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     

Scientific dissent and public policy

The temptation to silence dissenters whose non-mainstream views negatively affect public policies is powerful. However, silencing dissent, no matter how scientifically unsound it might be, can cause the public to mistrust science in general.Dissent is crucial for the advancement of science. Disagreement is at the heart of peer review and is important for uncovering unjustified assumptions, flawed methodologies and problematic reasoning. Enabling and encouraging dissent also helps to generate alternative hypotheses, models and explanations. Yet, despite the importance of dissent in science, there is growing concern that dissenting voices have a negative effect on the public perception of science, on policy-making and public health. In some cases, dissenting views are deliberately used to derail certain policies. For example, dissenting positions on climate change, environmental toxins or the hazards of tobacco smoke [1,2] seem to laypeople as equally valid conflicting opinions and thereby create or increase uncertainty. Critics often use legitimate scientific disagreements about narrow claims to reinforce the impression of uncertainty about general and widely accepted truths; for instance, that a given substance is harmful [3,4]. This impression of uncertainty about the evidence is then used to question particular policies [1,2,5,6].The negative effects of dissent on establishing public polices are present in cases in which the disagreements are scientifically well-grounded, but the significance of the dissent is misunderstood or blown out of proportion. A study showing that many factors affect the size of reef islands, to the effect that they will not necessarily be reduced in size as sea levels rise [7], was simplistically interpreted by the media as evidence that climate change will not have a negative impact on reef islands [8].In other instances, dissenting voices affect the public perception of and motivation to follow public-health policies or recommendations. For example, the publication of a now debunked link between the measles, mumps and rubella vaccine and autism [9], as well as the claim that the mercury preservative thimerosal, which was used in childhood vaccines, was a possible risk factor for autism [10,11], created public doubts about the safety of vaccinating children. Although later studies showed no evidence for these claims, doubts led many parents to reject vaccinations for their children, risking the herd immunity for diseases that had been largely eradicated from the industrialized world [12,13,14,15]. Many scientists have therefore come to regard dissent as problematic if it has the potential to affect public behaviour and policy-making. However, we argue that such concerns about dissent as an obstacle to public policy are both dangerous and misguided.Whether dissent is based on genuine scientific evidence or is unfounded, interested parties can use it to sow doubt, thwart public policies, promote problematic alternatives and lead the public to ignore sound advice. In response, scientists have adopted several strategies to limit these negative effects of dissent—masking dissent, silencing dissent and discrediting dissenters. The first strategy aims to present a united front to the public. Scientists mask existing disagreements among themselves by presenting only those claims or pieces of evidence about which they agree [16]. Although there is nearly universal agreement among scientists that average global temperatures are increasing, there are also legitimate disagreements about how much warming will occur, how quickly it will occur and the impact it might have [7,17,18,19]. As presenting these disagreements to the public probably creates more doubt and uncertainty than is warranted, scientists react by presenting only general claims [20].A second strategy is to silence dissenting views that might have negative consequences. This can take the form of self-censorship when scientists are reluctant to publish or publicly discuss research that might—incorrectly—be used to question existing scientific knowledge. For example, there are genuine disagreements about how best to model cloud formation, water vapour feedback and aerosols in general circulation paradigms, all of which have significant effects on the magnitude of global climate change predictions [17,19]. Yet, some scientists are hesitant to make these disagreements public, for fear that they will be accused of being denialists, faulted for confusing the public and policy-makers, censured for abating climate-change deniers, or criticized for undermining public policy [21,22,23,24].…there is growing concern that dissenting voices can have a negative effect on the public perception of science, on policy-making and public healthAnother strategy is to discredit dissenters, especially in cases in which the dissent seems to be ideologically motivated. This could involve publicizing the financial or political ties of the dissenters [2,6,25], which would call attention to their probable bias. In other cases, scientists might discredit the expertise of the dissenter. One such example concerns a 2007 study published in the Proceedings of the National Academy of Sciences USA, which claimed that cadis fly larvae consuming Bt maize pollen die at twice the rate of flies feeding on non-Bt maize pollen [26]. Immediately after publication, both the authors and the study itself became the target of relentless and sometimes scathing attacks from a group of scientists who were concerned that anti-GMO (genetically modified organism) interest groups would seize on the study to advance their agenda [27]. The article was criticized for its methodology and its conclusions, the Proceedings of the National Academy of Sciences USA was criticized for publishing the article and the US National Science Foundation was criticized for funding the study in the first place.Public policies, health advice and regulatory decisions should be based on the best available evidence and knowledge. As the public often lack the expertise to assess the quality of dissenting views, disagreements have the potential to cast doubt over the reliability of scientific knowledge and lead the public to question relevant policies. Strategies to block dissent therefore seem reasonable as a means to protect much needed or effective health policies, advice and regulations. However, even if the public were unable to evaluate the science appropriately, targeting dissent is not the most appropriate strategy to prevent negative side effects for several reasons. Chiefly, it contributes to the problems that the critics of dissent seek to address, namely increasing the cacophony of dissenting voices that only aim to create doubt. Focusing on dissent as a problematic activity sends the message to policy-makers and the public that any dissent undermines scientific knowledge. Reinforcing this false assumption further incentivizes those who seek merely to create doubt to thwart particular policies. Not surprisingly, think-tanks, industry and other organizations are willing to manufacture dissent simply to derail policies that they find economically or ideologically undesirable.Another danger of targeting dissent is that it probably stifles legitimate crucial voices that are needed for both advancing science and informing sound policy decisions. Attacking dissent makes scientists reluctant to voice genuine doubts, especially if they believe that doing so might harm their reputations, damage their careers and undermine prevailing theories or policies needed. For instance, a panel of scientists for the US National Academy of Sciences, when presenting a risk assessment of radiation in 1956, omitted wildly different predictions about the potential genetic harm of radiation [16]. They did not include this wide range of predictions in their final report precisely because they thought the differences would undermine confidence in their recommendations. Yet, this information could have been relevant to policy-makers. As such, targeting dissent as an obstacle to public policy might simply reinforce self-censorship and stifle legitimate and scientifically informed debate. If this happens, scientific progress is hindered.Second, even if the public has mistaken beliefs about science or the state of the knowledge of the science in question, focusing on dissent is not an effective way to protect public policy from false claims. It fails to address the presumed cause of the problem—the apparent lack of understanding of the science by the public. A better alternative would be to promote the public''s scientific literacy. If the public were educated to better assess the quality of the dissent and thus disregard instances of ideological, unsupported or unsound dissent, dissenting voices would not have such a negative effect. Of course, one might argue that educating the public would be costly and difficult, and that therefore, the public should simply listen to scientists about which dissent to ignore and which to consider. This is, however, a paternalistic attitude that requires the public to remain ignorant ‘for their own good''; a position that seems unjustified on many levels as there are better alternatives for addressing the problem.Moreover, silencing dissent, rather than promoting scientific literacy, risks undermining public trust in science even if the dissent is invalid. This was exemplified by the 2009 case of hacked e-mails from a computer server at the University of East Anglia''s Climate Research Unit (CRU). After the selective leaking of the e-mails, climate scientists at the CRU came under fire because some of the quotes, which were taken out of context, seemed to suggest that they were fudging data or suppressing dissenting views [28,29,30,31]. The stolen e-mails gave further ammunition to those opposing policies to reduce greenhouse emissions as they could use accusations of data ‘cover up'' as proof that climate scientists were not being honest with the public [29,30,31]. It also allowed critics to present climate scientists as conspirators who were trying to push a political agenda [32]. As a result, although there was nothing scientifically inappropriate revealed in the ‘climategate'' e-mails, it had the consequence of undermining the public''s trust in climate science [33,34,35,36].A significant amount of evidence shows that the ‘deficit model'' of public understanding of science, as described above, is too simplistic to account correctly for the public''s reluctance to accept particular policy decisions [37,38,39,40]. It ignores other important factors such as people''s attitudes towards science and technology, their social, political and ethical values, their past experiences and the public''s trust in governmental institutions [41,42,43,44]. The development of sound public policy depends not only on good science, but also on value judgements. One can agree with the scientific evidence for the safety of GMOs, for instance, but still disagree with the widespread use of GMOs because of social justice concerns about the developing world''s dependence on the interests of the global market. Similarly, one need not reject the scientific evidence about the harmful health effects of sugar to reject regulations on sugary drinks. One could rationally challenge such regulations on the grounds that informed citizens ought to be able to make free decisions about what they consume. Whether or not these value judgements are justified is an open question, but the focus on dissent hinders our ability to have that debate.Focusing on dissent as a problematic activity sends the message to policy-makers and the public that any dissent undermines scientific knowledgeAs such, targeting dissent completely fails to address the real issues. The focus on dissent, and the threat that it seems to pose to public policy, misdiagnoses the problem as one of the public misunderstanding science, its quality and its authority. It assumes that scientific or technological knowledge is the only relevant factor in the development of policy and it ignores the role of other factors, such as value judgements about social benefits and harms, and institutional trust and reliability [45,46]. The emphasis on dissent, and thus on scientific knowledge, as the only or main factor in public policy decisions does not give due attention to these legitimate considerations.Furthermore, by misdiagnosing the problem, targeting dissent also impedes more effective solutions and prevents an informed debate about the values that should guide public policy. By framing policy debates solely as debates over scientific facts, the normative aspects of public policy are hidden and neglected. Relevant ethical, social and political values fail to be publicly acknowledged and openly discussed.Controversies over GMOs and climate policies have called attention to the negative effects of dissent in the scientific community. Based on the assumption that the public''s reluctance to support particular policies is the result of their inability to properly understand scientific evidence, scientists have tried to limit dissenting views that create doubt. However, as outlined above, targeting dissent as an obstacle to public policy probably does more harm than good. It fails to focus on the real problem at stake—that science is not the only relevant factor in sound policy-making. Of course, we do not deny that scientific evidence is important to the develop.ment of public policy and behavioural decisions. Rather, our claim is that this role is misunderstood and often oversimplified in ways that actually contribute to problems in developing sound science-based policies.? Open in a separate windowInmaculada de Melo-MartínOpen in a separate windowKristen Intemann     

