Listening to public concerns about human life extension

The public view of life-extension technologies is more nuanced than expected and researchers must engage in discussions if they hope to promote awareness and acceptanceThere is increasing research and commercial interest in the development of novel interventions that might be able to extend human life expectancy by decelerating the ageing process. In this context, there is unabated interest in the life-extending effects of caloric restriction in mammals, and there are great hopes for drugs that could slow human ageing by mimicking its effects (Fontana et al, 2010). The multinational pharmaceutical company GlaxoSmithKline, for example, acquired Sirtris Pharmaceuticals in 2008, ostensibly for their portfolio of drugs targeting ‘diseases of ageing''. More recently, the immunosuppressant drug rapamycin has been shown to extend maximum lifespan in mice (Harrison et al, 2009). Such findings have stoked the kind of enthusiasm that has become common in media reports of life-extension and anti-ageing research, with claims that rapamycin might be “the cure for all that ails” (Hasty, 2009), or that it is an “anti-aging drug [that] could be used today” (Blagosklonny, 2007).Given the academic, commercial and media interest in prolonging human lifespan—a centuries-old dream of humanity—it is interesting to gauge what the public thinks about the possibility of living longer, healthier lives, and to ask whether they would be willing to buy and use drugs that slow the ageing process. Surveys that have addressed these questions, have given some rather surprising results, contrary to the expectations of many researchers in the field. They have also highlighted that although human life extension (HLE) and ageing are topics with enormous implications for society and individuals, scientists have not communicated efficiently with the public about their research and its possible applications.Given the academic, commercial and media interest in prolonging human lifespan […] it is interesting to gauge what the public thinks about the possibility of living longer, healthier lives…Proponents and opponents of HLE often assume that public attitudes towards ageing interventions will be strongly for or against, but until now, there has been little empirical evidence with which to test these assumptions (Lucke & Hall, 2005). We recently surveyed members of the public in Australia and found a variety of opinions, including some ambivalence towards the development and use of drugs that could slow ageing and increase lifespan. Our findings suggest that many members of the public anticipate both positive and negative outcomes from this work (Partridge 2009a, b, 2010; Underwood et al, 2009).In a community survey of public attitudes towards HLE we found that around two-thirds of a sample of 605 Australian adults supported research with the potential to increase the maximum human lifespan by slowing ageing (Partridge et al, 2010). However, only one-third expressed an interest in using an anti-ageing pill if it were developed. Half of the respondents were not interested in personally using such a pill and around one in ten were undecided.Some proponents of HLE anticipate their research being impeded by strong public antipathy (Miller, 2002, 2009). Richard Miller has claimed that opposition to the development of anti-ageing interventions often exists because of an “irrational public predisposition” to think that increased lifespans will only lead to elongation of infirmity. He has called this “gerontologiphobia”—a shared feeling among laypeople that while research to cure age-related diseases such as dementia is laudable, research that aims to intervene in ageing is a “public menace” (Miller, 2002).We found broad support for the amelioration of age-related diseases and for technologies that might preserve quality of life, but scepticism about a major promise of HLE—that it will delay the onset of age-related diseases and extend an individual''s healthy lifespan. From the people we interviewed, the most commonly cited potential negative personal outcome of HLE was that it would extend the number of years a person spent with chronic illnesses and poor quality of life (Partridge et al, 2009a). Although some members of the public envisioned more years spent in good health, almost 40% of participants were concerned that a drug to slow ageing would do more harm than good to them personally; another 13% were unsure about the benefits and costs (Partridge et al, 2010).…it might be that advocates of HLE have failed to persuade the public on this issueIt would be unwise to label such concerns as irrational, when it might be that advocates of HLE have failed to persuade the public on this issue. Have HLE researchers explained what they have discovered about ageing and what it means? Perhaps the public see the claims that have been made about HLE as ‘too good to be true‘.Results of surveys of biogerontologists suggest that they are either unaware or dismissive of public concerns about HLE. They often ignore them, dismiss them as “far-fetched”, or feel no responsibility “to respond” (Settersten Jr et al, 2008). Given this attitude, it is perhaps not surprising that the public are sceptical of their claims.Scientists are not always clear about the outcomes of their work, biogerontologists included. Although the life-extending effects of interventions in animal models are invoked as arguments for supporting anti-ageing research, it is not certain that these interventions will also extend healthy lifespans in humans. Miller (2009) reassuringly claims that the available evidence consistently suggests that quality of life is maintained in laboratory animals with extended lifespans, but he acknowledges that the evidence is “sparse” and urges more research on the topic (Miller, 2009). In the light of such ambiguity, researchers need to respond to public concerns in ways that reflect the available evidence and the potential of their work, without becoming apostles for technologies that have not yet been developed. An anti-ageing drug that extends lifespan without maintaining quality of life is clearly undesirable, but the public needs to be persuaded that such an outcome can be avoided.The public is also concerned about the possible adverse side effects of anti-ageing drugs. Many people were bemused when they discovered that members of the Caloric Restriction Society experienced a loss of libido and loss of muscle mass as a result of adhering to a low-calorie diet to extend their longevity—for many people, such side effects would not be worth the promise of some extra years of life. Adverse side effects are acknowledged as a considerable potential challenge to the development of an effective life-extending drug in humans (Fontana et al, 2010). If researchers do not discuss these possible effects, then a curious public might draw their own conclusions.Adverse side effects are acknowledged as a considerable potential challenge to the development of an effective life-extending drug in humansSome HLE advocates seem eager to tout potential anti-ageing drugs as being free from adverse side effects. For example, Blagosklonny (2007) has argued that rapamycin could be used to prevent age-related diseases in humans because it is “a non-toxic, well tolerated drug that is suitable for everyday oral administration” with its major “side-effects” being anti-tumour, bone-protecting, and mimicking caloric restriction effects. By contrast, Kaeberlein & Kennedy (2009) have advised the public against using the drug because of its immunosuppressive effects.Aubrey de Grey has called for scientists to provide more optimistic timescales for HLE on several occasions. He claims that public opposition to interventions in ageing is based on “extraordinarily transparently flawed opinions” that HLE would be unethical and unsustainable (de Grey, 2004). In his view, public opposition is driven by scepticism about whether HLE will be possible, and that concerns about extending infirmity, injustice or social harms are simply excuses to justify people''s belief that ageing is ‘not so bad'' (de Grey, 2007). He argues that this “pro-ageing trance” can only be broken by persuading the public that HLE technologies are just around the corner.Contrary to de Grey''s expectations of public pessimism, 75% of our survey participants thought that HLE technologies were likely to be developed in the near future. Furthermore, concerns about the personal, social and ethical implications of ageing interventions and HLE were not confined to those who believed that HLE is not feasible (Partridge et al, 2010).Juengst et al (2003) have rightly pointed out that any interventions that slow ageing and substantially increase human longevity might generate more social, economic, political, legal, ethical and public health issues than any other technological advance in biomedicine. Our survey supports this idea; the major ethical concerns raised by members of the public reflect the many and diverse issues that are discussed in the bioethics literature (Partridge et al, 2009b; Partridge & Hall, 2007).When pressed, even enthusiasts admit that a drastic extension of human life might be a mixed blessing. A recent review by researchers at the US National Institute on Aging pointed to several economic and social challenges that arise from longevity extension (Sierra et al, 2009). Perry (2004) suggests that the ability to slow ageing will cause “profound changes” and a “firestorm of controversy”. Even de Grey (2005) concedes that the development of an effective way to slow ageing will cause “mayhem” and “absolute pandemonium”. If even the advocates of anti-ageing and HLE anticipate widespread societal disruption, the public is right to express concerns about the prospect of these things becoming reality. It is accordingly unfair to dismiss public concerns about the social and ethical implications as “irrational”, “inane” or “breathtakingly stupid” (de Grey, 2004).The breadth of the possible implications of HLE reinforces the need for more discussion about the funding of such research and management of its outcomes ( Juengst et al, 2003). Biogerontologists need to take public concerns more seriously if they hope to foster support for their work. If there are misperceptions about the likely outcomes of intervention in ageing, then biogerontologists need to better explain their research to the public and discuss how their concerns will be addressed. It is not enough to hope that a breakthrough in human ageing research will automatically assuage public concerns about the effects of HLE on quality of life, overpopulation, economic sustainability, the environment and inequities in access to such technologies. The trajectories of other controversial research areas—such as human embryonic stem cell research and assisted reproductive technologies (Deech & Smajdor, 2007)—have shown that “listening to public concerns on research and responding appropriately” is a more effective way of fostering support than arrogant dismissal of public concerns (Anon, 2009).Biogerontologists need to take public concerns more seriously if they hope to foster support for their work? Open in a separate windowBrad PartridgeOpen in a separate windowJayne LuckeOpen in a separate windowWayne Hall     

The academic birth rate. Production and reproduction of the research work force, and its effect on innovation and research misconduct

