Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A(Cnp1) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A(Cnp1) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle

Editorial

By their very nature, constitutions are intergenerational documents. With rare exceptions, they are meant to endure for many generations. They establish the basic institutions of government, enshrine the fundamental values of a people, and place certain questions beyond the reach of simple majorities. Constitutions, especially written ones, are often intentionally made difficult to modify. Inevitably, constitutions raise important questions of intergenerational justice. When one generation enshrines its values in a constitution, and makes it difficult to amend the constitution, does it deprive future generations of the sovereignty each generation should be able to exercise? It might well not make a difference if those future generations share the values of their ancestors, but what if they do not? What if future generations see some important provisions of the constitution as not merely inconvenient, but as morally wrong, or even as a threat to their well-being? Of course, if enough people share this view, the constitution can be changed – but what if the division falls short of the supermajority needed to amend the constitution? This is the dilemma created by constitutions, particularly written constitutions which require supermajorities to alter their provisions. In our judgment there is no perfect solution to this dilemma. Rather, every solution represents a balancing of interests and risks. On the one hand, constitutions are valuable precisely because they remove some questions from the hands of electoral majorities. The institutions of government and the basic rights of individuals and communities are among the matters commonly protected by constitutions against the impact of day-to-day politics. Future generations benefit to the extent that constitutions establish just and stable institutions which can adapt and change peacefully to changing needs and circumstances. On the other hand, constitutions, like people, can age poorly. The values enshrined in a nation’s constitution can be ethically wrong when adopted (for example, the protection of the slave trade written into the U.S. Constitution). Time can also demonstrate that some provisions of a constitution are unwise. Technological change may also alter the effects of some provisions. (Consider the difference between the right to bear a 1790 firearm, and the right to bear an automatic weapon in 2010.) And the values of a people can change, too. To some extent, all of these sources of discontent with a nation’s constitution may be inevitable. The framers of a nation’s constitution are not all-wise and all-seeing, and even if they were, the constitution that fits a nation in its youth may be quite different from that which fits it two centuries later. The question, then, is how future generations can adapt to their constitution, and how they can adapt their constitution to their needs. This, in essence, is the problem we posed to the authors who submitted articles for this issue of the Intergenerational Justice Review. How do you balance the importance of placing some questions beyond the control of a simple majority in a written constitution, with the need to preserve for future generations the ability to adapt it to their changing needs? The answers our authors give in this issue of the IGJR vary. Two of them take as their starting point the disagreement between Thomas Jefferson and James Madison concerning the desirability of revising the U.S. Constitution every generation; and another addresses those concerns in the concluding section. Iñigo González-Ricoy’s opening article focuses on the legitimacy of constitutional provisions aimed at advancing future generations’ interests. He argues that the dilemma of future generations being constrained by the choices of their ancestors can be reduced considerably, at least with respect to those constitutional provisions that seek to advance the needs and interests of future generations. Legitimacy concerns may be addressed further through the use of sunset clauses and regular constitutional conventions. Our second article, by Shai Agmon, argues that Jefferson’s proposal that a constitution be re-authorised every 19 years is unsatisfactory because it fails to fulfil its own normative aspirations. It produces two groups of people who will end up living under laws to which they did not give their consent: (a) citizens who reach the voting age after the re-enactment process; (b) citizens who did not assent to being obliged by the majority vote’s results. In Agmon’s view, the existence of significant numbers of citizens who have not consented to the laws undermines any consent-based rationale for adopting a Jeffersonian approach. In our closing article, Michael Rose rejects the Jeffersonian argument that the self-determination of future generations is impeded by lasting constitutions. Rather, he argues that a demand for future generations’ full self-determination is both self-contradictory, and impossible to achieve. Instead, we should employ an attitude of “reflective paternalism” towards future generations by introducing their interests into today’s decision-making process, and by ensuring that the constitution itself provides for democratic self-determination. No doubt, more research is needed on the best ways to incorporate protections for the rights and interests of future generations into constitutions. Future research should also examine how the lessons we have learned from trying to protect the environment can be applied to the circumstances of future generations. The goal is a very practical one: to discover what constitutional provisions can best protect the rights of future generations. Bruce Auerbach (Albright College) Antony Mason (IF) Markus Rutsche (University of St. Gallen) Jörg Tremmel (University of Tübingen

Novel Technique of Craniospinal Axis Proton Therapy with the Spot-Scanning System: Avoidance of Patching Multiple Fields and Optimized Ventral Dose Distribution