Evolution and control of H5N1

The evolutionary dynamics of the H5N1 virus present a challenge for conventional control measures. Efforts must consider the regional aspects of endemic H5N1.The H5N1 virus has spread across Asia, Europe and Africa, and has infected birds in several endemic areas, including China, Indonesia, Vietnam and Egypt. H5N1 outbreaks pose a massive threat for the poultry industry and, ultimately, for human health [1]. However, the rapid spread of the virus also offers the opportunity to study and learn from its dynamics in the wild. The insights gained should inform new public health policies and preventive actions against a possible pandemic.Progress in influenza research has been impressive. In particular, the application of reverse genetics has led to the identification of mutations and reassortment changes that determine virus virulence. Perhaps the most significant results come from the two now infamous studies, published in Nature and Science, about the generation of recombinant H5N1 viruses that are transmissible in ferrets [2,3]. These advances show that we are steadily elucidating influenza virus at the molecular level. By contrast, our understanding of the dynamics of highly pathogenic influenza virus in the environment remains limited [4,5].Highly pathogenic avian influenza (HPAI) is an important poultry disease. The major reservoir of the virus is wild waterfowl, and infected birds are usually asymptomatic as a result of long-term evolutionary adaptation [1,6]. After transmission from wild waterfowl to poultry, however, avian influenza viruses occasionally become highly pathogenic and can cause mortalities of up to 100% within 48 h of infection. The standard method for controlling an HPAI outbreak is the testing and culling of all infected poultry, and the setting up of a concentric control area around the infected flock.The HPAI H5N1 virus, circulating in Eurasia and Africa, emerged in China around 1997 [1] but it only infected terrestrial birds at the time. Continuous transmission in poultry eventually allowed the virus to evolve, resulting in large outbreaks in China in 2005 with high mortality in wild waterfowl. The virus spread rapidly, probably though migratory birds, to Central Asia, Europe, the Middle East and Africa. Such ‘east to west'' movements of H5N1 viruses over comparably long distances have not since been recorded. Moreover, migrating wildfowl have begun to spread the virus intermittently between Asia and Siberia [7]. This H5N1 lineage is the longest-circulating HPAI virus that has been reported, and it has reached epizootic levels in both domestic and wild bird populations.…the challenge is to understand the evolution of H5N1 to better predict new strains that could become a serious threat for human healthOne of the striking characteristics of the H5N1 lineage, in contrast with other HPAI, is its infectivity toward mammals. H5N1 can be directly transmitted from birds to humans and cause severe disease, although it has a significantly lower transmissibility than seasonal influenza viruses [1]. So far, 608 cases of human H5N1 infections have been reported with 59% mortality [5]. Most human infections have resulted from close contact with H5N1-infected poultry or poultry products, and no sustained human–human transmission has as yet been documented. Nonetheless, a potential H5N1 pandemic remains a great concern for public health.The viruses that caused the five influenza pandemics since 1900 arose by two mechanisms: reassortment among avian, human and swine influenza viruses, and accumulation of mutations in an avian influenza virus [1,8]. Triple reassortment between avian H5N1, swine H3N1 and H1N1 viruses, and double reassortment between avian H5N1 and H9N2 viruses has already been reported in Asia, which raises concerns about new reassortment viruses that could infect humans [9,10]. Meanwhile, research has identified some 80 genetic mutations that could increase infectivity of avian influenza viruses in mammals, and thus potentially facilitate avian influenza evolution to generate a pandemic strain [8,11]. H5N1 strains with some of these mutations have often been found in bird populations [5] and in human H5N1 strains [12]. Indeed, specific mutations that could confer switching in receptor-binding specificity were reported in H5N1-infected patients in Thailand [13]. The two controversial studies published in Nature and Science also showed how a handful of mutations might enable the H5N1 virus to be transmitted between humans [2,3]. Pathogenic variants of the H5N1 virus with a higher pandemic potential could naturally evolve; the challenge is to understand the evolution of H5N1 to better predict new strains that could become a serious threat for human health.…continuous replication of H5N1 virus in Egypt has provided a valuable opportunity to study the impact of genetic evolution on phenotypic variation without reassortmentThe evolutionary dynamics of the Egyptian H5N1 strains provide clues to understanding the pandemic potential of H5N1. The virus was introduced only once in Egypt, in early 2006, and spread among a variety of bird species, including chickens, ducks, turkeys, geese and quail [14]. The virus rapidly evolved to form a phylogenetically distinct clade that has since diverged into multiple sublineages [15]. Thus, continuous replication of H5N1 virus in Egypt has provided a valuable opportunity to study the impact of genetic evolution on phenotypic variation without reassortment.After diversification in local bird populations, some new H5 sublineages have emerged in Egypt with a higher affinity for human-type receptors. Indeed, since their emergence in 2008, almost all human H5N1 strains in Egypt have been phylogenetically grouped into these new sublineages, which can be transmitted to humans with a higher efficacy than other avian influenza viruses. This might explain why, since 2009, Egypt has had the highest number of human cases of H5N1 infection, with more than 50% of the cases worldwide [5]. Fortunately, these Egyptian H5N1 sublineages still do not have binding affinity for receptors in the upper respiratory tract and, therefore, do not sustain transmission in humans. However, it increases the risk of H5N1 variants that are better adapted to humans after viral replication in infected patients.…Egypt is regarded as the country with the highest H5N1 pandemic potential worldwideThe Egyptian H5N1 sublineages are also diversifying antigenically in the field, as some are no longer crossreactive to other co-circulating sublineages [15]. Moreover, faint traces of species-specific evolutionary changes have been detected [16], implying a change in their host species. It shows that the H5N1 virus has undergone significant diversification in Egypt during the past seven years. Of greater concern, however, are Egyptian H5N1 strains that carry mammalian influenza virus type PB2 and have lost the N-linked 158 glycosylation site in the top region of haemagglutinin [15,17], both of which can potentially facilitate viral transmission to humans. The genetic diversification of H5N1 virus in Egypt represents an increasing pandemic potential, and Egypt is regarded as the country with the highest H5N1 pandemic potential worldwide [18].A similar situation exists in other geographical areas. Multiple clades and sublineages of H5N1 are co-circulating in Asia, occasionally enabling reassortment events within and beyond the viral subtypes in the field [19,20]. Several H5N1 strains with enhanced binding affinity to human-type receptors have been reported in Indonesia [12]. Similarly, avian and swine H5N1 strains with an altered receptor-binding preference have been isolated sporadically in Indonesia and Laos [21,22]. As in other areas, distinct groups of H5N1 viruses are circulating amongst themselves and with other avian influenza viruses, generating diverse viral phenotypes in nature. The evolutionary dynamics of H5N1 might even accelerate in the wild. H5N1 viruses diverge genetically in ducks [23]; they can transfer the virus over long distances by migration. Thus, the H5N1 virus has established a complex life cycle in nature with accelerated evolutionary dynamics. The pandemic threat of H5N1 remains a serious concern and might be increasing.Control measures based on isolating and culling are still the gold standard for controlling the early phase of an H5N1 outbreak, and worked against the H5N1 outbreaks in Hong Kong in 1997 and in Thailand in 2004 [4]. However, this measure failed in several countries and made H5N1 endemic. Cross-border circulation of H5N1 further complicates implementation of a classical control strategy based on culling in the infected area.In response, public health officials in several countries, including Egypt and Indonesia, advocate poultry vaccination as a preventive or adjunct control measure [1]. Although vaccination does not completely prevent infections, its proper use can help to control avian influenza outbreaks by reducing virus transmission from infected animals. However, it can also increase vaccine-driven evolution among avian influenza viruses. The endemic status of H5N1, which can cause devastating local epidemics, puts pressure on health officers to use a vaccine or a vaccination strategy that might eventually increase selective pressure and thereby accelerate H5N1 evolution. Given the high mutability and diversity of circulating viruses, it seems best to avoid using a vaccine based on a strain from a different geographical area because there would only be a partial antigen match; such a heterologous vaccine would only be effective in the short term compared with a homologous vaccine. During past control of H5N1 epidemics using imported vaccines, escape mutants have emerged within about a year of the start of vaccination, which made the epidemic even worse [14]. When a vaccination strategy is implemented in an endemic area, the vaccine seed strain should be selected from the same geographical area to try to get the longest possible protection. Vaccine seed virus selection must be periodically revised to produce well-matched and efficacious vaccines.Close communication and workshops hold the greatest potential for controlling the H5N1 virusIn most cases, H5 vaccine for an endemic area comes from a foreign supplier. It would be necessary to enable foreign manufacturers to produce customized H5 vaccines based on epidemic strains from different areas. The best approach might be a plasmid-based reverse genetics system to construct vaccine seed viruses [1]. In egg-based production, which is the basis of flu vaccine production, the seed virus needs to be adapted for high growth. This time-consuming step carries the risk of antigenic changes during vaccine production. Yet, advances in influenza reverse genetics have led to the development of cell culture systems to produce recombinant viruses, which would enable rapid genetic mutagenesis and reassortment. Once reverse genetics generates a virus genome that is well adapted to growth in cell culture, the haemagglutinin and neuraminidase genes can be easily interchanged with those of other influenza viruses. In addition, virus growth in cell culture can shorten production time, which increases the probability of selecting a seed virus antigenically appropriate for the upcoming flu season, and enables a rapid increase in production if necessary [24].A control strategy imposed without consideration of regional customs will not be successfulGiven the zoonotic risks of influenza viruses to both humans and animals, the establishment of a vaccine production system applicable to both human and animal infections is an urgent issue. The capacity of vaccine production needs to be flexible for seasonal, pre-pandemic and pandemic vaccines. Advances in genetic engineering facilitate in vitro control of human- and avian-type receptor expression on cultured cells, which should allow both human and avian influenza viruses to grow in the same system. As vaccine production capacity based on cell culture develops, commercial production of H5N1 vaccines tailored to each geographical area should become possible. In addition, emergency vaccination guidelines, such