Universities have been churning out PhD students to reap financial and other rewards for training biomedical scientists. This deluge of cheap labour has created unhealthy competition, which encourages scientific misconduct.Most developed nations invest a considerable amount of public money in scientific research for a variety of reasons: most importantly because research is regarded as a motor for economic progress and development, and to train a research workforce for both academia and industry. Not surprisingly, governments are occasionally confronted with questions about whether the money invested in research is appropriate and whether taxpayers are getting the maximum value for their investments.…questions about the size and composition of the research workforce have historically been driven by concerns that the system produces an insufficient number of scientistsThe training and maintenance of the research workforce is a large component of these investments. Yet discussions in the USA about the appropriate size of this workforce have typically been contentious, owing to an apparent lack of reliable data to tell us whether the system yields academic ‘reproduction rates'' that are above, below or at replacement levels. In the USA, questions about the size and composition of the research workforce have historically been driven by concerns that the system produces an insufficient number of scientists. As Donald Kennedy, then Editor-in-Chief of Science, noted several years ago, leaders in prestigious academic institutions have repeatedly rung alarm bells about shortages in the science workforce. Less often does one see questions raised about whether too many scientists are being produced or concerns about unintended consequences that may result from such overproduction. Yet recognizing that resources are finite, it seems reasonable to ask what level of competition for resources is productive, and at what level does competition become counter-productive.Finding a proper balance between the size of the research workforce and the resources available to sustain it has other important implications. Unhealthy competition—too many people clamouring for too little money and too few desirable positions—creates its own problems, most notably research misconduct and lower-quality, less innovative research. If an increasing number of scientists are scrambling for jobs and resources, some might begin to cut corners in order to gain a competitive edge. Moreover, many in the science community worry that every publicized case of research misconduct could jeopardize those resources, if politicians and taxpayers become unwilling to invest in a research system that seems to be riddled with fraud and misconduct.The biomedical research enterprise in the USA provides a useful context in which to examine the level of competition for resources among academic scientists. My thesis is that the system of publicly funded research in the USA as it is currently configured supports a feedback system of institutional incentives that generate excessive competition for resources in biomedical research. These institutional incentives encourage universities to overproduce graduate students and postdoctoral scientists, who are both trainees and a cheap source of skilled labour for research while in training. However, once they have completed their training, they become competitors for money and positions, thereby exacerbating competitive pressures.Questions raised about whether too many scientists are being produced or concerns about the unintended consequences of such overproduction are less commonThe resulting scarcity of resources, partly through its effect on peer review, leads to a shunting of resources away from both younger researchers and the most innovative ideas, which undermines the effectiveness of the research enterprise as a whole. Faced with an increasing number of grant applications and the consequent decrease in the percentage of projects that can be funded, reviewers tend to ‘play it safe'' and favour projects that have a higher likelihood of yielding results, even if the research is conservative in the sense that it does not explore new questions. Resource scarcity can also introduce unwanted randomness to the process of determining which research gets funded. A large group of scientists, led by a cancer biologist, has recently mounted a campaign against a change in a policy of the National Institutes of Health (NIH) to allow only one resubmission of an unfunded grant proposal (Wadman, 2011). The core of their argument is that peer reviewers are likely able to distinguish the top 20% of research applications from the rest, but that within that top 20%, distinguishing the top 5% or 10% means asking peer reviewers for a level of precision that is simply not possible. With funding levels in many NIH institutes now within that 5–10% range, the argument is that reviewers are being forced to choose at random which excellent applications do and do not get funding. In addition to the inefficiency of overproduction and excessive competition in terms of their costs to society and opportunity costs to individuals, these institutional incentives might undermine the integrity and quality of science, and reduce the likelihood of breakthroughs.My colleagues and I have expressed such concerns about workforce dynamics and related issues in several publications (Martinson, 2007; Martinson et al, 2005, 2006, 2009, 2010). Early on, we observed that, “missing from current analyses of scientific integrity is a consideration of the wider research environment, including institutional and systemic structures” (Martinson et al, 2005). Our more recent publications have been more specific about the institutional and systemic structures concerned. It seems that at least a few important leaders in science share these concerns.In April 2009, the NIH, through the National Institute of General Medical Sciences (NIGMS), issued a request for applications (RFA) calling for proposals to develop computational models of the research workforce (http://grants.nih.gov/grants/guide/rfa-files/RFA-GM-10-003.html). Although such an initiative might be premature given the current level of knowledge, the rationale behind the RFA seems irrefutable: “there is a need to […] pursue a systems-based approach to the study of scientific workforce dynamics.” Roughly four decades after the NIH appeared on the scene, this is, to my knowledge, the first official, public recognition that the biomedical workforce tends not to conform nicely to market forces of supply and demand, despite the fact that others have previously made such arguments.Early last year, Francis Collins, Director of the NIH, published a PolicyForum article in Science, voicing many of the concerns I have expressed about specific influences that have led to growth rates in the science workforce that are undermining the effectiveness of research in general, and biomedical research in particular. He notes the increasing stress in the biomedical research community after the end of the NIH “budget doubling” between 1998 and 2003, and the likelihood of further disruptions when the American Recovery and Reinvestment Act of 2009 (ARRA) funding ends in 2011. Arguing that innovation is crucial to the future success of biomedical research, he notes the tendency towards conservatism of the NIH peer-review process, and how this worsens in fiscally tight times. Collins further highlights the ageing of the NIH workforce—as grants increasingly go to older scientists—and the increasing time that researchers are spending in itinerant and low-paid postdoctoral positions as they stack up in a holding pattern, waiting for faculty positions that may or may not materialize. Having noted these challenging trends, and echoing the central concerns of a 2007 Nature commentary (Martinson, 2007), he concludes that “…it is time for NIH to develop better models to guide decisions about the optimum size and nature of the US workforce for biomedical research. A related issue that needs attention, though it will be controversial, is whether institutional incentives in the current system that encourage faculty to obtain up to 100% of their salary from grants are the best way to encourage productivity.”Similarly, Bruce Alberts, Editor-in-Chief of Science, writing about incentives for innovation, notes that the US biomedical research enterprise includes more than 100,000 graduate students and postdoctoral fellows. He observes that “only a select few will go on to become independent research scientists in academia”, and argues that “assuming that the system supporting this career path works well, these will be the individuals with the most talent and interest in such an endeavor” (Alberts, 2009).His editorial is not concerned with what happens to the remaining majority, but argues that even among the select few who manage to succeed, the funding process for biomedical research “forces them to avoid risk-taking and innovation”. The primary culprit, in his estimation, is the conservatism of the traditional peer-review system for federal grants, which values “research projects that are almost certain to ‘work''”. He continues, “the innovation that is essential for keeping science exciting and productive is replaced by […] research that has little chance of producing the breakthroughs needed to improve human health.”If an increasing number of scientists are scrambling for jobs and resources, some might begin to cut corners in order to gain a competitive edgeAlthough I believe his assessment of the symptoms is correct, I think he has misdiagnosed the cause, in part because he has failed to identify which influence he is concerned with from the network of influences in biomedical research. To contextualize the influences of concern to Alberts, we must consider the remaining majority of doctorally trained individuals so easily dismissed in his editorial, and further examine what drives the dynamics of the biomedical research workforce.Labour economists might argue that market forces will always balance the number of individuals with doctorates with the number of appropriate jobs for them in the long term. Such arguments would ignore, however, the typical information asymmetry between incoming graduate students, whose knowledge about their eventual job opportunities and career options is by definition far more limited than that of those who run the training programmes. They would also ignore the fact that universities are generally not confronted with the externalities resulting from overproduction of PhDs, and have positive financial incentives that encourage overproduction. During the past 40 years, NIH ‘extramural'' funding has become crucial for graduate student training, faculty salaries and university overheads. For their part, universities have embraced NIH extramural funding as a primary revenue source that, for a time, allowed them to implement a business model based on the interconnected assumptions that, as one of the primary ‘outputs'' or ‘products'' of the university, more doctorally trained individuals are always better than fewer, and because these individuals are an excellent source of cheap, skilled labour during their training, they help to contain the real costs of faculty research.“…the current system has succeeded in maximizing the amount of research […] it has also degraded the quality of graduate training and led to an overproduction of PhDs…”However, it has also made universities increasingly dependent on NIH funding. As recently documented by the economist Paula Stephan, most faculty growth in graduate school programmes during the past decade has occurred in medical colleges, with the majority—more than 70%—in non-tenure-track positions. Arguably, this represents a shift of risk away from universities and onto their faculty. Despite perennial cries of concern about shortages in the research workforce (Butz et al, 2003; Kennedy et al, 2004; National Academy of Sciences et al, 2005) a number of commentators have recently expressed concerns that the current system of academic research might be overbuilt (Cech, 2005; Heinig et al, 2007; Martinson, 2007; Stephan, 2007). Some explicitly connect this to structural arrangements between the universities and NIH funding (Cech, 2005; Collins, 2007; Martinson, 2007; Stephan, 2007).In 1995, David Korn pointed out what he saw as some problematic aspects of the business model employed by Academic Medical Centers (AMCs) in the USA during the past few decades (Korn, 1995). He noted the reliance of AMCs on the relatively low-cost, but highly skilled labour represented by postdoctoral fellows, graduate students and others—who quickly start to compete with their own professors and mentors for resources. Having identified the economic dependence of the AMCs on these inexpensive labour pools, he noted additional problems with the graduate training programmes themselves. “These programs are […] imbued with a value system that clearly indicates to all participants that true success is only marked by the attainment of a faculty position in a high-profile research institution and the coveted status of principal investigator on NIH grants.” Pointing to “more than 10 years of severe supply/demand imbalance in NIH funds”, Korn concluded that, “considering the generative nature of each faculty mentor, this enterprise could only sustain itself in an inflationary environment, in which the society''s investment in biomedical research and clinical care was continuously and sharply expanding.” From 1994 to 2003, total funding for biomedical research in the USA increased at an annual rate of 7.8%, after adjustment for inflation. The comparable rate of growth between 2003 and 2007 was 3.4% (Dorsey et al, 2010). These observations resonate with the now classic observation by Derek J. de Solla Price, from more than 30 years before, that growth in science frequently follows an exponential pattern that cannot continue indefinitely; the enterprise must eventually come to a plateau (de Solla Price, 1963).In May 2009, echoing some of Korn''s observations, Nobel laureate Roald Hoffmann caused a stir in the US science community when he argued for a “de-coupling” of the dual roles of graduate students as trainees and cheap labour (Hoffmann, 2009). His suggestion was to cease supporting graduate students with faculty research grants, and to use the money instead to create competitive awards for which graduate students could apply, making them more similar to free agents. During the ensuing discussion, Shirley Tilghman, president of Princeton University, argued that “although the current system has succeeded in maximizing the amount of research performed […] it has also degraded the quality of graduate training and led to an overproduction of PhDs in some areas. Unhitching training from research grants would be a much-needed form of professional ‘birth control''” (Mervis, 2009).The greying of the NIH research workforce is another important driver of workforce dynamics, and it is integrally linked to the fate of young scientistsAlthough the issue of what I will call the ‘academic birth rate'' is the central concern of this analysis, the ‘academic end-of-life'' also warrants some attention. The greying of the NIH research workforce is another important driver of workforce dynamics, and it is integrally linked to the fate of young scientists. A 2008 news item in Science quoted then 70-year-old Robert Wells, a molecular geneticist at Texas A&M University, “‘if I and other old birds continue to land the grants, the [young scientists] are not going to get them.” He worries that the budget will not be able to support “the 100 people ‘I''ve trained […] to replace me''” (Kaiser, 2008). While his claim of 100 trainees might be astonishing, it might be more astonishing that his was the outlying perspective. The majority of senior scientists interviewed for that article voiced intentions to keep doing science—and going after NIH grants—until someone forced them to stop or they died.Some have looked at the current situation with concern, primarily because of the threats it poses to the financial and academic viability of universities (Korn, 1995; Heinig et al, 2007; Korn & Heinig, 2007), although most of those who express such concerns have been distinctly reticent to acknowledge the role of universities in creating and maintaining the situation. Others have expressed concerns about the differential impact of extreme competition and meagre job prospects on the recruitment, development and career survival of young and aspiring scientists (Freeman et al, 2001; Kennedy et al, 2004; Martinson et al, 2006; Anderson et al, 2007a; Martinson, 2007; Stephan, 2007). There seems to be little disagreement, however, that the system has generated excessively high competition for federal research funding, and that this threatens to undermine the very innovation and production of knowledge that is its raison d''etre.The production of knowledge in science, particularly of the ‘revolutionary'' variety, is generally not a linear input–output process with predictable returns on investment, clear timelines and high levels of certainty (Lane, 2009). On the contrary, it is arguable that “revolutionary science is a high risk and long-term endeavour which usually fails” (Charlton & Andras, 2008). Predicting where, when and by whom breakthroughs in understanding will be produced has proven to be an extremely difficult task. In the face of such uncertainty, and denying the realities of finite resources, some have argued that the best bet is to maximize the number of scientists, using that logic to justify a steady-state production of new PhDs, regardless of whether the labour market is sending signals of increasing or decreasing demand for that supply. Only recently have we begun to explore the effects of the current arrangement on the process of knowledge production, and on innovation in particular (Charlton & Andras, 2008; Kolata, 2009).…most of those who express such concerns have been reticent to acknowledge the role of universities themselves in creating and maintaining the situationBruce Alberts, in the above-mentioned editorial, points to several initiatives launched by the NIH that aim to get a larger share of NIH funding into the hands of young scientists with particularly innovative ideas. These include the “New Innovator Award,” the “Pioneer Award” and the “Transformational R01 Awards”. The proportion of NIH funding dedicated to these awards, however, amounts to “only 0.27% of the NIH budget” (Alberts, 2009). Such a small proportion of the NIH budget does not seem likely to generate a large amount of more innovative science. Moreover, to the extent that such initiatives actually succeed in enticing more young investigators to become dependent on NIH funds, any benefit these efforts have in terms of innovation may be offset by further increases in competition for resources that will come when these new ‘innovators'' reach the end of this specialty funding and add to the rank and file of those scrapping for funds through the standard mechanisms.Our studies on research integrity have been mostly oriented towards understanding how the influences within which academic scientists work might affect their behaviour, and thus the quality of the science they produce (Anderson et al, 2007a, 2007b; Martinson et al, 2009, 2010). My colleagues and I have focused on whether biomedical researchers perceive fairness in the various exchange relationships within their work systems. I am persuaded by the argument that expectations of fairness in exchange relationships have been hard-wired into us through evolution (Crockett et al, 2008; Hsu et al, 2008; Izuma et al, 2008; Pennisi, 2009), with the advent of modern markets being a primary manifestation of this. Thus, violations of these expectations strike me as potentially corrupting influences. Such violations might be prime motivators for ill will, possibly engendering bad-faith behaviour among those who perceive themselves to have been slighted, and therefore increasing the risk of research misconduct. They might also corrupt the enterprise by signalling to talented young people that biomedical research is an inhospitable environment in which to develop a career, possibly chasing away some of the most talented individuals, and encouraging a selection of characteristics that might not lead to optimal effectiveness, in terms of scientific innovation and productivity (Charlton, 2009).To the extent that we have an ecology with steep competition that is fraught with high risks of career failure for young scientists after they incur large costs of time, effort and sometimes financial resources to obtain a doctoral degree, why would we expect them to take on the additional, substantial risks involved in doing truly innovative science and asking risky research questions? And why, in such a cut-throat setting, would we not anticipate an increase in corner-cutting, and a corrosion of good scientific practice, collegiality, mentoring and sociability? Would we not also expect a reduction in high-risk, innovative science, and a reversion to a more career-safe type of ‘normal'' science? Would this not reduce the effectiveness of the institution of biomedical research? I do not claim to know the conditions needed to maximize the production of research that is novel, innovative and conducted with integrity. I am fairly certain, however, that putting scientists in tenuous positions in which their careers and livelihoods would be put at risk by pursuing truly revolutionary research is one way to insure against it.     

