above for the analysis of the yeast deletion collection apply equally to the transposon mutant collections, including the observation that phenotypes are often very strongly background-dependent.

# Other genome-wide mutant collections and their uses

Several databases have been developed to catalog the subcellular localizations of yeast proteins as identified by fluorescence microscopy. The yeast protein localization database \[65,66\] describes the results obtained using a library of yeast genes fused to a green fluorescent protein (GFP) reporter. The TRIPLES database \[55,56\] includes the use of the transposon-insertion libraries to determine protein localization for 5,504 insertions. The yeast GFP fusion localization database \[67,68\] presents the localization of 4,156 proteins into 22 distinct subcellular locations, as determined using a library of GFP-tagged proteins compared with reference strains expressing proteins of known localization tagged with red fluorescent protein (the strains used are available from Invitrogen \[9\]).

In a recent study \[69\], an exhaustive global analysis of protein expression in yeast was reported. Each open reading frame was marked by an insertion cassette consisting of a modified version of the tandem affinity purification (TAP) tag, a yeast selectable marker to drive homologous recombination, and regions homologous to yeast genes. The level of expression of each protein was determined by very sensitive western-blotting analysis that could detect less than 50 protein molecules per cell. The level of expression of 4,251 gene products was identified in exponential growth conditions \[69\].

Like the sequencing of the yeast genome, the construction of the single and double deletion libraries and that of the major transposon insertion library has required a considerable investment by the international yeast research community. The three original papers \[4,33,52\] presenting the libraries have a total of 83 authors, funded by public agencies from the USA, from the European Union and from Canada. Was this effort worthwhile?

Over the last five years, the three seminal papers have been cited more than 950 times \[6\]. We have found over 50 experimental papers reporting on different phenotypic screens carried out with these genome-wide libraries. More than 100 experimental conditions have been tested, representing near a million individual mutant screens. We estimate that more than 5,000 novel phenotypic traits have been assigned to yeast genes of known or unknown molecular functions. This undoubtedly represents a considerable amount of progress towards the ultimate goal of a full description of the functions and interactions of all the molecular components of a basic eukaryotic cell. Several recent improvements of the use of the original genome-wide mutant libraries have been reported, such as the DART \[64\] and SLAM \[35\] approaches described above. New genome-wide libraries using more sensitive protein tagging are being developed \[69\] for global measurements of protein levels or subcellular expression. In all this work, yeast continues to be a very convenient test-bed for the development of novel high-throughput tools that rely on the availability of the complete genome sequence.

But a word of caution is appropriate. As was the case for other high-throughput tools that were also pioneered using yeast, such as two-hybrid protein-interaction screening, DNA-hybridization microarrays and proteomic analyses, the information provided by large screens of genome-wide libraries contains an appreciable number of false-positive and false-negative data points. It is therefore essential to confirm each result by several independent approaches. Ultimately, we will have to return to 'reductionist' biochemical approaches to demonstrate fully the molecular function suggested by a large primary screen.

Finally, there is an urgent need to build databases to collect and organize all the data obtained from all the large screening approaches. The SGD \[49\] and the Munich information center for protein sequences (MIPS) database \[70\] are making good progress in this respect. The authors of these databases should, however, be encouraged to further develop procedures that take into account the difficult assessment of the uncertainties associated with much of the data. Here again, yeast is expected to become a pioneer.

## Figures and Tables

Screens performed so far on the yeast deletion strain collection and transposon-insertion collection

| Condition, treatment or phenotype | Number of genes responding to the treatments\* | References |
|----|----|----|
| **Phenotype screens of the deletion collection** |  |  |
| Fitness after growth on minimal medium, high salt, low salt, galactose, sorbitol, pH 8, heat shock, cold shock, or hydrogen peroxide | ? | \[10\] |
| Growth on non-fermentable carbon substrates | 466 | \[11\] |
| Response to DNA-damaging agents, ionizing radiation, UV radiation, cisplatin, or hydrogen peroxide | \>170 | \[13-16\] |
| Chemical DNA damage (methane sulfonate sensitivity) | 103 | \[17\] |
| Enhanced sporulation proficiency | 102 | \[18,19\] |
| Sporulation defects | 261 | \[18,19\] |
| Post-germination defects | 158 | \[18,19\] |
| Genes essential for sporulation | 334 | \[18,19\] |
| Saline response | About 500 | \[12\] |
| Suppression of the accumulation of mutations | 33 | \[20\] |
| Non-homologous DNA end-joining | About 30 | \[21\] |
| Ty1 retrotransposition cycle | 101 | \[22\] |
| Response to the introduction of human Huntingtin or α-synuclein fragments (unfolded protein response) | 52/86 | \[23\] |
| Karmellae assembly | 120 | \[24\] |
| Bipolar bud-site selection | 127 | \[25\] |
| Cell-size distribution | 500 | \[26,27\] |
| Altered cell morphology | ? | \[28\] |
| Meiotic chromosomal segregation | ? | \[29\] |
| Vacuolar protein sorting | 146 | \[30\] |
| Host genes affecting replication of a positive-strand RNA virus | 100 | \[31\] |
| Glycogen storage (low, high) | 324, 242 | \[32\] |
|  |  |  |
| **Sensitivity of the deletion collection to drugs or chemicals** |  |  |
| Rapamycin | 106 | \[43\] |
| PS-341 | ? | \[38\] |
| Bleomycin (hypersensitivity, resistance) | 231, 5 | \[37,40\] |
| Amoxillin, penicillin, rifampin, vancomycin, (oxy)tetracycline, or gentamicin | 23 | \[42\] |
| Anticancer, antifungal agents, statins, alverine citrate, dyclonine, or nystatin | About 10 | \[10,41\] |
| K1 killer toxin, hygromycin B, calcofluor or sodium dodecyl sulfate | 268 | \[44\] |
| 6-Azauracil | 42 | \[45\] |
| Wortmannin (resistance, sensitivity) | 591, 476 | \[39\] |
|  |  |  |
| **Phenotypes of transposon-insertion libraries** |  |  |
| Phenotypes under 20 conditions, expression levels and subcellular localization (TRIPLES database) | ? | \[55\] |
| High salt | 31 | \[58\] |
| Survival of hypo-osmotic shock and growth at 15°C | 10 | \[58\] |
| Ethanol tolerance and cell-wall modifications | 5 | \[59\] |
| Nitrogen starvation, invasive or filamentous growth and sporulation | About 105 | \[60\] |

\*The number of open reading frames responding to the relevant screen is reported whenever this information could be deduced from the relevant article; ? indicates that the information could not be deduced.