as pre-pandemic vaccine stockpiling, expanding and accelerating vaccine production and setting vaccination priorities, should be formulated in a business–government partnership, to ensure pandemic preparation. There is no guarantee that the H5N1 virus will be the next pandemic influenza strain. However, exploring options for versatile vaccine manufacturing is a key to controlling zoonotic influenza viruses, including H5N1.The complexity of H5N1 ecology also makes control of endemic H5N1 by vaccination a complex task. The problem is that antigenically different groups of viruses, which are not crossreactive, are often co-circulating in endemic areas. Circulation of viruses in each sublineage is not restricted in terms of geography or host species, which complicates efforts to use a vaccine produced against antigens from a single virus strain [15]. Of greater concern, H5N1 virus infects a variety of bird species [1], which means the vaccination targets have expanded. Bird species differ in their optimal vaccination protocol—for example, the single vaccination used routinely in chickens does not induce an adequate immune response in turkeys, which require multi-dose vaccination at an older age [25]. Furthermore, rearing many bird species and their hybrid breeds in uncontrolled confinement is common in H5N1 endemic countries, especially in rural areas. Therefore, the immunogenicity of existing vaccines is probably inadequate to protect all target species with a single vaccination scheme. Endemic H5N1 already forces public health officials to redefine vaccine development policy to improve both vaccine immunogenicity and vaccination regime.Unfortunately, it is unlikely that science will ever produce a clear answer as to when, where and how the next pandemic influenza virus will emergeToday, there are numerous techniques that could overcome these problems by increasing immunogenic potency and crossreactivity. Innovative vaccine formats—multivalent, universal, nasal and synthetic vaccines—possibly coupled with the use of adjuvants, could improve the global vaccine supply [24]. These new technologies should be applied as soon as possible. Nevertheless, no single technique can probably resolve the underlying complexity of H5N1 dynamics. Over-reliance on vaccination might therefore only worsen the situation. Vaccination can help control endemic H5N1 only when administered as part of an integrated control programme that includes surveillance, culling, restricting host movement and enhanced quarantine and biosecurity.The complex evolutionary dynamics of the H5N1 virus are challenging host species barriers and the ecology brings H5N1 into close proximity to humans [1]. The close link between the virus and humans is a multifaceted phenomenon that can affect health in myriad ways. Thus, we need to redefine control strategies to address the nature of H5N1 dynamics. Surveillance is the basis of infection control in the field. Wild birds and their predators should be included as surveillance targets, thereby expanding the H5N1 host species range. Another drawback is the fact that epidemiological studies focus mainly on virus genotyping. Although genetic data is informative, the diversity of H5N1 viruses makes characterization based only on genetic traits difficult. Characterization of viral phenotypes—antigenicity, receptor-binding preference, pathogenicity and transmissibility—is equally important for investigating the evolutionary dynamics of H5N1 viruses in nature. We would need techniques to determine easily viral phenotype, in particular new rapid diagnostic systems that can be used for timely epidemiological investigations and rapid infection control measures [1]. For example, portable kits that can determine virus receptor specificity would allow field testing of whether a particular avian influenza virus strain has adapted to human-type receptors, thereby adding a new dimension for characterizing and assessing H5N1 outbreaks.Our perception of H5N1 control should change from short-term hunting to long-term controlThe large-scale slaughter of all known and suspected infected birds in H5N1 endemic countries is hugely expensive in terms of execution costs and compensation for lost poultry. Financial assistance from international organizations might be needed to promote the thorough implementation of such a policy. However, H5N1 endemic countries are not all poor nations and some have already built a certain level of technology infrastructure. Thus, transfer of epidemiological skills and concepts to local health officers and scientists is a priority. Overseas collaborations between technologically developed countries and their institutions, and H5N1 endemic countries and their institutions, should be established at a functional level. Close communication and workshops hold the greatest potential for controlling the H5N1 virus. Such projects supported by governments and funding agencies would encourage establishment of bilateral and multilateral relationships between developed countries and the developing countries, which are the epicentres of H5N1 outbreaks. Sharing information about risk and risk management is one of the key methods for reducing the threat of future H5N1 epidemics.Globalization has had major benefits for international travel and trade, and sharing of information. The improvements in information technology have dramatically increased the speed and ease of data flow [26]. Intelligence networks facilitate instantaneous sharing of information and enable global warnings about potential hazards as well as problem-solving. Moreover, collaborative research centres, which have been established on reciprocal bases between scientifically advanced countries and institutes and overseas partner countries and institutes in Asia, Africa and Latin America, are important players in information networking—for instance the Institute Pasteur Network, the Mahidol Oxford Tropical Medicine Research Unit and Japan Initiative for Global Research Network on Infectious Diseases. Linking such laboratory-based networks should be the next step. This would have a profound synergistic effect by maximizing research capacity, human resources and geographic coverage to build a robust global-scale network for infection control.However, regional socio-cultural issues can be a significant concern for virus control wherever accepted values and scientific understanding might differ. Multiple local and regional factors—customs, religion, politics and economics—can affect H5N1 control in an area. Successful implementation of an H5N1 control strategy depends largely on mutual understanding and consideration of local idiosyncrasies.Some examples from Egypt show how regional identity can be closely linked with local public health initiatives. Egypt is an Islamic nation and bird meat is an important source of animal protein, and the only source in some rural areas [14]. A large proportion of Egyptian households in rural areas raise poultry. Although broiler and layer chickens are raised under modern hygienic controls on commercial farms, backyard birds are raised in open uncontrolled farms, leaving them free to interact with other birds (Fig 1A). The poultry meat trade depends mainly on live bird markets in traditional bazaars (Fig 1B), because of a preference for freshly slaughtered poultry. Pigeon towers are built on farms, backyards and roofs throughout villages to raise pigeons for eating. Generally, birds in Egypt are raised in proximity to humans (Fig 1C), which presents an increasing risk of human H5N1 infection in Egypt and establishment of endemic H5N1 in birds nationwide.Open in a separate windowFigure 1Socio-cultural traditions in rearing birds for food in Egypt. (A) Free rearing of backyard birds. (B) Live birds at a downtown market. (C) An example of the intertwined relationship between birds and humans.Such regional identity is inseparable from socio-cultural contexts, making fundamental change virtually impossible. Although there are many scenarios in which a local public health system could be improved by food safety standards and veterinary inspection or short-term closing of live bird markets for virus clearance, H5N1 control measures have to be implemented whilst respecting the intrinsic socio-cultural traditions in the region. A control strategy imposed without consideration of regional customs will not be successful. It is the local health officers and scientists who are best suited to address the enormous complexity and breadth of issues required for H5N1 control. They also experience H5N1 outbreaks in their area on a regular basis and have a great incentive to be involved in infection control. Therefore, it is important to include local expertise in planning and implementing a control strategy.Science in an area such as infectious disease research can no longer be viewed as independent of societal needs…Science is frequently looked at as if it can produce a ‘silver bullet'' to solve every problem. Early success in vaccine and antibiotic development also created a false sense of optimism that scientific methods could eliminate the risk of infection. However, the reality has turned out to be different—some infectious diseases remain uncontrollable and far from eradication. Given the mutable and diversifying nature of avian influenza viruses, there is a significant possibility that different avian influenza subtypes and strains do not follow a single evolutionary pathway. Unfortunately, it is unlikely that science will ever produce a clear answer as to when, where and how the next pandemic influenza virus will emerge. Our perception of H5N1 control should change from short-term hunting to long-term control. The ecology of H5N1 virus brings it into close proximity to humans. The most important strategy is to minimize contact between terrestrial poultry and wild waterfowl to segregate H5N1 in poultry, because H5N1 spread would be uncontrollable if it established a stable equilibrium in waterfowl. For example, H5N1 viruses in Siberia have not been consistently isolated each year from carcasses and faeces of wildfowl migrating from Asia [7]. This implies that H5N1 circulation in the wild still largely depends on occasional introduction from poultry. It is possible that trials to limit H5N1 infection in poultry would lead to a reduction in viral spread and a dwindling evolutionary path in nature. Infection control policy must abandon fixed strategies in favour of flexible ones to keep pace with the evolutionary dynamics of pathogens such as H5N1 (Fig 2).Open in a separate windowFigure 2Changing dynamics of H5N1 virus in the field. Endemic H5N1 virus diversifies in nature, making traditional control measures extremely difficult.Today''s infection control strategy is becoming largely dependent on the reliability and accuracy of information networking. However, the vast flood of scientific information can hide erroneous information and easily mislead the public [26]. Of greater concern, globalization has prompted the centralization of capital and resources, which can lead to an overemphasis on certain research topics. As a consequence, research projects are often short term, without consideration of effects that might have a long-term social impact [27]. This has led to a debate about whether to limit publication of certain types of research or keep scientific information completely accessible. There is probably no easy answer to this. Our global society needs a more mature approach to support research projects that are accurate reflections of societal needs in public health. At the same time, the increasing links between science and society put more pressure on science to play a greater role in society. This is a serious dilemma—how to use science to solve societal problems whilst maintaining its autonomy [27]. Science in an area such as infectious disease research can no longer be viewed as independent of societal needs; we need to establish a balance between the pursuit of independent basic research and its application for solving clinical problems and crises.? Open in a separate windowYohei WatanabeOpen in a separate windowKazuyoshi IkutaOpen in a separate windowMadiha S Ibrahim     