Reply to J.N. Perry et al

The authors of “The anglerfish deception” respond to the criticism of their article.EMBO reports (2012) advanced online publication; doi: 10.1038/embor.2012.70EMBO reports (2012) 13 2, 100–105; doi: 10.1038/embor.2011.254Our respondents, eight current or former members of the EFSA GMO panel, focus on defending the EFSA''s environmental risk assessment (ERA) procedures. In our article for EMBO reports, we actually focused on the proposed EU GMO legislative reform, especially the European Commission (EC) proposal''s false political inflation of science, which denies the normative commitments inevitable in risk assessment (RA). Unfortunately the respondents do not address this problem. Indeed, by insisting that Member States enjoy freedom over risk management (RM) decisions despite the EFSA''s central control over RA, they entirely miss the relevant point. This is the unacknowledged policy—normative commitments being made before, and during, not only after, scientific ERA. They therefore only highlight, and extend, the problem we identified.The respondents complain that we misunderstood the distinction between RA and RM. We did not. We challenged it as misconceived and fundamentally misleading—as though only objective science defined RA, with normative choices cleanly confined to RM. Our point was that (i) the processes of scientific RA are inevitably shaped by normative commitments, which (ii) as a matter of institutional, policy and scientific integrity must be acknowledged and inclusively deliberated. They seem unaware that many authorities [1,2,3,4] have recognized such normative choices as prior matters, of RA policy, which should be established in a broadly deliberative manner “in advance of risk assessment to ensure that [RA] is systematic, complete, unbiased and transparent” [1]. This was neither recognized nor permitted in the proposed EC reform—a central point that our respondents fail to recognize.In dismissing our criticism that comparative safety assessment appears as a ‘first step'' in defining ERA, according to the new EFSA ERA guidelines, which we correctly referred to in our text but incorrectly referenced in the bibliography [5], our respondents again ignore this widely accepted ‘framing'' or ‘problem formulation'' point for science. The choice of comparator has normative implications as it immediately commits to a definition of what is normal and, implicitly, acceptable. Therefore the specific form and purpose of the comparison(s) is part of the validity question. Their claim that we are against comparison as a scientific step is incorrect—of course comparison is necessary. This simply acts as a shield behind which to avoid our and others'' [6] challenge to their self-appointed discretion to define—or worse, allow applicants to define—what counts in the comparative frame. Denying these realities and their difficult but inevitable implications, our respondents instead try to justify their own particular choices as ‘science''. First, they deny the first-step status of comparative safety assessment, despite its clear appearance in their own ERA Guidance Document [5]—in both the representational figure (p.11) and the text “the outcome of the comparative safety assessment allows the determination of those ‘identified'' characteristics that need to be assessed [...] and will further structure the ERA” (p.13). Second, despite their claims to the contrary, ‘comparative safety assessment'', effectively a resurrection of substantial equivalence, is a concept taken from consumer health RA, controversially applied to the more open-ended processes of ERA, and one that has in fact been long-discredited if used as a bottleneck or endpoint for rigorous RA processes [7,8,9,10]. The key point is that normative commitments are being embodied, yet not acknowledged, in RA science. This occurs through a range of similar unaccountable RA steps introduced into the ERA Guidance, such as judgement of ‘biological relevance'', ‘ecological relevance'', or ‘familiarity''. We cannot address these here, but our basic point is that such endless ‘methodological'' elaborations of the kind that our EFSA colleagues perform, only obscure the institutional changes needed to properly address the normative questions for policy-engaged science.Our respondents deny our claim concerning the singular form of science the EC is attempting to impose on GM policy and debate, by citing formal EFSA procedures for consultations with Member States and non-governmental organizations. However, they directly refute themselves by emphasizing that all Member State GM cultivation bans, permitted only on scientific grounds, have been deemed invalid by EFSA. They cannot have it both ways. We have addressed the importance of unacknowledged normativity in quality assessments of science for policy in Europe elsewhere [11]. However, it is the ‘one door, one key'' policy framework for science, deriving from the Single Market logic, which forces such singularity. While this might be legitimate policy, it is not scientific. It is political economy.Our respondents conclude by saying that the paramount concern of the EFSA GMO panel is the quality of its science. We share this concern. However, they avoid our main point that the EC-proposed legislative reform would only exacerbate their problem. Ignoring the normative dimensions of regulatory science and siphoning-off scientific debate and its normative issues to a select expert panel—which despite claiming independence faces an EU Ombudsman challenge [12] and European Parliament refusal to discharge their 2010 budget, because of continuing questions over conflicts of interests [13,14]—will not achieve quality science. What is required are effective institutional mechanisms and cultural norms that identify, and deliberatively address, otherwise unnoticed normative choices shaping risk science and its interpretive judgements. It is not the EFSA''s sole responsibility to achieve this, but it does need to recognize and press the point, against resistance, to develop better EU science and policy.     

Of mice and men

Thomas Erren and colleagues point out that studies on light and circadian rhythmicity in humans have their own interesting pitfalls, of which all researchers should be mindful.We would like to compliment, and complement, the recent Opinion in EMBO reports by Stuart Peirson and Russell Foster (2011), which calls attention to the potential obstacles associated with linking observations on light and circadian rhythmicity made on nocturnal mice to diurnally active humans. Pitfalls to consider include that qualitative extrapolations from short-lived rodents to long-lived humans, quantitative extrapolations of very different doses (Gold et al, 1992), and the varying sensitivities of each species to experimental optical radiation as a circadian stimulus (Bullough et al, 2006) can all have a critical influence on an experiment. Thus, Peirson & Foster remind us that “humans are not big mice”. We certainly agree, but we also thought it worthwhile to point out that human studies have their own interesting pitfalls, of which all researchers should be mindful.Many investigations with humans—such as testing the effects of different light exposures on alertness, cognitive performance, well-being and depression—can suffer from what has been coined as the ‘Hawthorne effect''. The term is derived from a series of studies conducted at the Western Electric Company''s Hawthorne Works near Chicago, Illinois, between 1924 and 1932, to test whether the productivity of workers would change with changing illumination levels. One important punch line was that productivity increased with almost any change that was made at the workplaces. One prevailing interpretation of these findings is that humans who know that they are being studied—and in most investigations they cannot help but notice—might exhibit responses that have little or nothing to do with what was intended as the experiment. Those who conduct circadian biology studies in humans try hard to eliminate possible ‘Hawthorne effects'', but every so often, all they can do is to hope for the best and expect the Hawthorne effect to be insignificant.Even so, and despite the obstacles to circadian experiments with both mice and humans, the wealth of information from work in both species is indispensable. To exemplify, in the last handful of years alone, experimental research in mice has substantially contributed to our understanding of the retinal interface between visible light and circadian circuitry (Chen et al, 2011); has shown that disturbances of the circadian systems through manipulations of the light–dark cycles might accelerate carcinogenesis (Filipski et al, 2009); and has suggested that perinatal light exposure—through an imprinting of the stability of circadian systems (Ciarleglio et al, 2011)—might be related to a human''s susceptibility to mood disorders (Erren et al, 2011a) and internal cancer developments later in life (Erren et al, 2011b). Future studies in humans must now examine whether, and to what extent, what was found in mice is applicable to and relevant for humans.The bottom line is that we must be aware of, and first and foremost exploit, evolutionary legacies, such as the seemingly ubiquitous photoreceptive clockwork that marine and terrestrial vertebrates—including mammals such as mice and humans—share (Erren et al, 2008). Translating insights from studies in animals to humans (Erren et al, 2011a,b), and vice versa, into testable research can be a means to one end: to arrive at sensible answers to pressing questions about light and circadian clockworks that, no doubt, play key roles in human health and disease. Pitfalls, however, abound on either side, and we agree with Peirson & Foster that they have to be recognized and monitored.     

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.Introduction of new diagnostic assays in the clinical setting requires an operating pipeline to efficiently translate putative biomarkers into validated biomarkers. Despite the discovery platforms'' capacity to generate well populated lists of candidate biomarkers, very few proteins reach the patient bedside as fully fledged “FDA-approved” biomarkers. This is largely because of divergences between analytical needs and performances of the techniques available for candidate biomarker evaluation (1, 2).Candidate biomarker evaluation is a major process of the biomarker pipeline, positioned downstream of the biomarker discovery phase and necessary before clinical validation. Candidate evaluation aims to select, among hundreds of putative biomarkers, those of clinical relevance. Evaluation phase combines two steps which respectively consist in: (1) confirming a difference between physiological and pathological concentrations in biofluids (the so-called “qualification phase”) and (2) assessing the specificity of candidate biomarkers (the so-called “verification phase”) (1). Currently, because of its high throughput and high sensitivity, quantitative ELISA is the preferred assay format for studies evaluating biomarkers. However, as most candidates are likely to fail as relevant biomarkers, developing ELISA tests (with high quality antibodies) for all candidates is a financial burden for the diagnostics industry (3).Thus, there exists an urgent need to develop analytical methods capable of reliable candidate evaluation, at high throughput and reasonable cost. Selected Reaction Monitoring (SRM)¹ mass spectrometry combined with stable isotope dilution (SID-SRM) has shown promise as a solution to this technological hurdle (4, 5). MS analysis in SRM mode offers the unique possibility to specifically and simultaneously monitor the signatures of hundreds of target peptides generated by trypsin digestion of proteins. Combined with isotope-labeled quantification standards (6), SRM can provide quantitative data for each protein targeted (5).Recently, in an effort to demonstrate the potential of SID-SRM for candidate biomarker evaluation, a multilaboratory study was set up to assess its analytical performances and potential transferability (7). Exogenous proteins, seven in all, were added to unfractionated plasma samples. The spiked samples were analyzed by eight independent laboratories using SRM and isotope-labeled peptides as standards. The results obtained clearly demonstrated the capacity of SID-SRM to specifically and precisely quantify protein biomarkers in plasma. However, the results also revealed that the protein digestion rate was highly variable between laboratories. This variability had a significant effect on peptide recovery and on the accuracy of protein quantification. As suggested by the authors, this type of bias could be avoided if properly folded isotope-labeled protein standards were used as quantification standards (7, 8).In 2007, we developed the PSAQ™ (Protein Standard Absolute Quantification) method, which uses full-length isotope-labeled proteins as internal standards for absolute quantitative MS analysis. We demonstrated that, in contrast with peptide standards, adding isotope-labeled proteins before sample digestion enables accurate protein quantification, even for proteins resistant to trypsin digestion (9, 10). In addition, we, and others, have shown that this type of protein standard (“PSAQ standard”) also corrects for protein losses that may occur during sample handling prior to trypsin digestion and liquid chromatography (LC)-MS analysis (11–17). This latter feature is a particular advantage for MS analysis of blood biomarkers. Indeed, as plasma/serum are highly complex matrices and display a huge dynamic range, sample prefractionation must be performed to detect low-abundance protein biomarkers (4).In this study, we have tested a combination of the PSAQ strategy with SRM (PSAQ-SRM) for quantification of cardiovascular biomarkers in serum samples. Selected biomarkers include LDH-B, CKMB, myoglobin, and troponin I. For some of these validated biomarkers, a comparison of PSAQ-SRM data and ELISA results was performed on samples from patients having suffered myocardial infarction.     