A structural road map to unveil basal body composition and assembly

EMBO J 31 3, 552–562 (2012); published online December132011The Basal Body (BB) acts as the template for the axoneme, the microtubule-based structure of cilia and flagella. Although several proteins were recently implicated in both centriole and BB assembly and function, their molecular mechanisms are still poorly characterized. In this issue of The EMBO journal, Li and coworkers describe for the first time the near-native structure of the BB at 33 Å resolution obtained by Cryo-Electron Microscopy analysis of wild-type (WT) isolated Chlamydomonas BBs. They identified several uncharacterized non-tubulin structures and variations along the length of the BB, which likely reflect the binding and function of numerous macromolecular complexes. These complexes are expected to define BB intrinsic properties, such as its characteristic structure and stability. Similarly to the high-resolution structures of ribosome and nuclear pore complexes, this study will undoubtedly contribute towards the future analysis of centriole and BB biogenesis, maintenance and function.The microtubule (MT)-based structure of the cilium/flagellum grows from the distal part of the Basal Body (BB), which in many animal cells develops from the mature centriole in the centrosome. Electron microscopic (EM) images of chemically fixed resin-embedded centrioles and basal bodies (CBBs) suggest that their ultrastructure is similar, and that their key components are MTs. The mechanisms underlying the organization of CBB MTs, comprising highly stable closed and open MTs, are likely to hold many surprises as they are remarkably different from other microtubular structures in the cell. Additionally, non-MT-based structures are also part of the CBB, including a cartwheel in the proximal lumen region that reinforces CBB symmetry (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011).Several centriole components and BB proteins were identified by comparative and/or functional genomics and proteomics studies of purified CBBs (reviewed in Azimzadeh and Marshall, 2010 and Carvalho-Santos et al, 2011). Advances in our understanding of the molecular mechanisms of CBB assembly depend on high-resolution comparative studies of wild-type (WT) and mutant structures, as well as characterization of the localization of molecular complexes within the small CBB structure. Despite the existence of beautiful ultrastructure data acquired from chemically fixed specimens (Geimer and Melkonian, 2004; Ibrahim et al, 2009), high-resolution structures of native CBBs were missing. Using electron cryo-tomography and 3D subtomogram averaging, Li et al (2012) solved the structure of the near-native BB triplet at 33 Å resolution. A pseudo-atomic model of the tubulin protofilaments at the core of the triplets was built by fitting the atomic structure of α/β-tubulin monomers into the BB tomograms.The 3D density map reveals several additional densities that represent non-tubulin proteins attached, both internally and externally, to all triplet MTs, some linking MTs inside the triplets and/or MTs in consecutive triplets (Li et al, 2012; for a summary, see Li et al, 2012; Geimer and Melkonian, 2004; Ibrahim et al, 2009), but with less detail and complexity. The authors speculate that some of the additional densities present at the A- and B-tubule inner wall might correspond to proteins of the tektin family, probably conferring rigidity to the BB triplet (Amos, 2008).

Table 1

Characteristics of the non-α/β-tubulin structures reported in Li et al (2012) in this issue of The EMBO journal

The health risks of ART

Assisted reproductive technologies enable subfertile couples to have children. But there are health risks attached for both mothers and children that need to be properly understood and managed.Assisted reproductive technology (ART) has become a standard intervention for couples with infertility problems, especially as ART is highly successful and overall carries low risks [1,2]. The number of infants born following ART has increased steadily worldwide, with more than 5,000,000 so far [3]. In industrialized countries, 1–4% of newborns have been conceived by using ART [4,5], probably owing to the fact that couples frequently delay childbearing until their late 30s, when fertility decreases in both men and women [2]. Considering the possibility that male fertility might be declining, as Richard Sharpe has discussed in this series [6], it is likely that ART will be even more widely used in the future. Yet, as the rate of ART and the total number of pregnancies has increased, it has become apparent that ART is associated with potential risks to the mother and fetus. The most commonly cited health problems pertain to multiple gestation pregnancies and multiple births. More recently, however, concerns about the risks of birth defects and genetic disorders have been raised. There are questions about whether the required manipulations and the artificial environments of gametes and embryos are potentially creating short- and long-term health risks in mothers and children by interfering with epigenetic reprogramming.Notwithstanding, ART represents a tremendous achievement in human reproductive medicine. The birth of Louise Brown, the first ‘test tube baby'' in 1978, was the result of the collaborative work of embryologist Robert Edwards and gynaecologist Patrick Steptoe [7]. This success was a culmination of many years of work at universities and clinics worldwide. An initial lack of support, as well as criticism from ethicists and the church, delayed the opening of the first in vitro fertilization (IVF) clinic in Bourn Hall near Cambridge until 1980. By 1986, 1,000 children conceived by IVF at Bourn Hall had been born [8]. In 2010, Edwards received the Nobel Prize in Medicine for the development of IVF. Regrettably, Steptoe had passed away in 1988 and could not share the honour.…as the rate of ART and the total number of pregnancies has increased, it has become apparent that ART is associated with potential risks to mother and fetusOver the next decades, many improvements in IVF procedures were made to reduce the risks of adverse effects and increase success rates, including controlled ovarian stimulation, timed ovulation induction, ultrasound-guided egg retrieval, cryopreservation of embryos and intracytoplasmic sperm injection (ICSI)—a technique in which a single sperm cell is injected into an oocyte using a microneedle. In addition, there were further improvements such as assisted hatching and in media composition, such as sequential media, which allow the in vitro culture of the embryo to reach the blastocyst stage [8].Current IVF procedures involve multiple steps including ovarian stimulation and monitoring, oocyte retrieval from the ovary, fertilization in vitro and embryo transfer to the womb. Whereas the first IVF cycles, including the conception of Louise Brown, used natural ovulatory cycles, which result in the retrieval of one or two oocytes, most IVF cycles performed today rely on controlled ovarian stimulation using injectable gonadotropins—follicle stimulating hormone and luteinizing hormone—in supraphysiological concentrations for 10–14 days, followed by injection of human chorionic gonadotropin (hCG) 38–40 h before egg retrieval to trigger ovulation. This updated protocol makes it possible to grow multiple follicles and to retrieve 10–20 oocytes in one IVF cycle, thereby increasing the number of eggs available for fertilization.Post-retrieval, the embryologist places an egg and sperm together in a test tube for fertilization. Alternatively, a single sperm cell can be injected into an egg by using ICSI. This procedure was initially developed for couples with poor sperm quality [9], but has become the predominant fertilization technique used in many IVF clinics worldwide [8]. The developing embryos are monitored by microscopy, and viable embryos are transferred into the woman''s womb for implantation. Louise Brown, as with many embryos today, was transferred three days after egg retrieval, at approximately the eight-cell stage. However, using sequential media, many clinics advocate culturing embryos until day five when they reach the blastocyst stage. The prolonged culture period allows self-selection of the most viable embryos for transfer and increases the chance of a viable pregnancy. Excess embryos can be cryopreserved and transferred at a later date by using a procedure known as frozen embryo transfer (FET). In this article we use the term ART to refer to IVF procedures with or without ICSI and FET.