Autophagy--alias self-eating--appetite and ageing

Two articles—one published online in January and in the March issue EMBO reports—implicate autophagy in the control of appetite by regulating neuropeptide production in hypothalamic neurons. Autophagy decline with age in POMC neurons induces obesity and metabolic syndrome.Kaushik et al. EMBO reports, this issue doi:10.1038/embor.2011.260Macroautophagy, which I will call autophagy, is a critical process that degrades bulk cytoplasm, including organelles, protein oligomers and a range of selective substrates. It has been linked with diverse physiological and disease-associated functions, including the removal of certain bacteria, protein oligomers associated with neurodegenerative diseases and dysfunctional mitochondria [1]. However, the primordial role of autophagy—conserved from yeast to mammals—appears to be its ability to provide nutrients to starving cells by releasing building blocks, such as amino acids and free fatty acids, obtained from macromolecular degradation. In yeast, autophagy deficiency enhances death in starvation conditions [2], and in mice it causes death from starvation in the early neonatal period [3,4]. Two recent articles from the Singh group—one of them in this issue of EMBO reports—also implicate autophagy in central appetite regulation [5,6].Autophagy seems to decline with age in the liver [7], and it has thus been assumed that autophagy declines with age in all tissues, but this has not been tested rigorously in organs such as the brain. Conversely, specific autophagy upregulation in Caenorhabditis elegans and Drosophila extends lifespan, and drugs that induce autophagy—but also perturb unrelated processes, such as rapamycin—promote longevity in rodents [8].Autophagy literally means self-eating, and it is therefore interesting to see that this cellular ‘self-eating'' has systemic roles in mammalian appetite control. The control of appetite is influenced by central regulators, including various hormones and neurotransmitters, and peripheral regulators, including hormones, glucose and free fatty acids [9]. Autophagy probably has peripheral roles in appetite and energy balance, as it regulates lipolysis and free fatty acid release [10]. Furthermore, Singh and colleagues have recently implicated autophagy in central appetite regulation [5,6].The arcuate nucleus in the hypothalamus has received extensive attention as an integrator and regulator of energy homeostasis and appetite. Through its proximity to the median eminence, which is characterized by an incomplete blood–brain barrier, these neurons rapidly sense metabolic fluctuations in the blood. There are two different neuronal populations in the arcuate nucleus, which appear to have complementary effects on appetite (Fig 1). The proopiomelanocortin (POMC) neurons produce the neuropeptide precursor POMC, which is cleaved to form α-melanocyte stimulating hormone (α-MSH), among several other products. The α-MSH secreted from these neurons activates melanocortin 4 receptors on target neurons in the paraventricular nucleus of the hypothalamus, which ultimately reduce food intake. The second group of neurons contain neuropeptide Y (NPY) and Agouti-related peptide (AgRP). Secreted NPY binds to downstream neuronal receptors and stimulates appetite. AgRP blocks the ability of α-MSH to activate melanocortin 4 receptors [11]. Furthermore, AgRP neurons inhibit POMC neurons [9].Open in a separate windowFigure 1Schematic diagram illustrating the complementary roles of POMC and NPY/AgRP neurons in appetite control. AgRP, Agouti-related peptide; MC4R, melanocortin 4 receptor; α-MSH, α-melanocyte stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin.The first study from Singh''s group started by showing that starvation induces autophagy in the hypothalamus [5]. This finding alone merits some comment. Autophagy is frequently assessed by using phosphatidylethanolamine-conjugated Atg8/LC3 (LC3-II), which is specifically associated with autophagosomes and autolysosomes. LC3-II levels on western blot and the number of LC3-positive vesicles strongly correlate with the number of autophagosomes [1]. To assess whether LC3-II formation is altered by a perturbation, its level can be assessed in the presence of lysosomal inhibitors, which inhibit LC3-II degradation by blocking autophagosome–lysosome fusion [12]. Therefore, differences in LC3-II levels in response to a particular perturbation in the presence of lysosomal inhibitors reflect changes in autophagosome synthesis. An earlier study using GFP-LC3 suggested that autophagy was not upregulated in the brains of starved mice, compared with other tissues where this did occur [13]. However, this study only measured steady state levels of autophagosomes and was performed before the need for lysosomal inhibitors was appreciated. Subsequent work has shown rapid flux of autophagosomes to lysosomes in primary neurons, which might confound analyses without lysosomal inhibitors [14]. Thus, the data of the Singh group—showing that autophagy is upregulated in the brain by a range of methods including lysosomal inhibitors [5]—address an important issue in the field and corroborate another recent study that examined this question by using sophisticated imaging methods [15].“…decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with age”Singh and colleagues then analysed mice that have a specific knockout of the autophagy gene Atg7 in AgRP neurons [5]. Although fasting increases AgRP mRNA and protein levels in normal mice, these changes were not seen in the knockout mice. AgRP neurons provide inhibitory signals to POMC neurons, and Kaushik and colleagues found that the AgRP-specific Atg7 knockout mice had higher levels of POMC and α-MSH, compared with the normal mice. This indicated that starvation regulates appetite in a manner that is partly dependent on autophagy. The authors suggested that the peripheral free fatty acids released during starvation induce autophagy by activating AMP-activated protein kinase (AMPK), a known positive regulator of autophagy. This, in turn, enhances degradation of hypothalamic lipids and increases endogenous intracellular free fatty acid concentrations. The increased intracellular free fatty acids upregulate AgRP mRNA and protein expression. As AgRP normally inhibits POMC/α-MSH production in target neurons, a defect in AgRP responses in the autophagy-null AgRP neurons results in higher α-MSH levels, which could account for the decreased mouse bodyweight.In follow-up work, Singh''s group have now studied the effects of inhibiting autophagy in POMC neurons, again using Atg7 deletion [6]. These mice, in contrast to the AgRP autophagy knockouts, are obese. This might be accounted for, in part, by an increase in POMC preprotein levels and its cleavage product adrenocorticotropic hormone in the knockout POMC neurons, which is associated with a failure to generate α-MSH. Interestingly, these POMC autophagy knockout mice have impaired peripheral lipolysis in response to starvation, which the authors suggest might be due to reduced central sympathetic tone to the periphery from the POMC neurons. In addition, POMC-neuron-specific Atg7 knockout mice have impaired glucose tolerance.This new study raises several interesting issues. How does the autophagy defect in the POMC neurons alter the cleavage pattern of POMC? Is this modulated within the physiological range of autophagy activity fluctuations in response to diet and starvation? Importantly, in vivo, autophagy might fluctuate similarly (or possibly differently) in POMC and AgRP neurons in response to diet and/or starvation. Given the tight interrelation of these neurons, how does this affect their overall response to appetite regulation in wild-type animals?Finally, the study also shows that hypothalamic autophagosome formation is decreased in older mice. To my knowledge, this is the first such demonstration of this phenomenon in the brain. The older mice phenocopied aspects of the POMC-neuron autophagy null mice—increased hypothalamic POMC preprotein and ACTH and decreased α-MSH, along with similar adiposity and lipolytic defects, compared with young mice. These data are provocative from several perspectives. In the context of metabolism, it is tantalizing to consider that decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with ageing. Again, this model considers the POMC neurons in isolation, and it would be important to understand how reduced autophagy in aged AgRP neurons counterbalances this situation. In a more general sense, the data strongly support the concept that neuronal autophagy might decline with age.Autophagy is a major clearance route for many mutant, aggregate-prone intracytoplasmic proteins that cause neurodegenerative disease, such as tau (Alzheimer disease), α-synuclein (Parkinson disease), and huntingtin (Huntington disease), and the risk of these diseases is age-dependent [1]. Thus, it is tempting to suggest that the dramatic age-related risks for these diseases could be largely due to decreased neuronal capacity of degrading these toxic proteins. Neurodegenerative pathology and age-related metabolic abonormalities might be related—some of the metabolic disturbances that occur in humans with age could be due to the accumulation of such toxic proteins. High levels of these proteins are seen in many people who do not have, or who have not yet developed, neurodegenerative diseases, as many of them start to accumulate decades before any sign of disease. These proteins might alter metabolism and appetite either directly by affecting target neurons, or by influencing hormonal and neurotransmitter inputs into such neurons.     

Suicide among children and adolescents in Canada: trends and sex differences, 1980�C2008

Background:

Suicide is the second leading cause of death for young Canadians (10–19 years of age) — a disturbing trend that has shown little improvement in recent years. Our objective was to examine suicide trends among Canadian children and adolescents.

Methods:

We conducted a retrospective analysis of standardized suicide rates using Statistics Canada mortality data for the period spanning from 1980 to 2008. We analyzed the data by sex and by suicide method over time for two age groups: 10–14 year olds (children) and 15–19 year olds (adolescents). We quantified annual trends by calculating the average annual percent change (AAPC).

Results:

We found an average annual decrease of 1.0% (95% confidence interval [CI] −1.5 to −0.4) in the suicide rate for children and adolescents, but stratification by age and sex showed significant variation. We saw an increase in suicide by suffocation among female children (AAPC = 8.1%, 95% CI 6.0 to 10.4) and adolescents (AAPC = 8.0%, 95% CI 6.2 to 9.8). In addition, we noted a decrease in suicides involving poisoning and firearms during the study period.