Science & Society Series on Sex and Science

Sex is the greatest invention of all time: not only has sexual reproduction facilitated the evolution of higher life forms, it has had a profound influence on human history, culture and society. This series explores our attempts to understand the influence of sex in the natural world, and the biological, medical and cultural aspects of sexual reproduction, gender and sexual pleasure.Embryos can also be screened for chromosomal aneuploidies—missing or extra chromosomes—by preimplantation genetic diagnosis (PGD) when indicated and when available. PGD can also be used to test fertile couples at increased risk of genetic disorders. To perform PGD, a single cell is obtained from three-day-old embryos for molecular testing, for example sequencing for inherited monogenic disorders or fluorescent in situ hybridization for chromosomal abnormalities [8]. Only embryos with a normal chromosomal constitution, and without the genetic disorder in question, would then be transferred into the woman''s womb.Despite tremendous progress during the past three decades, people undertaking ART still face a considerable risk of failure to achieve parenthood. The rate of clinical pregnancies in Bourn Hall between 1980 and 1985 was 24% and 14% in women younger and older than 40 years, respectively [10]. The reported rates for clinical pregnancies and live births vary by country; the average delivery rate is 22.4%, 23.3% and 17.1% for IVF, ICSI and FET cycles, respectively [11]. According to the last Centers for Disease Control and Prevention report in 2009, the average live-birth rate was 35% per fresh ART cycle, although it sharply declines with age, from 45% among women younger than 35 years to 7% among women older than 42 years [5]. The reasons include poor response to ovarian stimulation, ovarian hyperstimulation syndrome and failure of eggs to fertilize. However, these failures occur in only a minority of patients and the success rate of egg retrieval and fertilization leading to embryo transfer is a remarkable 90% [12].Implantation remains the least understood process and is a key rate-limiting step in ART. Poor embryo quality is considered to be the main cause of implantation failure and it reflects a high incidence of chromosomal aneuploidies, which increases with maternal age [13]. One obvious solution to improve implantation rates is to transfer more embryos. However, this also increases the risk of multiple births, and related morbidity and mortality in newborns. An alternative approach is to select for good-quality embryos by culturing them to the blastocyst stage, because it seems that aneuploid embryos arrest by this stage and that blactocysts are more likely to have a normal chromosomal complement. There is ongoing research aimed at identifying viable embryos through PGD and metabolic profiling [13].Despite tremendous progress during the past three decades, people undertaking ART still face a considerable risk of failure to achieve parenthoodIt has also been suggested that failure to implant could be caused by the inability of the embryo to hatch out of a glycoprotein layer surrounding the embryo, known as the ‘zona pellucida''; this layer hardens if the embryo is cultured or frozen. Assisted hatching by rupturing the zona pellucida before embryo transfer does increase clinical pregnancy rates, especially for thawed embryos [13]. Another factor linked to the failure of implantation is endometrial receptivity. The endometrium consists of multi-layered mucosa cells in the inner wall of the uterus, which undergoes coordinated remodelling during the menstrual cycle and there is a specific time window when it is receptive to embryo implantation. Several research studies have identified molecular biomarkers of poor endometrial receptivity, showing that prostaglandins, cell adhesion molecules, mucins and cytokines are important [13].When it comes to health risks for mothers and infants, the use of ART increases the risk of multiple births, including higher rates of caesarian sections, prematurity, low birth weight, infant death and disability. More recently, concerns regarding elevated risks of birth defects, genetic abnormalities, neurodevelopmental disorders and imprinting disorders have been reported; however, not all are substantiated. There are still many unanswered questions regarding the potential short- and long-term health risks of ART for women and children, and there are tremendous challenges in studying the safety of ART procedures. Apart from the subset of individuals undergoing ART for social reasons—single parents or same sex couples—most patients are subfertile couples. Subfertility, defined as a failure to conceive naturally after 12 months of unprotected intercourse, affects 8–20% of couples [2], and it can occur for a variety of unknown or known reasons including maternal factors—endocrine, hormonal, endometriosis and blocked fallopian tubes—and paternal factors such as spermatogenesis abnormalities.Most studies have assessed the risks of ART by comparing the outcomes of ART-conceived pregnancies to naturally conceived pregnancies. There is emerging evidence that underlying maternal or paternal subfertility might be an important factor in obstetric, neonatal and childhood outcomes in the ART population. Therefore, to determine the specific health risks associated with the ART process itself, the outcomes of ART-conceived pregnancies should be assessed in comparison with naturally conceived pregnancies in subfertile parents, which is methodologically difficult. Alternatively, studying the health risks of ART in fertile couples—for instance, same-sex couples and couples at risk of genetic disorders—would be informative, but the number of such couples is relatively small.Women who undergo ART are at risk of ovarian hyperstimulation syndrome (OHSS). OHSS is a complication of ovulation induction resulting in enlargement of ovaries and retention of fluids leading to various secondary complications, which normally resolve within two weeks, but can persist if pregnancy occurs. Patients with OHSS can be offered embryo cryopreservation and frozen embryo transfer when symptoms resolve. Moderate forms of OHSS occur in 5% of patients undergoing ART; 2% of patients require hospitalization. Death occurs with an incidence of approximately 3 per 100,000 ART cycles [14]. OHSS is predominantly caused by human chorionic gonadotropin injection used for inducing final oocyte maturation and ovulation. Research is focused on optimizing alternative stimulation protocols [14].The use of supraphysiological concentrations of hormones during ovarian stimulation has also raised concerns that ART can increase cancer risks linked to hormonal fluctuations. These include breast, ovarian, endometrial, cervical and colon cancers, as well as melanoma. Studies evaluating the risks of cervical cancers, colon cancers and melanoma have not demonstrated increased risks for women undergoing ART [1]. The data for breast, ovarian and endometrial cancer is more complex, however, and more research is required to conclusively determine whether there is an increased risk.The perinatal and obstetric risks of ART are most significantly influenced by multiple pregnancies. These are at a more than 60% risk of low birth weight or premature delivery [2], and related risks of pregnancy complications such as gestational diabetes, abnormal placentation and hypertensive disorders [1]. Multiple pregnancies occur in 1% of naturally conceived pregnancies and 25–50% of ART pregnancies, owing to multiple embryo transfer. In the Western world, about 30–50% of all twin pregnancies result from ART [2]. Whilst double or triple embryo transfer is still common, the development of cryopreservation techniques and extended blastocyst culture has increased the use of single embryo transfer (SET), especially for younger women. Many European countries and the province of Quebec, in Canada, where ART is publicly funded, have adopted a policy of SET, which has dramatically decreased the incidence of multiple pregnancies. In Belgium and Quebec, SET policies have reduced multiple pregnancies from 19% to 3% and from 27% to 6%, respectively. It has been argued that SET results in a lower live-birth rate than a double-embryo transfer, but this is almost completely overcome by an additional single frozen embryo cycle [2].