Interpretation:

Our results show that suicide rates in Canada are increasing among female children and adolescents and decreasing among male children and adolescents. Limiting access to lethal means has some potential to mitigate risk. However, suffocation, which has become the predominant method for committing suicide for these age groups, is not amenable to this type of primary prevention.Suicide was ranked as the second leading cause of death among Canadians aged 10–34 years in 2008.¹ It is recognized that suicidal behaviour and ideation is an important public health issue among children and adolescents; disturbingly, suicide is a leading cause of Canadian childhood mortality (i.e., among youths aged 10–19 years).^2,3Between 1980 and 2008, there were substantial improvements in mortality attributable to unintentional injury among 10–19 year olds, with rates decreasing from 37.7 per 100 000 to 10.7 per 100 000; suicide rates, however, showed less improvement, with only a small reduction during the same period (from 6.2 per 100 000 in 1980 to 5.2 per 100 000 in 2008).¹Previous studies that looked at suicides among Canadian adolescents and young adults (i.e., people aged 15–25 years) have reported rates as being generally stable over time, but with a marked increase in suicides by suffocation and a decrease in those involving firearms.² There is limited literature on self-inflicted injuries among children 10–14 years of age in Canada and the United States, but there appears to be a trend toward younger children starting to self-harm.^3,4 Furthermore, the trend of suicide by suffocation moving to younger ages may be partly due to cases of the “choking game” (self-strangulation without intent to cause permanent harm) that have been misclassified as suicides.^5–7Risk factors for suicidal behaviour and ideation in young people include a psychiatric diagnosis (e.g., depression), substance abuse, past suicidal behaviour, family factors and other life stressors (e.g., relationships, bullying) that have complex interactions.⁸ A suicide attempt involves specific intent, plans and availability of lethal means, such as firearms,⁹ elevated structures¹⁰ or substances.¹¹ The existence of “pro-suicide” sites on the Internet and in social media¹² may further increase risk by providing details of various ways to commit suicide, as well as evaluations ranking these methods by effectiveness, amount of pain involved and length of time to produce death.^13–15Our primary objective was to present the patterns of suicide among children and adolescents (aged 10–19 years) in Canada.     

The future of molecular evolution

Antony Dean explores the past, present and future of evolutionary theory and our continuing efforts to explain biological patterns in terms of molecular processes and mechanisms.There are just two questions to be asked in evolution: how are things related, and what makes them differ? Lamarck was the first biologist—he invented the word—to address both. In his Philosophie Zoologique (1809) he suggested that the relationships among species are better described by branching trees than by a simple ladder, that new species arise gradually by descent with modification and that they adapt to changing environments through the inheritance of acquired characteristics. Much that Lamarck imagined has since been superseded. Following Wallace and Darwin, we now envision that species belong to a single highly branched tree and that natural selection is the mechanism of adaptation. Nonetheless, to Lamarck we owe the insight that pattern is produced by process and that both need mechanistic explanation.Questions of pattern, process and mechanism pervade the modern discipline of molecular evolution. The field was established when Zuckerkandl & Pauling (1965) noted that haemoglobins evolve at a roughly constant rate. Their “molecular evolutionary clock” forever changed our view of evolutionary history. Not only were seemingly intractable relationships resolved—for example, whales are allies of the hippopotamus—but also the eubacterial origins of eukaryotic organelles were firmly established and a new domain of life was discovered: the Archaea.Yet, different genes sometimes produce different trees. Golding & Gupta (1995) resolved two-dozen conflicting protein trees by suggesting that Eukarya arose following massive horizontal gene transfer between Bacteria and Archaea. Whole genome sequencing has since revealed so many conflicts that horizontal gene transfer seems characteristic of prokaryote evolution. In higher animals—where horizontal transfer is sufficiently rare that the tree metaphor remains robust—rapid and inexpensive whole genome sequencing promises to provide a wealth of data for population studies. The patterns of migration, admixture and divergence of species will be soon addressed in unprecedented detail.Sequence analyses are also used to infer processes. A constant molecular clock originally buttressed the neutral theory of molecular evolution (Kimura, 1985). The clock has since proven erratic, while the neutral theory now serves as a null hypothesis for statistical tests of ‘selection''. In truth, most tests are also sensitive to demographic changes. The promise of ultra-high throughput sequencing to provide genome-wide data should help dissect selection, which targets particular genes, from demography, which affects all the genes in a genome, although weak selection and ancient adaptations will remain undetected.In the functional synthesis (Dean & Thornton, 2007), molecular biology provides the experimental means to test evolutionary inferences decisively. For example, site-directed mutagenesis can be used to introduce putatively selected mutations into reconstructed ancestral sequences, the gene products are then expressed and purified and their functional properties determined in vitro. In microbial species, homologous recombination is used routinely to replace wild-type with engineered genes, enabling organismal phenotypes and fitnesses to be determined in vivo. The vision of Zuckerkandl & Pauling (1965) that by “furnishing probable structures of ancestral proteins, chemical paleogenetics will in the future lead to deductions concerning molecular functions as they were presumably carried out in the distant evolutionary past” is now a reality.If experimental tests of evolutionary inferences open windows on past mechanisms, directed evolution focuses on the mechanisms without attempting historical reconstruction. Today''s ‘fast-forward'' molecular breeding experiments use mutagenic PCR to generate vast libraries of variation and high throughput screens to identify rare novel mutants (Romero & Arnold, 2009; Khersonsky & Tawfik, 2010). Among numerous topics explored are: the role of intragenic recombination in furthering adaptation, the number and location of mutations in protein structures, the necessity—or lack thereof—of broadening substrate specificity before a new function is acquired, the evolution of robustness, and the alleged trade-off between stability and catalytic efficiency. Few, however, have approached the detail found in those classic studies of evolved β-galactosidase (Hall, 2003) that revealed how the free-energy profile of an enzyme-catalysed reaction evolved. Even further removed from natural systems are catalytic RNAs that, by combining phenotype and genotype within the same molecule, allow evolution to proceed in a lifeless series of chemical reactions. Recently, two RNA enzymes that catalyse each other''s synthesis were shown to undergo self-sustained exponential amplification (Lincoln & Joyce, 2009). Competition for limiting tetranucleotide resources favours mutants with higher relative fitness—faster replication—demonstrating that adaptive evolution can occur in a chemically defined abiotic genetic system.Lamarck was the first to attempt a coherent explanation of biological patterns in terms of processes and mechanisms. That his legacy can still be discerned in the vibrant field of molecular evolution would no doubt please him as much as it does us in promising extraordinary advances in our understanding of the mechanistic basis of molecular adaptation.     

Stem-cell tourism and scientific responsibility. Stem-cell researchers are in a unique position to curb the problem of stem-cell tourism

Stem-cell tourism exploits the hope of patients desperate for therapies and cures. Scientists have both a special responsibility and a unique role to play in addressing this problem.During the past decade, thousands of patients with a variety of diseases unresponsive to conventional treatment have gone abroad to receive stem-cell therapies. This phenomenon, commonly referred to as ‘stem-cell tourism'', raises significant ethical concerns, because patients often receive treatments that are not only unproven, but also unregulated, potentially dangerous or even fraudulent (Kiatpongsan & Sipp, 2009; Lindvall & Hyun, 2009). Stem-cell clinics have sprung up in recent years to take advantage of desperate patients who have exhausted other alternatives (Ryan et al, 2010). These clinics usually advertise their services directly to consumers through the Internet, make extravagant claims about the benefits, downplay the risks involved and charge hefty fees of US $20,000 or more for treatments (Lau et al, 2008; Regenberg et al, 2009).Stem-cell tourism is regarded as ethically problematic because patients receive unproven therapies from untrustworthy sourcesWith a few exceptions—such as the use of bone-marrow haematopoietic cells to treat leukaemia—novel stem-cell therapies are often unproven in clinical trials (Lindvall & Hyun, 2009). Even well-proven therapies can lead to tumour formation, tissue rejection, autoimmunity, permanent disability and death (Gallagher & Forrest, 2007; Murphy & Blazar, 1999). The risks of unproven and unregulated therapies are potentially much worse (Barclay, 2009).In this commentary, we argue that stem-cell scientists have a unique and important role to play in addressing the problem of stem-cell tourism. Stem-cell scientists should carefully examine all requests to provide cell lines and other materials, and share them only with responsible investigators or clinicians. They should require recipients of stem cells to sign material transfer agreements (MTAs) that describe how the cells may be used, and to provide documentation about their scientific or medical qualifications.In discussing these ethical and regulatory issues, it is important to distinguish between stem-cell tourism and other types of travel to receive medical treatment including stem-cell therapy. Stem-cell tourism is regarded as ethically problematic because patients receive unproven therapies from untrustworthy sources. Other forms of travel usually do not raise troubling ethical issues (Lindvall & Hyun, 2009). Many patients go to other countries to receive proven stem-cell therapies—such as haematopoietic cells to treat leukaemia—from responsible physicians. Other patients obtain unproven stem-cell treatments by participating in scientifically valid, legally sanctioned clinical trials, or by receiving ethically responsible, innovative medical care (Lindvall & Hyun, 2009). In some cases, patients need to travel because the therapy is approved in only some countries; by way of example, on 1 July, Korea was the first country that approved the clinical use of adult stem cells to treat heart attack victims (Heejung & Yi, 2011).…even when regulations are in place, unscrupulous individuals might still evade these rulesAny medical innovation is ethically responsible when it is based on animal studies or other research that guarantee evidence of safety and clinical efficacy. Adequate measures must also be taken to protect patients from harm, such as clinical monitoring, follow-up, exclusion of individuals who are likely to be harmed or are unlikely to benefit, use of only clinical-grade stem cells, careful attention to dosing strategies and informed consent (Lindvall & Hyun, 2009).Many of the articles examining the ethics of stem-cell tourism have focused on the need for more regulatory oversight and education to prevent harm (Lindvall & Hyun, 2009; Caplan & Levine, 2010; Cohen & Cohen, 2010; Zarzeczny & Caulfield, 2010). We agree that additional regulations are needed, as there is little oversight of stem-cell research or therapy at present. Although most countries have regulations for conducting research with human subjects, as well as medical malpractice and licensing laws, these provide general guidance and do not directly address stem-cell therapy.Regulations have significant limitations, however. First, regulations apply intra-nationally, not internationally. If a country passes laws designed to oversee therapy and research, these laws would not apply in another nation. Physicians and investigators who do not want to adhere to these rules can simply move to another country that has a permissive legal environment. International agreements can help to close this regulatory gap, but there will still be countries that do not accept or abide by these agreements. Second, even when regulations are in place, unscrupulous individuals might still evade these rules (Resnik, 1999).Educating patients about the risks of unproven therapies can also help to address the problem of stem-cell tourism. However, education too has significant limitations, since many people will remain ignorant of the dangers of unproven therapies, or they will simply ignore warnings and prudent advice. For many years, cancer patients have travelled to foreign countries to receive unconventional and unproven treatments, despite educational campaigns and media reports discussing the dangers of these therapies. Since the 1970s, thousands of patients have travelled to cancer clinics in Mexico to receive medical treatments not available in the USA (Moss, 2005).Education for physicians on the dangers of unproven stem-cell therapies can be helpful, but this strategy also has limitations, since many will not receive this education or will choose to ignore it. Additionally, responsible physicians might still find it difficult to persuade their patients not to receive an unproven therapy, especially when conventional treatments have failed. The history of cancer treatment offers important lessons here, since many oncologists have tried, unsuccessfully, to convince their patients not travel to foreign countries to receive questionable treatments (Moss, 2005).Since regulation and education have significant shortcomings, it is worth considering another strategy for dealing with the problem of stem-cell tourism, one that focuses on the social responsibilities of stem-cell scientists.Many codes of ethics adopted by scientific associations include provisions relating to social responsibilities (Shamoo & Resnik, 2009). For example, the Code of Ethics of the American Society for Biochemistry and Molecular Biology states that “investigators will promote and follow practices that enhance the public interest or well-being” (American Society of Microbiology, 2011). Social responsibilities in science include an obligation to avoid causing harm and an obligation to benefit the public (Shamoo & Resnik, 2009).…education too has significant limitations, since many people will remain ignorant of the dangers of unproven therapies, or they will simply ignore warnings and prudent adviceThere are two distinct rationales for social responsibility. First, scientists should be accountable to the public since the public provides scientists with funding, facilities and staff (Shamoo & Resnik, 2009). Second, stem-cell scientists are uniquely positioned to exercise their social responsibilities and take effective action pertaining to stem-cell tourism. They understand the science behind stem-cell research, including the potential for harm and the likely clinical efficacy. This knowledge can be used to evaluate the scientific validity of the different uses of stem cells, especially clinical uses. Stem-cell scientists also have control over cell lines and other materials that they may or may not choose to share with other researchers or physicians.Many of the private clinics that offer stem-cell treatments are relatively small and often depend on acquiring resources from scientists working in the field. The materials they might require could include adult, embryonic and fetal stem-cell lines; vectors that can be used to induce pluripotency in isolated adult cells; genes, DNA and RNA sequences; antibodies; purified protein products, such as growth factors; and special cocktails, media or extracellular matrices to culture specific stem-cell types.Social responsibilities in science include an obligation to avoid causing harm and an obligation to benefit the publicOne way in which stem-cell scientists can help to address the problem of stem-cell tourism is to refuse to share cell lines or other materials with physicians or investigators whom they believe might be behaving irresponsibly. To decide whether someone who requests materials is a responsible individual, stem-cell scientists should ask recipients to supply documentation, such as a CV, website, a research or clinical protocol, or clinical trial number, as evidence of their work and expertise in stem cells. This would ensure that the stem cells and other materials are going to be used in the course of responsible biomedical research, a legally sanctioned clinical trial, or in responsible medical innovation. If the recipients provide insufficient documentation, scientists should refuse to honour their requests for materials.Stem-cell scientists should also require recipients to sign MTAs that describe what will be done with the material supplied. MTAs are contracts governing the transfer of materials between organizations and typically include a variety of terms and conditions, such as the purposes for which the materials may be used—commercial or academic research, for example—modification of the materials, transfers to third parties, intellectual property rights, and compliance with legal, regulatory and other policies (Rodriguez, 2005).To help address the problem of stem-cell tourism, MTAs should state whether the materials will be used in humans, and under what conditions. If the stem cells are not clinical grade, the MTA should state that they will not be transplanted into humans, unless the recipients have a well-developed and legally sanctioned procedure—approved by the Food and Drug Administration or other relevant agency—for verifying the quality of the cells and performing the necessary changes to make them acceptable for human use. For example, the recipients could test the cells for viral and bacterial infections, mutations, chemical impurities or other factors that would compromise their clinical utility in an attempt to develop clinical grade cell lines.In addition, the MTA could stipulate that scientists must follow the ethical Guidelines for Clinical Translation of Stem Cells set forth by the International Society for Stem Cell Research (Hyun et al, 2008). These guidelines set forth various preclinical and clinical conditions for stem-cell interventions. Describing such conditions might help to deter unscrupulous individuals from using stem cells for scientifically and ethically questionable practices. By evaluating a recipient''s qualifications and intended uses of stem-cell lines and other reagents, scientists demonstrate social responsibility and uphold public trust when sharing materials.Stem-cell scientists also have control over cell lines and other materials that they may or may not choose to share with other researchers or physiciansSince an MTA is a type of contract between institutions, there is legal recourse if it is broken. A plaintiff could sue a defendant that violates an MTA for breach of contract. Also, if the aggrieved party is a funding agency, it could withhold research funding from the offending party. The onus is on the plaintiff—the scientist and scientific organization providing the materials—to file a lawsuit against the defendants for breach of contract and this requires the scientist or others in the organization to follow-up and ensure that the materials transferred are being used in compliance with the conditions set forth in the MTA.Some might object to our proposal because it violates the principle of scientific openness, which is an integral part of the ethos of science (Shamoo & Resnik, 2009). Scientists have an obligation to share data, reagents, cell lines, methods and other research tools because sharing is vital to the progress of science. Many granting agencies and journals also have policies that require scientists to make data and materials available to other scientists on request (Shamoo & Resnik, 2009).Although openness is vital to the ethical practice of science, it can be superseded by other important factors, such as protecting the privacy and confidentiality of human research subjects, safeguarding proprietary or classified research, securing intellectual property or scientific priority, or preventing bioterrorism (Shamoo & Resnik, 2009). We consider tackling the problem of stem-cell tourism to be a sufficiently important reason for refusing to share research materials in some situations.Although openness is vital to the ethical practice of science, it can be superseded by other important factors…Some might also object to our proposal on the grounds that it places unnecessary burdens on already overworked scientists, or that unscrupulous scientists and physicians will find alternative ways to obtain stem cells, even if investigators refuse to share them.We recognize the need to avoid burdening researchers unnecessarily with administrative work, but we think that verifying the qualifications of a recipient and reviewing a protocol is a reasonable burden. If principal investigators do not wish to shoulder this responsibility, they can ask a postdoctoral fellow or another senior member of the laboratory or faculty to help them. Far from being a waste of time and effort, taking some simple steps to determine whether requests for stem cells come from responsible physicians or investigators can be an important part of the scientific community''s response to stem-cell tourism.A month before his death in 1963, former US President John F. Kennedy (1917-1963) made an address at the Centennial Convocation of the National Academy of Sciences in which he said: “If scientific discovery has not been an unalloyed blessing, if it has conferred on mankind the power not only to create but also to annihilate, it has at the same time provided humanity with a supreme challenge and a supreme testing.” Stem-cell scientists can rise to this challenge and address the problem of stem-cell tourism by ensuring that the products of their research are controlled responsibly and shared wisely with genuine investigators or clinicians through the use of MTAs. Doing so should help to deter fraudulent scientists or physicians from exploiting patients who travel to foreign countries in their desperate search for cures.? Open in a separate windowZubin MasterOpen in a separate windowDavid B Resnik     