…there are tremendous challenges in studying the safety of ART proceduresThe question of whether ART increases the risks of pregnancy complications, including prematurity and low birth weight in singletons, remains unresolved; several studies have found an increased risk, but others have not replicated these findings [1,2]. It has been suggested that the fertility history of patients undergoing ART is an important factor, as there is an association between the length of time to conception and prematurity and birth weight [15]. Prematurity and low birth weight are also known to be associated with long-term health effects, including adult onset coronary artery disease, hypertension, obesity and type 2 diabetes [16,17].Various studies have also reported a higher incidence of congenital anomalies in ART-conceived children, with a suggested 30% increase of malformations [2]. However, this is another risk that might be attributable to parental subfertility, as a study comparing children conceived by ART to subfertile parents and children conceived naturally to subfertile parents did not find any significant difference in the congenital anomaly rate [2]. Findings from another study of the risks of birth defects in children conceived naturally to women with and without a history of subfertility compared with children conceived with the assistance of ART also suggest that it is subfertility, rather than ART, that is associated with an increased risk of birth defects [18].Several studies reported an increased risk of cerebral palsy and other neurological abnormalities in children conceived by ART [2]. But again, these findings are mainly attributed to complications resulting from multiple pregnancies including prematurity and low birth weight. The increased utilization of SET is therefore expected to result in fewer multiple pregnancies, which should result in a concomitant decrease in neurological complications. Further evidence that neurological complications in ART children are not exclusively related to ART came from studies that have assessed neurodevelopmental outcomes, such as locomotion, cognition, language and behavioural development of ART children in comparison with naturally conceived children. These analyses did not reveal any differences when adjusted for confounding factors of low birth weight and prematurity. In a similar vein, numerous studies have investigated whether there is an increased incidence of autism in ART-conceived children, but these have been inconclusive [19].There are potential concerns regarding the fertility of ART children. However, this requires future studies as most of this population is younger than 30 years of age. There is some evidence that boys conceived through ICSI have an increased rate of genital anomalies [2] and that males with severe infertility, such as low sperm counts, are more likely to carry chromosomal abnormalities, which could be passed on to their children conceived through ICSI [15].It has also been suggested that there might be an increased risk of cancers in ART-conceived offspring. Although multiple studies have identified no such risk, a large Swedish study reported a marginally increased risk of cancer, including haematologic, eye, nervous system, solid tumours and histiocytosis [2]. Similarly to other ART-related adverse health outcomes, it has been suggested that the increased risk of cancer could be attributed to prematurity, a recognized risk factor for cancer, rather than to the ART procedure itself. Further long-term studies are required to determine if there is truly an increased risk of adult cancers in ART offspring.…there remain unanswered questions about both the health risks associated with ART and the potential mechanisms that could account for these findingsOne thing is clear from the available evidence to date: there remain unanswered questions about both the health risks associated with ART and the potential mechanisms that could account for these findings. One possible explanation is that the exposure of gametes and preimplantation embryos to the various steps of ART might affect growth and development of offspring through dysregulation of epigenetic pathways [20]. In addition, there is evidence that genetic and epigenetic alterations might be inherited from the gametes of subfertile parents, which would reinforce assertions that subfertility itself might play a role in ART-related health outcomes [1,20].Epigenetics refers to heritable changes in gene expression without alterations to the underlying DNA sequence. DNA methylation and modifications of histones are epigenetic modifications that determine active against repressive conformation of chromatin structure, thereby regulating gene expression and driving essential processes such as embryonic development, fetal organ development, cell differentiation and tissue-specific gene expression [21]. Genomic imprinting is a type of epigenetic gene regulation that uses epigenetic marks to silence specifically one of the parental alleles. There are approximately 100 known imprinted genes in humans [22]. Most imprinted genes are found in clusters across the genome and are regulated by parent-specific DNA methylation and histone modification marks at cis-acting imprinting centres, as well as non-coding RNAs. Most of the known imprinted genes have functions related to growth and behaviour; disruption of the normally programmed parental expression of imprinted genes can therefore result in disorders related to growth and neurodevelopment.Gametogenesis and embryogenesis are important stages of mammalian development that require genome-wide epigenetic reprogramming. During spermatogenesis, protamines replace most histone proteins to create a highly compacted DNA. Establishment of DNA methylation imprints at paternally methylated imprinting centres is complete in males at the time of birth. In females, the establishment of maternally methylated imprinting centres begins during puberty and is almost complete in ovulated oocytes. After fertilization, the paternal genome undergoes rapid active DNA demethylation in which protamines are replaced by histones, whilst the maternal genome is passively demethylated, so that DNA methylation patterns are lost through cell divisions. Although, the whole genome undergoes demethylation, parent-specific DNA methylation is maintained at imprinting centres. Subsequently, the genome is remethylated and cell-type-specific epigenetic patterns are established as embryonic development proceeds. The parent-specific DNA methylation at imprinting centres is maintained in somatic cells, but it is erased and re-established in the gametes starting a new cycle of imprinting (Fig 1; [23]). As the establishment and maintenance of imprinting marks coincides in timing with important stages of ART, such as oocyte maturation under supraphysiological hormone concentrations and embryo culture, it has been proposed that ART can lead to imprinting errors [24].Open in a separate windowFigure 1Life cycle of genomic imprinting and assisted reproductive technology. Erasure, re-establishment and maintenance of genomic imprinting occur during gametogenesis and preimplantation embryo development. Blue and red solid lines show paternal and maternal methylation at imprinting centres through gametogenesis and early stages of preimplantation development. Imprinting marks are erased at early stages of gametogenesis. Re-establishment of imprinting occurs throughout gametogenesis, but finishes much later in oocytes compared with sperm. During preimplantation development, both maternal and paternal imprinting marks are maintained whilst the rest of the genome is demethylated. The paternal genome is demethylated rapidly and actively (dashed blue line) whilst the maternal genome is demethylated at a slower rate passively through cell division (dashed red line). Various steps of assisted reproductive technology such as ovarian stimulation, ovulation induction, gamete and embryo manipulation and culturing create unusual environments for gametes and embryos and thus, can interfere with proper establishment of imprinting marks in oocytes or maintenance of imprinting marks in embryos. Subfertility can be associated with epigenetic errors in imprinting erasure and/or establishment in both oocytes and sperm. Adapted from [23].In 2001, the first evidence that genomic imprinting can be perturbed during ART procedures came from studying sheep fetuses derived from in vitro cultured embryos that presented with large offspring syndrome (LOS; [25]). LOS occurs sporadically in cattle and sheep conceived by IVF and is characterized by a 20–30% increase in birth weight frequently accompanied by congenital anomalies and placental dysfunction [24]. Owing to phenotypic similarities of LOS to the human overgrowth disorder Beckwith–Wiedemann syndrome (BWS), which is caused by the dysregulation of gene expression within an imprinted cluster on chromosome 11p15.5, the authors hypothesized that genes from the orthologous cluster in sheep or a closely related pathway could be dysregulated in LOS. They tested expression of the insulin-like growth factor 2 (IGF2) gene known to be overexpressed in BWS, and the IGF2R receptor gene, which is involved in clearance of IGF2 from the circulation. IGF2R is imprinted in sheep but not in humans. In sheep with LOS, no differences for IGF2 were found, but reduced expression of IGF2R was observed after loss of DNA methylation at the imprinting centre for this gene [25].In the following decade, several studies provided further evidence that children conceived by ART might be at increased risk of imprinting disorders. The strongest case has been made for BWS and Angelman syndrome. BWS is the most common human overgrowth syndrome characterized by prenatal and postnatal overgrowth, congenital anomalies and tumour predisposition [26]. Angelman syndrome is a neurodevelopmental disorder characterized by microcephaly, severe intellectual disability and a unique behavioural profile including frequent laughter, smiling and excitability [27]. Multiple case reports from various countries indicate an increased frequency of BWS and Angelman syndrome in ART children (3–10-fold) compared with the general population. However, two cohort studies failed to replicate this association [28]. The low incidence of both BWS (1 in 13,700) and Angelman syndrome (1 in 15,000) in the general population [28] makes epidemiological studies difficult—the two cohort studies reported 2,492 and 6,052 ART children, respectively, and are probably underpowered to detect an increased risk of BWS and Angelman syndrome. However, even if there might be increased relative risks for these syndromes in ART children, the absolute risks in this population remain low.The molecular causes of BWS and Angelman syndrome are heterogeneous. They include genomic (deletion, uniparental disomy and gene mutation) and epigenetic (loss of imprinting due to aberrant DNA methylation) alterations at imprinted gene clusters on chromosomes 11p5.5 and 15q11–q13, respectively. These alterations occur with specific frequencies for each of the two disorders [26,27]. Results of molecular testing in children with these syndromes and conceived using