Aiming high,but investing little

The French government has ambitious goals to make France a leading nation for synthetic biology research, but it still needs to put its money where its mouth is and provide the field with dedicated funding and other support.Synthetic biology is one of the most rapidly growing fields in the biological sciences and is attracting an increasing amount of public and private funding. France has also seen a slow but steady development of this field: the establishment of a national network of synthetic biologists in 2005, the first participation of a French team at the International Genetically Engineered Machine competition in 2007, the creation of a Master''s curriculum, an institute dedicated to synthetic and systems biology at the University of Évry-Val-d''Essonne-CNRS-Genopole in 2009–2010, and an increasing number of conferences and debates. However, scientists have driven the field with little dedicated financial support from the government.Yet the French government has a strong self-perception of its strengths and has set ambitious goals for synthetic biology. The public are told about a “new generation of products, industries and markets” that will derive from synthetic biology, and that research in the field will result in “a substantial jump for biotechnology” and an “industrial revolution”[1,2]. Indeed, France wants to compete with the USA, the UK, Germany and the rest of Europe and aims “for a world position of second or third”[1]. However, in contrast with the activities of its competitors, the French government has no specific scheme for funding or otherwise supporting synthetic biology[3]. Although we read that “France disposes of strong competences” and “all the assets needed”[2], one wonders how France will achieve its ambitious goals without dedicated budgets or detailed roadmaps to set up such institutions.In fact, France has been a straggler: whereas the UK and the USA have published several reports on synthetic biology since 2007, and have set up dedicated governing networks and research institutions, the governance of synthetic biology in France has only recently become an official matter. The National Research and Innovation Strategy (SNRI) only defined synthetic biology as a “priority” challenge in 2009 and created a working group in 2010 to assess the field''s developments, potentialities and challenges; the report was published in 2011[1].At the same time, the French Parliamentary Office for the Evaluation of Scientific and Technological Choices (OPECST) began a review of the field “to establish a worldwide state of the art and the position of our country in terms of training, research and technology transfer”. Its 2012 report entitled The Challenges of Synthetic Biology[2] assessed the main ethical, legal, economic and social challenges of the field. It made several recommendations for a “controlled” and “transparent” development of synthetic biology. This is not a surprise given that the development of genetically modified organisms and nuclear power in France has been heavily criticized for lack of transparency, and that the government prefers to avoid similar future controversies. Indeed, the French government seems more cautious today: making efforts to assess potential dangers and public opinion before actually supporting the science itself.Both reports stress the necessity of a “real” and “transparent” dialogue between science and society and call for “serene […] peaceful and constructive” public discussion. The proposed strategy has three aims: to establish an observatory, to create a permanent forum for discussion and to broaden the debate to include citizens[4]. An Observatory for Synthetic Biology was set up in January 2012 to collect information, mobilize actors, follow debates, analyse the various positions and organize a public forum. Let us hope that this observatory—unlike so many other structures—will have a tangible and durable influence on policy-making, public opinion and scientific practice.Many structural and organizational challenges persist, as neither the National Agency for Research nor the National Centre for Scientific Research have defined the field as a funding priority and public–private partnerships are rare in France. Moreover, strict boundaries between academic disciplines impede interdisciplinary work, and synthetic biology is often included in larger research programmes rather than supported as a research field in itself. Although both the SNRI and the OPECST reports make recommendations for future developments—including setting up funding policies and platforms—it is not clear whether these will materialize, or when, where and what size of investments will be made.France has ambitious goals for synthetic biology, but it remains to be seen whether the government is willing to put ‘meat to the bones'' in terms of financial and institutional support. If not, these goals might come to be seen as unrealistic and downgraded or they will be replaced with another vision that sees synthetic biology as something that only needs discussion and deliberation but no further investment. One thing is already certain: the future development of synthetic biology in France is a political issue.     

Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months

Background:

The gut microbiota is essential to human health throughout life, yet the acquisition and development of this microbial community during infancy remains poorly understood. Meanwhile, there is increasing concern over rising rates of cesarean delivery and insufficient exclusive breastfeeding of infants in developed countries. In this article, we characterize the gut microbiota of healthy Canadian infants and describe the influence of cesarean delivery and formula feeding.

Methods:

We included a subset of 24 term infants from the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort. Mode of delivery was obtained from medical records, and mothers were asked to report on infant diet and medication use. Fecal samples were collected at 4 months of age, and we characterized the microbiota composition using high-throughput DNA sequencing.

Results:

We observed high variability in the profiles of fecal microbiota among the infants. The profiles were generally dominated by Actinobacteria (mainly the genus Bifidobacterium) and Firmicutes (with diverse representation from numerous genera). Compared with breastfed infants, formula-fed infants had increased richness of species, with overrepresentation of Clostridium difficile. Escherichia–Shigella and Bacteroides species were underrepresented in infants born by cesarean delivery. Infants born by elective cesarean delivery had particularly low bacterial richness and diversity.

Interpretation:

These findings advance our understanding of the gut microbiota in healthy infants. They also provide new evidence for the effects of delivery mode and infant diet as determinants of this essential microbial community in early life.The human body harbours trillions of microbes, known collectively as the “human microbiome.” By far the highest density of commensal bacteria is found in the digestive tract, where resident microbes outnumber host cells by at least 10 to 1. Gut bacteria play a fundamental role in human health by promoting intestinal homeostasis, stimulating development of the immune system, providing protection against pathogens, and contributing to the processing of nutrients and harvesting of energy.^1,2 The disruption of the gut microbiota has been linked to an increasing number of diseases, including inflammatory bowel disease, necrotizing enterocolitis, diabetes, obesity, cancer, allergies and asthma.¹ Despite this evidence and a growing appreciation for the integral role of the gut microbiota in lifelong health, relatively little is known about the acquisition and development of this complex microbial community during infancy.³Two of the best-studied determinants of the gut microbiota during infancy are mode of delivery and exposure to breast milk.^4,5 Cesarean delivery perturbs normal colonization of the infant gut by preventing exposure to maternal microbes, whereas breastfeeding promotes a “healthy” gut microbiota by providing selective metabolic substrates for beneficial bacteria.^3,5 Despite recommendations from the World Health Organization,⁶ the rate of cesarean delivery has continued to rise in developed countries and rates of breastfeeding decrease substantially within the first few months of life.^7,8 In Canada, more than 1 in 4 newborns are born by cesarean delivery, and less than 15% of infants are exclusively breastfed for the recommended duration of 6 months.^9,10 In some parts of the world, elective cesarean deliveries are performed by maternal request, often because of apprehension about pain during childbirth, and sometimes for patient–physician convenience.¹¹The potential long-term consequences of decisions regarding mode of delivery and infant diet are not to be underestimated. Infants born by cesarean delivery are at increased risk of asthma, obesity and type 1 diabetes,¹² whereas breastfeeding is variably protective against these and other disorders.¹³ These long-term health consequences may be partially attributable to disruption of the gut microbiota.^12,14Historically, the gut microbiota has been studied with the use of culture-based methodologies to examine individual organisms. However, up to 80% of intestinal microbes cannot be grown in culture.^3,15 New technology using culture-independent DNA sequencing enables comprehensive detection of intestinal microbes and permits simultaneous characterization of entire microbial communities. Multinational consortia have been established to characterize the “normal” adult microbiome using these exciting new methods;¹⁶ however, these methods have been underused in infant studies. Because early colonization may have long-lasting effects on health, infant studies are vital.^3,4 Among the few studies of infant gut microbiota using DNA sequencing, most were conducted in restricted populations, such as infants delivered vaginally,¹⁷ infants born by cesarean delivery who were formula-fed¹⁸ or preterm infants with necrotizing enterocolitis.¹⁹Thus, the gut microbiota is essential to human health, yet the acquisition and development of this microbial community during infancy remains poorly understood.³ In the current study, we address this gap in knowledge using new sequencing technology and detailed exposure assessments²⁰ of healthy Canadian infants selected from a national birth cohort to provide representative, comprehensive profiles of gut microbiota according to mode of delivery and infant diet.     

Elixirs of death

Substandard and fake drugs are increasingly threatening lives in both the developed and developing world, but governments and industry are struggling to improve the situation.When people take medicine, they assume that it will make them better. However many patients cannot trust their drugs to be effective or even safe. Fake or substandard medicine is a major public health problem and it seems to be growing. More than 200 heart patients died in Pakistan in 2012 after taking a contaminated drug against hypertension [1]. In 2006, cough syrup that contained diethylene glycol as a cheap substitute for pharmaceutical-grade glycerin was distributed in Panama, causing the death of at least 219 people [2,3]. However, the problem is not restricted to developing countries. In 2012, more than 500 patients came down with fungal meningitis and several dozens died after receiving contaminated steroid injections from a compounding pharmacy in Massachusetts [4]. The same year, a fake version of the anti-cancer drug Avastin, which contained no active ingredient, was sold in the USA. The drug seemed to have entered the country through Turkey, Switzerland, Denmark and the UK [5].…many patients cannot trust their drugs to be effective or even safeThe extent of the problem is not really known, as companies and governments do not always report incidents [6]. However, the information that is available is alarming enough, especially in developing countries. One study found that 20% of antihypertensive drugs collected from pharmacies in Rwanda were substandard [7]. Similarly, in a survey of anti-malaria drugs in Southeast Asia and sub-Saharan Africa, 20–42% were found to be either of poor quality or outright fake [8], whilst 56% of amoxicillin capsules sampled in different Arab countries did not meet the US Pharmacopeia requirements [9].Developing countries are particularly susceptible to substandard and fake medicine. Regulatory authorities do not have the means or human resources to oversee drug manufacturing and distribution. A country plagued by civil war or famine might have more pressing problems—including shortages of medicine in the first place. The drug supply chain is confusingly complex with medicines passing through many different hands before they reach the patient, which creates many possible entry points for illegitimate products. Many people in developing countries live in rural areas with no local pharmacy, and anyway have little money and no health insurance. Instead, they buy cheap medicine from street vendors at the market or on the bus (Fig 1; [2,10,11]). “People do not have the money to buy medicine at a reasonable price. But quality comes at a price. A reasonable margin is required to pay for a quality control system,” explained Hans Hogerzeil, Professor of Global Health at Groningen University in the Netherlands. In some countries, falsifying medicine has developed into a major business. The low risk of being detected combined with relatively low penalties has turned falsifying medicine into the “perfect crime” [2].Open in a separate windowFigure 1Women sell smuggled, counterfeit medicine on the Adjame market in Abidjan, Ivory Coast, in 2007. Fraudulent street medecine sales rose by 15–25% in the past two years in Ivory Coast.Issouf Sanogo/AFP Photo/Getty Images.There are two main categories of illegitimate drugs. ‘Substandard'' medicines might result from poor-quality ingredients, production errors and incorrect storage. ‘Falsified'' medicine is made with clear criminal intent. It might be manufactured outside the regulatory system, perhaps in an illegitimate production shack that blends chalk with other ingredients and presses it into pills [10]. Whilst falsified medicines do not typically contain any active ingredients, substandard medicine might contain subtherapeutic amounts. This is particularly problematic when it comes to anti-infectious drugs, as it facilitates the emergence and spread of drug resistance [12]. A sad example is the emergence of artemisinin-resistant Plasmodium strains at the Thai–Cambodia border [8] and the Thai–Myanmar border [13], and increasing multidrug-resistant tuberculosis might also be attributed to substandard medication [11].Many people in developing countries live in rural areas with no local pharmacy, and anyway have little money and no health insuranceEven if a country effectively prosecutes falsified and substandard medicine within its borders, it is still vulnerable to fakes and low-quality drugs produced elsewhere where regulations are more lax. To address this problem, international initiatives are urgently required [10,14,15], but there is no internationally binding law to combat counterfeit and substandard medicine. Although drug companies, governments and NGOs are interested in good-quality medicines, the different parties seem to have difficulties coming to terms with how to proceed. What has held up progress is a conflation of health issues and economic interests: innovator companies and high-income countries have been accused of pushing for the enforcement of intellectual property regulations under the guise of protecting quality of medicine [14,16].The concern that intellectual property (IP) interests threaten public health dates back to the ‘Trade-Related Aspects of Intellectual Property Rights (TRIPS) Agreement'' of the World Trade Organization (WTO), adopted in 1994, to establish global protection of intellectual property rights, including patents for pharmaceuticals. The TRIPS Agreement had devastating consequences during the acquired immunodeficiency syndrome epidemic, as it blocked patients in developing countries from access to affordable medicine. Although it includes flexibility, such as the possibility for governments to grant compulsory licenses to manufacture or import a generic version of a patented drug, it has not always been clear how these can be used by countries [14,16,17].In response to public concerns over the public health consequences of TRIPS, the Doha Declaration on the TRIPS Agreement and Public Health was adopted at the WTO''s Ministerial Conference in 2001. It reaffirmed the right of countries to use TRIPS flexibilities and confirmed the primacy of public health over the enforcement of IP rights. Although things have changed for the better, the Doha Declaration did not solve all the problems associated with IP protection and public health. For example, anti-counterfeit legislation, encouraged by multi-national pharmaceutical industries and the EU, threatened to impede the availability of generic medicines in East Africa [14,16,18]. In 2008–2009, European customs authorities seized shipments of legitimate generic medicines in transit from India to other developing countries because they infringed European IP laws [14,16,17]. “We''re left with decisions being taken based on patents and trademarks that should be taken based on health,” commented Roger Bate, a global health expert and resident scholar at the American Enterprise Institute in Washington, USA. “The health community is shooting themselves in the foot.”Conflating health care and IP issues are reflected in the unclear use of the term ‘counterfeit'' [2,14]. “Since the 1990s the World Health Organization (WHO) has used the term ‘counterfeit'' in the sense we now use ‘falsified'',” explained Hogerzeil. “The confusion started in 1995 with the TRIPS agreement, through which the term ‘counterfeit'' got the very narrow meaning of trademark infringement.” As a consequence, an Indian generic, for example, which is legal in some countries but not in others, could be labelled as ‘counterfeit''—and thus acquire the negative connotation of bad quality. “The counterfeit discussion was very much used as a way to block the market of generics and to put them in a bad light,” Hogerzeil concluded.The rifts between the stakeholders have become so deep during the course of these discussions that progress is difficult to achieve. “India is not at all interested in any international regulation. And, unfortunately, it wouldn''t make much sense to do anything without them,” Hogerzeil explained. Indeed, India is a core player: not only does it have a large generics industry, but also the country seems to be, together with China, the biggest source of fake medical products [19,20]. The fact that India is so reluctant to react is tragically ironic, as this stance hampers the growth of its own generic companies like Ranbaxy, Cipla or Piramal. “I certainly don''t believe that Indian generics would lose market share if there was stronger action on public health,” Bate said. Indeed, stricter regulations and control systems would be advantageous, because they would keep fakers at bay. The Indian generic industry is a common target for fakers, because their products are broadly distributed. “The most likely example of a counterfeit product I have come across in emerging markets is a counterfeit Indian generic,” Bate said. Such fakes can damage a company''s reputation and have a negative impact on its revenues when customers stop buying the product.The WHO has had a key role in attempting to draft international regulations that would contain the spread of falsified and substandard medicine. It took a lead in 2006 with the launch of the International Medical Products Anti-Counterfeiting Taskforce (IMPACT). But IMPACT was not a success. Concerns were raised over the influence of multi-national drug companies and the possibility that issues on quality of medicines were conflated with the attempts to enforce stronger IP measures [17]. The WHO distanced itself from IMPACT after 2010. For example, it no longer hosts IMPACT''s secretariat at its headquarters in Geneva [2].‘Substandard'' medicines might result from poor quality ingredients, production errors and incorrect storage. ‘Falsified'' medicine is made with clear criminal intentIn 2010, the WHO''s member states established a working group to further investigate how to proceed, which led to the establishment of a new “Member State mechanism on substandard/spurious/falsely labelled/falsified/counterfeit medical products” (http://www.who.int/medicines/services/counterfeit/en/index.html). However, according to a publication by Amir Attaran from the University of Ottawa, Canada, and international colleagues, the working group “still cannot agree how to define the various poor-quality medicines, much less settle on any concrete actions” [14]. The paper''s authors demand more action and propose a binding legal framework: a treaty. “Until we have stronger public health law, I don''t think that we are going to resolve this problem,” Bate, who is one of the authors of the paper, said.Similarly, the US Food and Drug Administration (FDA) commissioned the Institute of Medicine (IOM) to convene a consensus committee on understanding the global public health implications of falsified and substandard pharmaceuticals [2]. Whilst others have called for a treaty, the IOM report calls on the World Health Assembly—the governing body of the WHO—to develop a code of practice such as a “voluntary soft law” that countries can sign to express their will to do better. “At the moment, there is not yet enough political interest in a treaty. A code of conduct may be more realistic,” Hogerzeil, who is also on the IOM committee, commented. Efforts to work towards a treaty should nonetheless be pursued, Bate insisted: “The IOM is right in that we are not ready to sign a treaty yet, but that does not mean you don''t start negotiating one.”Whilst a treaty might take some time, there are several ideas from the IOM report and elsewhere that could already be put into action to deal with this global health threat [10,12,14,15,19]. Any attempts to safeguard medicines need to address both falsified and substandard medicines, but the counter-measures are different [14]. Falsifying medicine is, by definition, a criminal act. To counteract fakers, action needs to be taken to ensure that the appropriate legal authorities deal with criminals. Substandard medicine, on the other hand, arises when mistakes are made in genuine manufacturing companies. Such mistakes can be reduced by helping companies do better and by improving quality control of drug regulatory authorities.Manufacturing pharmaceuticals is a difficult and costly business that requires clean water, high-quality chemicals, expensive equipment, technical expertise and distribution networks. Large and multi-national companies benefit from economies of scale to cope with these problems. But smaller companies often struggle and compromise in quality [2,21]. “India has 20–40 big companies and perhaps nearly 20,000 small ones. To me, it seems impossible for them to produce at good quality, if they remain so small,” Hogerzeil explained. “And only by being strict, can you force them to combine and to become bigger industries that can afford good-quality assurance systems.” Clamping down on drug quality will therefore lead to a consolidation of the industry, which is an essential step. “If you look at Europe and the US, there were hundreds of drug companies—now there are dozens. And if you look at the situation in India and China today, there are thousands and that will have to come down to dozens as well,” Bate explained.…innovator companies and high-income countries have been accused of pushing for the enforcement of intellectual property regulations under the guise of protecting […] medicineIn addition to consolidating the market by applying stricter rules, the IOM has also suggested measures for supporting companies that observe best practices [2]. For example, the IOM proposes that the International Finance Corporation and the Overseas Private Investment Corporation, which promote private-sector development to reduce poverty, should create separate investment vehicles for pharmaceutical manufacturers who want to upgrade to international standards. Another suggestion is to harmonize market registration of pharmaceutical products, which would ease the regulatory burden for generic producers in developing countries and improve the efficiency of regulatory agencies.Once the medicine leaves the manufacturer, controlling distribution systems becomes another major challenge in combatting falsified and substandard medicine. Global drug supply chains have grown increasingly complicated; drugs cross borders, are sold back and forth between wholesalers and distributers, and are often repackaged. Still, there is a main difference between developing and developed countries. In the latter case, relatively few companies dominate the market, whereas in poorer nations, the distribution system is often fragmented and uncontrolled with parallel schemes, too few pharmacies, even fewer pharmacists and many unlicensed medical vendors. Every transaction creates an opportunity for falsified or substandard medicine to enter the market [2,10,19]. More streamlined and transparent supply chains and stricter licensing requirements would be crucial to improve drug quality. “And we can start in the US,” Hogerzeil commented.…India is a core player: not only does it have a large generics industry, but the country also seems to be, together with China, the biggest source of fake medical productsDistribution could be improved at different levels, starting with the import of medicine. “There are states in the USA where the regulation for medicine importation is very lax. Anyone can import; private clinics can buy medicine from Lebanon or elsewhere and fly them in,” Hogerzeil explained. The next level would be better control over the distribution system within the country. The IOM suggests that state boards should license wholesalers and distributors that meet the National Association of Boards of Pharmacy accreditation standards. “Everybody dealing with medicine has to be licensed,” Hogerzeil said. “And there should be a paper trail of who buys what from whom. That way you close the entry points for illegal drugs and prevent that falsified medicines enter the legal supply chain.” The last level would be a track-and-trace system to identify authentic drugs [2]. Every single package of medicine should be identifiable through an individual marker, such as a 3D bar code. Once it is sold, it is ticked off in a central database, so the marker cannot be reused.According to Hogerzeil, equivalent measures at these different levels should be established in every country. “I don''t believe in double standards”, he said. “Don''t say to Uganda: ‘you can''t do that''. Rather, indicate to them what a cost-effective system in the West looks like and help them, and give them the time, to create something in that direction that is feasible in their situation.”Nigeria, for instance, has demonstrated that with enough political will, it is possible to reduce the proliferation of falsified and substandard medicine. Nigeria had been a major source for falsified products, but things changed in 2001, when Dora Akunyili was appointed Director General of the National Agency for Food and Drug Administration and Control. Akunyili has a personal motivation for fighting falsified drugs: her sister Vivian, a diabetic patient, lost her life to fake insulin in 1988. Akunyili strengthened import controls, campaigned for public awareness, clamped down on counterfeit operations and pushed for harsher punishments [10,19]. Paul Orhii, Akunyili''s successor, is committed to continuing her work [10]. Although there are no exact figures, various surveys indicate that the rate of bad-quality medicine has dropped considerably in Nigeria [10].China is also addressing its drug-quality problems. In a highly publicized event, the former head of China''s State Food and Drug Administration, Zheng Xiaoyu, was executed in 2007 after he was found guilty of accepting bribes to approve untested medicine. Since then, China''s fight against falsified medicine has continued. As a result of heightened enforcement, the number of drug companies in China dwindled from 5,000 in 2004 to about 3,500 this year [2]. Moreover, in July 2012, more than 1,900 suspects were arrested for the sale of fake or counterfeit drugs.Quality comes at a price, however. It is expensive to produce high-quality medicine, and it is expensive to control the production and distribution of drugs. Many low- and middle-income countries might not have the resources to tackle the problem and might not see quality of medicine as a priority. But they should, and affluent countries should help. Not only because health is a human right, but also for economic reasons. A great deal of time and money is invested into testing the safety and efficacy of medicine during drug development, and these resources are wasted when drugs do not reach patients. Falsified and substandard medicines are a financial burden to health systems and the emergence of drug-resistant pathogens might make invaluable medications useless. Investing in the safety of medicine is therefore a humane and an economic imperative.     