ART, reveal an excess of epigenetic compared with genetic molecular alterations. For example, loss of DNA methylation at imprinting centre 2 occurs in about 50% of BWS cases in the general population, whereas several studies found loss of DNA methylation at imprinting centre 2 in 96% (27/28) of BWS ART-conceived children. In Angelman syndrome, approximately 3% of cases in the general population have loss of methylation at 15q11–13, whereas 5 out of 19 (26%) Angelman syndrome children conceived by ART or naturally by parents with a history of subfertility had loss of DNA methylation at 15q11–13 (Fig 2).Open in a separate windowFigure 2Enrichment of epigenetic alterations in Beckwith–Wiedemann syndrome and Angelman syndrome after assisted reproductive technology. Loss of methylation (LOM) at imprinting centre 2 (IC2) on chromosome 11p15.5 contributes to 50% of Beckwith–Wiedemann syndrome (BWS) cases in the general population, whereas LOM at IC2 is found in 27 out of 28 cases (96%) in the BWS assisted reproductive technology (ART) population, which represents a 1.9-fold enrichment of this epigenetic defect. For Angelman syndrome (AS), methylation disruption at the 15q11–q13 imprinting centre contributes to 3% of AS cases, and in the AS ART and subfertility population it was found in 5 out of 19 cases (26%; eight fold enrichment). Data from the following publications were used for these calculations, BWS [31,32,33,34,35] AS [35,36].The data for loss of DNA methylation in Angelman syndrome cases conceived naturally by subfertile parents highlights the fact that epigenetic alterations could, at least in part, result from underlying parental subfertility. Indeed, several studies have shown that abnormalities of spermatogenesis, such as oligospermia (low sperm concentration), low sperm motility or abnormal sperm morphology are associated with altered DNA methylation at imprinted loci. These occur in both maternal and paternal alleles of imprinting centres in sperm and could be transmitted to offspring conceived by ART [26]. One study of chromosomally normal fetuses spontaneously aborted at six to nine weeks of gestation found that DNA methylation alterations at imprinted loci were sometimes inherited from sperm. Thus, it is possible that this dysregulation of imprinting in male gametes might be one cause of the association between imprinting disorders and ART.Studies of other known imprinted syndromes, such as Prader–Willi syndrome, Russell–Silver syndrome, maternal and paternal uniparental disomy of chromosome 14, pseudohypoparathyroidism type 1b and transient neonatal diabetes mellitus, have either not demonstrated an association with ART or have been inconclusive owing to their small size [29]. A link has also been suggested between ART and the newly defined ‘multiple maternal hypomethylation syndrome'', which clinically presents either as BWS or transient neonatal diabetes mellitus, and is associated with loss of DNA methylation at multiple maternally methylated imprinting centres; loss of methylation at paternal imprinting centres has not been reported so far. Thus, human imprinting disorders that have been observed with increased relative frequency in ART offspring are confined to loss of DNA methylation at maternally methylated imprinting centres, similar to epimutations of IGF2R in LOS. One could propose that ART has a greater impact on female than male gametes, as the eggs are subjected to more environmental exposures—supraphysiological doses of hormones—and more manipulation than the sperm. However, studies of mouse in vitro cultured embryos and ART-exposed human and mouse gametes suggest that ART can also be associated with either loss or gain of DNA methylation on both maternal and paternal alleles [23].Mouse models are a valuable method to investigate which stages of ART procedures can disrupt normal imprinting patterns. The advantage of using mouse models is the ability to investigate each of the parameters of ART—ovulation stimulation and embryo culturing—separately and at different stages of development. Furthermore, mouse models allow investigators to alter ART parameters, such as concentration of hormones or media for embryo culturing. Most importantly, studies in animal models have shown that ART procedures without the confounding factor of subfertility do have a negative impact on imprint regulation [23].The exposure of maturing oocytes from mice to abnormally high doses of gonadotropins has been suggested to alter imprint establishment. Yet, studies performed directly on superovulated oocytes are inconclusive, as not all of them have demonstrated increased rates of DNA methylation errors at imprint centres compared with spontaneously ovulated oocytes. Interestingly, studies of DNA methylation in mouse blastocysts harvested from superovulated mothers identified an increased rate of DNA methylation errors at imprint centres. This included loss of DNA methylation at the paternally methylated H19—the imprinting centre on human chromosome 11 and mouse chromosome 7 implicated in BWS and the related undergrowth Russell–Silver syndrome. It suggests that superovulation also impairs imprinting maintenance; probably by affecting the ability of the oocyte to synthesize and store sufficient maternal factors (RNA and proteins; [23]). In support of this hypothesis, four maternal effect proteins have been previously identified that are involved in imprinting maintenance in preimplantation embryos. It was also found that imprint errors arise in blastocysts in a dose-dependent manner—higher doses of hormones resulted in DNA methylation errors in a larger number of embryos [23].As the establishment and maintenance of imprinting marks coincides in timing with important stages of ART […] it has been proposed that ART can lead to imprinting errorsAnother factor that might contribute to imprinting errors is the micromanipulation of gametes during IVF and ICSI procedures. Evidence supporting this hypothesis includes the observation in mouse models that a higher number of IVF embryos—resulting from superovulation alone or superovulation and embryo culturing—have aberrant H19 DNA methylation compared with in vivo conceived embryos [23]. Media with varying compositions are used in ART clinics, and whilst all of the media are suboptimal for normal maintenance of all DNA imprints in mouse embryos, the number of embryos with aberrant DNA methylation at imprinting centres varies depending on the media [23]. Interestingly, it was also found that embryos with faster rates of development are more prone to loss of DNA methylation at imprinting centres [23].Though it is not yet clear how these findings relate to ART in humans, the mouse research is crucial for informing human studies about which variables should be addressed to optimize the safety and efficacy of ART procedures. Apart from ART itself, it has been shown that compromised fertility in mice results in loss or delay of DNA methylation acquisition in one of three tested imprinted genes. The compromised fertility is induced by genetic manipulation of a gene involved in communication between oocytes and surrounding follicular cells, which is crucial for proper oocyte maturation. The results suggest that the observed loss of DNA methylation could be caused by impaired transport of metabolites from follicular cells to oocytes, which is important for imprint establishment [23].Data linking dysregulation of imprinted loci and ART is limited to several imprinted gene clusters associated with clinically recognizable syndromes. However, there are more genes in the human genome that have been discovered to be, or are predicted to be, imprinted [22] but are not yet known to be associated with clinical phenotypes. Potentially, ART can lead to dysregulation of these imprinted genes, which might be another, as yet unrecognized factor contributing to neonatal and long-term health problems of ART-conceived children. At this point, it is also not clear whether epigenetic disruption during ART is limited to imprinted genes or has more global effects on the genome. The data for genome-wide DNA methylation analysis are limited in both human and mouse to individuals with no apparent disease phenotype. So far, these data have been inconclusive [23,28].One could propose that ART has a greater impact on female than male gametes, as the eggs are subjected to more environmental exposures […] and more manipulation than the spermDespite significant advances in the efficacy and success of ART procedures during the past few decades, the health risks, especially related to long-term outcomes in ART-conceived children, remain poorly understood. Moreover, the phenomena known as ‘fetal programming''—when maternal and in utero exposures can lead to various adult onset disease susceptibilities—have been suggested to be transmissible to the next generations, probably through epigenetic mechanisms [30]. In the case of ART procedures, the effect of ‘unusual'' environments during gametogenesis and early embryonic development on adult-onset disease and trans-generational inheritance is still not clear. Additional research is needed to elucidate the effects of ART on genome-wide epigenetic patterns and their link to human disease. As ART will continue to be an important medical intervention and the number of children born with the help of ART procedures will probably continue to rise in the future, it is crucial to understand the associated health risks and underlying molecular mechanisms of these technologies. This will increase the safety of this intervention and enable couples using ART to be fully informed regarding both present and future health-related risks.? Open in a separate windowDaria GrafodatskayaOpen in a separate windowCheryl CytrynbaumOpen in a separate windowRosanna Weksberg     

Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase

EMBO J 32 15, 2099–2112 doi:10.1038/emboj.2013.125; published online May312013Mutations in Parkin represent ∼50% of disease-causing defects in autosomal recessive-juvenile onset Parkinson''s disease (AR-JP). Recently, there have been four structural reports of autoinhibited forms of this RING-IBR-RING (RBR) ubiquitin ligase (E3) by the Gehring, Komander, Johnston and Shaw groups. The important advances from these studies set the stage for the next steps in understanding the molecular basis for Parkinson''s disease (PD).Regulated protein degradation requires that E3s and their access to substrates be exquisitely controlled. RBR family E3s provide striking examples of this regulation. The complex and compact structures of Parkin (Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) as well as another RBR E3, human homologue of Ariadne (HHARI) (Duda et al, 2013), demonstrate extraordinarily intricate inter-domain arrangements. These autoinhibited structures ensure that their functions are restricted until activated.Until recently, RBR E3s were believed to be a subclass of RING E3s, which allosterically activate E2 conjugated with ubiquitin (E2∼Ub). However, Wenzel et al (2011) determined that they are actually hybrid E3s, containing an E2 binding site in RING1 and a catalytic cysteine residue in the domain designated as RING2. The catalytic cysteine is an acceptor for an ubiquitin from RING1-bound E2∼Ub forming an intermediate (E3∼Ub) that leads to substrate or autoubiquitination. In this way, RBRs resemble HECT E3s, which also form catalytic intermediates in ubiquitination. There are 13 human RBR family E3s. Besides Parkin, two notable RBRs are HOIL-1 and HOIP, which form part of a complex integral to NF-κB activation (Wenzel and Klevit, 2012).In addition to causal roles in AR-JP, single allele mutations of Parkin are found in some sporadic cases of PD (references in Wauer and Komander, 2013). Mutations in the Parkin-associated kinase PINK1, which is upstream of Parkin, also account for a significant number of AR-JP cases (Hardy et al, 2009; Narendra et al, 2012; Lazarou et al, 2013). A number of diverse Parkin substrates have been postulated to be associated with PD. There is substantial evidence that one role for Parkin is at mitochondria. Once activated and recruited to damaged/depolarized mitochondria by PINK1, it ubiquitinates exposed mitochondrial proteins leading to both proteasomal degradation and mitophagy (Narendra et al, 2012; Sarraf et al, 2013). Parkin has also been implicated in cell surface signalling and as a tumour suppressor (see references in Wauer and Komander, 2013).Parkin encodes five structured domains, beginning with an N-terminal ubiquitin-like domain (UbLD) and followed by four domains that each bind two zinc (Zn) atoms (Figure 1A). The most N-terminal of the Zn-binding domains is RING0. C-terminal to this is the RBR, consisting of RING1, the IBR and RING2. The crystal structures of inactive Parkin from Riley et al (2013), Trempe et al (2013) and Wauer and Komander (2013) show remarkable congruity. Spatially, the IBR is at the complete opposite end of the molecule from RING2, to which it is connected by a partially unstructured ∼37 residue linker. This linker includes a two-turn helix, referred to as the repressor element of Parkin (REP) or tether, which binds and occludes the E2 binding face of RING1. RING1 occupies the central position in these structures, and RING0 separates RING1 from RING2 (Figure 1B and C). The latter contains the residue identified by Wenzel et al (2011), and confirmed by all three groups, to be the catalytic cysteine, C431. A lower resolution structure also includes the UbLD and places this domain adjacent to RING1 (Trempe et al, 2013). A second unstructured linker connects the UbLD and RING0. UbLDs are involved in a number of protein–protein interactions and small angle X-ray scattering confirms that this domain is integral to the core structure of Parkin (Spratt et al, 2013; Trempe et al, 2013). Biophysical characterization of Parkin and HHARI suggests that each is a monomer in solution.Open in a separate windowFigure 1Schematic and spatial representation of Parkin. (A) Primary structure and domain designations of Parkin, including the REP sequence within the otherwise unstructured IBR-RING2 linker. (B) Structural representation of full-length Parkin (PDB 4K95) highlighting the complex domain interactions in the three-dimensional structure, the catalytic C431 residue, and residue W403 within the REP, which plays a role in stabilizing the autoinhibited form of Parkin. (C) A model of Parkin with the E2 UbcH5B/Ube2D2 bound (devised using PDB 4K95 and PDB 4AP4 to mimic the position of an E2 bound to RING1) to illustrate the required displacement of UbLD and REP and the large distance between the E2∼Ub attachment site of the E2 and the catalytic active site of Parkin. Note that in this conformation the catalytic Cys within RING2 (C431) remains buried by RING0.RING1 is the only bona fide RING domain. All NMR and crystal structures of IBR domains from Parkin, HHARI and HOIP (PDB ID: 2CT7) are in good agreement. The Parkin and HHARI RING2s are structurally highly homologous and share a common Zn-coordinating arrangement with IBR domains. In contrast to the IBR and RING2, RING0 has a distinct arrangement of Zn-coordinating residues (Beasley et al, 2007; Duda et al, 2013; Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) (see Figure 1F of Trempe et al (2013) for the various Zn coordination arrangements).All of the Parkin crystal structures represent inactive forms of the E3. This is imposed by the quaternary positioning of the domains, which precludes activity in multiple ways. RING0 plays two obvious roles to maintain Parkin in an inactive state. RING0 shares an interface with RING2 and buries C431, making it unavailable as an ubiquitin acceptor. Moreover, RING0 intervenes between RING1 and RING2, creating an insurmountable separation of >50 Å between the active site Cys of an E2 bound to RING1 and C431 (Figure 1B and C). Thus, RING0 must be displaced for ubiquitin transfer to occur. Accordingly, deletion of RING0 results in a marked increase in Parkin autoubiquitination and in C431 reactivity (Riley et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013). In HHARI, these two inhibitory functions are fulfilled by the C-terminal Ariadne domain, which similarly interposes between RING1 and RING2 (Duda et al, 2013).Additional inhibition is provided by the REP, which binds to RING1 at the canonical RING-E2 binding site and prevents E2 binding. This provides at least a partial explanation for the impaired ability of Parkin to bind E2 when compared to HHARI, which lacks this element (Duda et al, 2013). A disease-associated REP mutant (A398T) at the RING1 interface increases autoubiquitination (Wauer and Komander, 2013). The significance of inhibition by REP-RING1 binding was verified by mutating a critical RING1-interacting REP residue (W403A). This increased autoubiquitination and E2 binding (Trempe et al, 2013). Consistent with the requirement for charging C431 with ubiquitin in mitochondrial translocation (Lazarou et al, 2013), Parkin association with depolarized mitochondria is accelerated with this mutation (Trempe et al, 2013). Interestingly, W403 also interacts with the C-terminal Val of Parkin within RING2, and could therefore potentially further stabilize the autoinhibited form of the protein (Riley et al, 2013), consistent with previous observations (Henn et al, 2005).The quaternary structure of full-length Parkin also suggests that displacement of its N-terminal UbLD must occur for full activation (Trempe et al, 2013). The positioning of the UbLD adjacent to RING1 indicates that it would provide a steric impediment to E2∼Ub binding (Figure 1B and C). Additionally, displacement of the UbLD could be important to relieve interactions with the IBR-RING2 linker, which, as suggested in a previous study (Chaugule et al, 2011), might help to maintain Parkin in an inactive state. Finally, the crystal structure of the full-length Parkin indicates that the UbLD is not available for interactions with other proteins. This would limit Parkin''s range of intermolecular interactions.RBR E3s have at least two domains critical for sequential ubiquitin transfer and full activity, RING1 and RING2. The RING1 of Parkin, as well as all other RBR E3s, is notable in lacking the basic residue in the second Zn coordinating loop (or its equivalent in U-box proteins), which has recently been implicated in RING-mediated transfer of Ub from E2∼Ub (Metzger et al, 2013). This suggests that other factors play compensatory roles in positioning ubiquitin for transfer from E2∼Ub to C431. A non-mutually exclusive possibility is that the lack of this basic residue in RING1 limits unwanted attack on the E2∼Ub linkage, thereby minimizing the unregulated ubiquitination. Turning to RING2, the area surrounding the active site C431 of Parkin is notable in that it includes a sequence recognizable as a catalytic triad, similar to that in deubiquitinating enzymes. The Cys-His-Glu grouping, found in Parkin and other RBR E3s, contributes to in vitro activity (Riley et al, 2013; Wauer and Komander, 2013). Interestingly, however, the Glu was dispensable in a cellular assay (Riley et al, 2013). This triad is conserved in HHARI, where an Asn between the Cys and His residues (found in a number of RBRs but not conserved in Parkin), was found to be important for catalysis (Duda et al, 2013).The advances made in these studies impart significant information about an important and clinically relevant E3. However, Parkin, as well as HHARI, has been captured in their inactive, unmodified forms. One obvious question is how does Parkin transition between inactive and active states. PINK1 is implicated in phosphorylating Parkin on its UbLD and potentially other sites, with evidence that phosphorylation contributes to Parkin activation (Narendra et al, 2012). How phosphorylation could contribute to protein interactions that might facilitate Parkin activation, potentially including Parkin oligomerization (Lazarou et al, 2013), is unknown. Regardless, it is evident that considerable unwinding of its quaternary structure must take place.While there is much work ahead to understand these processes, one important interface that must be disrupted for activation is that between the REP and RING1. It is intriguing to consider that such interruption might be associated with other alterations in the IBR-RING2 linker, potentially facilitating the movement of the UbLD from RING1 and contributing to activation. Related to activation is the all-important question of how Parkin recognizes and targets specific substrates. While the UbLD represents a potential site of interaction, most purported substrates are not known to have UbLD-interaction domains. Although interactions involving the UbLD could occur indirectly, through bridging molecules, there is also evidence that other regions of Parkin, including the RBR region, might recognize substrates either directly or indirectly (Tsai et al, 2003) and that some substrates may be phosphorylated by PINK1 (Narendra et al, 2012). Conformational changes induced by substrate interactions, particularly in the IBR RING2 linker, could, as above, represent an important aspect of activation.There are over 75 missense mutations of Parkin associated with AR-JP, most of these inactivate the protein, but there are also some that are activating (Wauer and Komander, 2013). Activating mutations presumably result in pathology at least partially as a consequence of increased autoubiquitination and degradation (e.g., A398T). The current studies help to provide a classification of missense mutations into those that affect (i) folding or stability, (ii) catalytic mechanism, and (iii) interactions between domains. Interdomain mutations might inactivate or contribute to constitutive activation leading to autoubiquitination and degradation.Finally, we know little about how the autosomal recessive and the much more prevalent sporadic forms of PD overlap in their molecular pathology. However, mitochondrial dysfunction is increasingly a common theme. Thus, with the structure of the inactive protein in hand, there is hope that we can begin to consider ways in which domain interactions might be altered in a controlled manner to activate, but not hyperactivate, this critical E3 and lessen the progression of PD.     

Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.