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy

EMBO J 31 5, 1062–1079 (2012); published online January172012In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).The textbook notion that apoptosis would always take place unrecognized by the immune system has recently been invalidated (Zitvogel et al, 2010; Galluzzi et al, 2012). Thus, in specific circumstances (in particular in response to anthracyclines, oxaliplatin, and γ irradiation), cancer cells can enter a lethal stress pathway linked to the emission of a spatiotemporally defined combination of signals that is decoded by the immune system to activate tumour-specific immune responses (Zitvogel et al, 2010). These signals include the pre-apoptotic exposure of intracellular proteins such as the endoplasmic reticulum (ER) chaperon CRT and the heat-shock protein HSP90 at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1 (Zitvogel et al, 2010). Together, these processes (and perhaps others) constitute the molecular determinants of ICD.In this issue of The EMBO Journal, Garg et al (2012) add hypericin-based PTD (Hyp-PTD) to the list of bona fide ICD inducers and convincingly link Hyp-PTD-elicited ICD to the functional activation of the immune system. Moreover, Garg et al (2012) demonstrate that Hyp-PDT stimulates ICD via signalling pathways that overlap with—but are not identical to—those elicited by anthracyclines, which constitute the first ICD inducers to be characterized (Casares et al, 2005; Zappasodi et al, 2010; Fucikova et al, 2011).Intrigued by the fact that the ER stress response is required for anthracycline-induced ICD (Panaretakis et al, 2009), Garg et al (2012) decided to investigate the immunogenicity of Hyp-PDT (which selectively targets the ER). Hyp-PDT potently stimulated CRT exposure and ATP release in human bladder carcinoma T24 cells. As a result, T24 cells exposed to Hyp-PDT (but not untreated cells) were engulfed by Mf4/4 macrophages and human dendritic cells (DCs), the most important antigen-presenting cells in antitumour immunity. Similarly, murine colon carcinoma CT26 cells succumbing to Hyp-PDT (but not cells dying in response to the unspecific ER stressor tunicamycin) were preferentially phagocytosed by murine JAWSII DCs, and efficiently immunized syngenic BALB/c mice against a subsequent challenge with living cells of the same type. Of note, contrarily to T24 cells treated with lipopolysaccharide (LPS) or dying from accidental necrosis, T24 cells exposed to Hyp-PDT activated DCs while eliciting a peculiar functional profile, featuring high levels of NO production and absent secretion of immunosuppressive interleukin-10 (IL-10) (Garg et al, 2012). Moreover upon co-culture with Hyp-PDT-treated T24 cells, human DCs were found to secrete high levels of IL-1β, a cytokine that is required for the adequate polarization of interferon γ (IFNγ)-producing antineoplastic CD8⁺ T cells (Aymeric et al, 2010). Taken together, these data demonstrate that Hyp-PDT induces bona fide ICD, eliciting an antitumour immune response.By combining pharmacological and genetic approaches, Garg et al (2012) then investigated the molecular cascades that are required for Hyp-PDT-induced CRT exposure and ATP release. They found that CRT exposure triggered by Hyp-PDT requires reactive oxygen species (as demonstrated with the ¹O₂ quencher L-histidine), class I phosphoinositide-3-kinase (PI3K) activity (as shown with the chemical inhibitor wortmannin and the RNAi-mediated depletion of the catalytic PI3K subunit p110), the actin cytoskeleton (as proven with the actin inhibitor latrunculin B), the ER-to-Golgi anterograde transport (as shown using brefeldin A), the ER stress-associated kinase PERK, the pro-apoptotic molecules BAX and BAK as well as the CRT cell surface receptor CD91 (as demonstrated by their knockout or RNAi-mediated depletion). However, there were differences in the signalling pathways leading to CRT exposure in response to anthracyclines (Panaretakis et al, 2009) and Hyp-PDT (Garg et al, 2012). In contrast to the former, the latter was not accompanied by the exposure of the ER chaperon ERp57, and did not require eIF2α phosphorylation (as shown with non-phosphorylatable eIF2α mutants), caspase-8 activity (as shown with the pan-caspase blocker Z-VAD.fmk, upon overexpression of the viral caspase inhibitor CrmA and following the RNAi-mediated depletion of caspase-8), and increased cytosolic Ca²⁺ concentrations (as proven with cytosolic Ca²⁺ chelators and overexpression of the ER Ca²⁺ pump SERCA). Moreover, Hyp-PDT induced the translocation of CRT at the cell surface irrespective of retrograde transport (as demonstrated with the microtubular poison nocodazole) and lipid rafts (as demonstrated with the cholesterol-depleting agent methyl-β-cyclodextrine). Of note, ATP secretion in response to Hyp-PDT depended on the ER-to-Golgi anterograde transport, PI3K and PERK activity (presumably due to their role in the regulation of secretory pathways), but did not require BAX and BAK (Garg et al, 2012). Since PERK can stimulate autophagy in the context of ER stress (Kroemer et al, 2010), it is tempting to speculate that autophagy is involved in Hyp-PDT-elicited ATP secretion, as this appears to be to the case during anthracycline-induced ICD (Michaud et al, 2011).Altogether, the intriguing report by Garg et al (2012) demonstrates that the stress signalling pathways leading to ICD depend—at least in part—on the initiating stimulus (Figure 1). Speculatively, this points to the coexistence of a ‘core'' ICD signalling pathway (which would be common to several, if not all, ICD inducers) with ‘private'' molecular cascades (which would be activated in a stimulus-dependent fashion). Irrespective of these details, the work by Garg et al (2012) further underscores the importance of anticancer immune responses elicited by established and experimental therapies.Open in a separate windowFigure 1Molecular mechanisms of immunogenic cell death (ICD). At least three processes underlie the immunogenicity of cell death: the pre-apoptotic exposure of calreticulin (CRT) at the cell surface, the secretion of ATP, and the post-apoptotic release of HMGB1. ICD can be triggered by multiple stimuli, including photodynamic therapy, anthracycline-based chemotherapy, and some types of radiotherapy. The signalling pathways elicited by distinct ICD inducers overlap, but are not identical. In red are indicated molecules and processes that—according to current knowledge—may be required for CRT exposure and ATP secretion in response to most, if not all, ICD inducers. The molecular determinants of the immunogenic release of HMGB1 remain poorly understood. ER, endoplasmic reticulum; P-eIF2α, phosphorylated eIF2α; PI3K, class I phosphoinositide-3-kinase; ROS, reactive oxygen species.