Background and Purpose:: Conventional craniospinal irradiation (CSI) is a complex procedure carrying a high risk of adverse side effects. Still, it is indispensable for cure in a number of pediatric brain tumors. In this study, the feasibility and the potential advantage of spot-scanning proton therapy for CSI are investigated. Material and Methods:: A boy (5.5 years of age) with a recurrent medulloblastoma received CSI with a single posterior field using the spot-scanning system at Paul Scherrer Institute. Dose distribution to the targets and the organs at risk, treatment time, reproducibility of patient positioning, toxicity (according to EORTC/RTOG score), and treatment outcome were evaluated. Results:: The plan achieved a homogeneous coverage of the target volume, even using a single field. The doses to the organs ventral to the target were minimized. During treatment, grade 1 skin reaction and grade 2 central nervous system toxicity were observed. After 2 months, the boy presented with a transitory fatigue. After 24 months, he is alive and free of disease. Growth hormones and thyroid hormones are reduced. Conclusion:: These results, based on a single patient, suggest that spot-scanning proton therapy for craniospinal treatment is feasible and safe. By applying a single dorsal field, difficulties of multiple-field patching can be avoided and the ventral dose spread can be minimize

Editorial

The global trend towards greater longevity means that the number of older voters is constantly increasing, and the proportional number of younger voters is decreasing. In many of the world’s democracies, older people vote more consistently and in greater numbers than their younger counterparts. The apparent reluctance of the young to exercise their right to vote only serves to reinforce this demographic trend. The result is that politicians tend to pander to the “Grey Vote”, and young people run the risk of being under-represented in parliament while seeing their issues overlooked by governments. In such a scenario, young people may be easier targets for unpopular government measures, such as the belt-tightening associated with austerity. The statistics make the case. In Germany’s 2013 general election, the average voting turnout was 72.4%. All of the age-cohorts above the age of 45 fell above this average, whereas all of the age-cohorts below 45 fell exactly on or below it. Turnout was highest amongst 60-70 year olds (almost 80%), whereas turnout amongst 18-21 year olds was below 65%. In the United Kingdom, turnout in 2015’s general election among those aged 18 to 24 was at a mere 43%, far below the average turnout of 66.1%. The participatory gap has widened over the decades and its last year’s figures were exceeded only in 2005, when youth turnout was a staggering 24 percentage points below that of the entire population. A recent article in The Economist (23 April 2016) suggests, however, that this is partly due to the fact that most British university students live in short-term accommodation and tend to move frequently, which makes it harder for them to register as voters in the first place. In either of these cases, would lowering the voting age make a difference? In Germany, where 16 year olds are eligible to vote in the local elections of some Länder (federate states), there is some evidence to suggest that a cohort who obtain their voting right at 16 will have a higher poll turnout over the course of their whole lives than a cohort who are not allowed to cast their first vote until a later age. In other words, early participation seems to set a trend for life. One possible way of reducing the median voting age could be the introduction of compulsory suffrage, which already exists in countries such as Belgium, Greece, Luxemburg, Cyprus, and Australia. However, this kind of imposed political legitimacy is seen by some to offend the principles of liberal democracy – even though it need not imply the imposition of legal sanctions against non-voters, as the case of Belgium and others demonstrates. The question of whether the democratic act of voting should be recast from a civic duty to an obligation is multi-faceted and will remain open to discussion for years to come. Additionally, measures to increase the electoral turnout of the younger age groups could aim at making the very act of voting easier, that is, more “user-friendly” – for example through e-voting. At any rate, it is hardly possible for the interests and preferences of a group, even with the very best intentions, to be better identified by a third-party than by the affected group itself. The paternalistic conception that men understand women’s needs better than women themselves, for instance, was successfully rejected by women during their long battle for the suffrage. As John Stuart Mill put it in his Considerations on Representative Government, the rulers and ruling classes are “under a necessity of considering the interests and wishes of those who have the suffrage; but of those who are excluded, it is in their option whether they will do so or not.” Therefore, the very idea of democracy is called into question if any group within it become sidelined, while others are favoured. There will be repercussions for political legitimacy if young people perceive themselves as being left out of the political process; hence remedies are needed to ensure that this does not happen. This issue of the Intergenerational Justice Review addresses the topic from two angles: it asks for the reasons why the electoral turnout of young voters is comparably low in the first place, and it discusses some possible solutions to the problem. In the first of two research articles, Charlotte Snelling asks for the potential of education in raising youth turnout. Aggregate increases in education do little to alter an individual’s relative status within the education system, she argues. Using the 2011 UK Citizens in Transition Survey, she suggests that education affects turnout by determining young people’s positioning within social networks. Some of these networks, however, are more politicised than others. Individuals with relatively lower educational status continue to be excluded from more politically engaged networks – irrespective of their educational attainment – and, as a result, they lack the mobilisation and greater sense of political efficacy required to vote. In short, the simple formula “more education leads to more political interest” turns out to be just that, a simplification. In the second article, Thomas Tozer discusses how to increase electoral turnout among the young. He considers two methods for doing so: compulsory voting and a scheme of financial incentives. The incentive scheme that he prefers would pay young people £30 if they attend an hour-long information session on the election, an hour-long discussion session, and then vote. Tozer argues that this proposed scheme is preferable to compulsory voting because it is more likely to lead young people to deliver reasoned and well-considered votes; and it does so, he holds, without violating individual liberty. In the review section, our authors discuss some of the most recent publications on voting and intergenerational justice. The research articles of this issue are the winning entries to the 2014/2015 Demography Prize, bestowed jointly by the Foundation for the Rights of Future Generations (FRFG) and the Intergenerational Foundation (IF). Please also consider our Call for Papers on “Constitutions and Intergenerational Justice”, printed at the end of this issue. Last but not least, we cordially invite you to visit our newly launched website at www.igjr.org. Whether electronically or in print – we wish you a rewarding and insightful read.  Antony Mason (IF) Hans-Ulrich Kramer (FRFG) Jörg Tremmel (University of Tübingen) Markus Rutsche (University of Tübingen

Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing

Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA, while avoiding the need for problematic template-switching (TS) reactions. Compared with prior efforts, our method involves neither random priming nor stochastic cDNA termination, thus enabling amplification of transcripts that were previously inaccessible via related click chemistry-based RNA sequencing techniques. A key modification involving the use of PCR primers containing two overhanging 3′-nucleotides substantially improved the read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where such modifications typically hinder amplification. This allowed us to more than double the possible insert size compared with the state-of-the art click chemistry-based technique, PAC-seq. Furthermore, our method performed on par with a commercially available PCR-cDNA RNA sequencing kit, as determined by Oxford Nanopore sequencing. Given the known advantages of PAC-seq, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts

Editorial

Among the many headlines that followed the 2018 midterm elections in the United States, one stood out as very good news for political representation: the election of Alexandria Ocasio-Cortez. Irrespective of one’s political leanings – Republican or Democrat – it is an extraordinary achievement for a 29-year-old woman and daughter to a Puerto Rican to be elected into the House of Representatives. Against a predominantly old, male, and white sitting Congress, her election means a step towards better representation of certain demographics in American policy-making processes. Ocasio-Cortez represents a minority around the world, as young-(er) people are notoriously underrepresented in political parties and trade unions – in fact, in political decision-making processes writ large. And this despite the fact that a 29-year-old will have to live a lot longer with the intended and unintended consequences of political decisions taken in 2018. In other words, while younger people are disproportionately affected by political decisions, they are at the same time heavily underrepresented in organisations and processes leading to the decisions. The first prize of the Intergenerational Justice Prize 2017/18 was awarded to Mona Lena Krook and Mary K. Nugent who contribute to this debate by arguing that lowering the eligibility age to run for office leads to better representation of the youngest and next-youngest cohorts in parliament. Drawing on data from 144 countries and 192 parliaments, they first show that the average “waiting period” for citizens – defined as the difference between the legal voting age and the legal age for holding office – is 5.3 years. By combining these data with MPs’ ages from around the world, they are able to show a strong correlation between eligibility age and MPs’ ages. In their words, the results show that “the average impact of reducing the minimum age to stand for office from 25 to 18 would be to increase the proportion of MPs under 45 by over 5 percentage points”. Based on literature on women and young people in politics, the authors attribute these effects to the mobilising character of a lower age requirement. It allows citizens to compete in elections at a younger age and this increases the probability of younger people being represented in parliament. Aksel Sundström and Daniel Stockemer, also among the winners of the Intergenerational Justice Prize 2017/18, take a closer look at the age of parliamentarians and investigate which political parties can help to foster the election of young parliamentarians, and to what extent. After all, political parties act as gatekeepers because they are in control of the list of candidates running for election. Drawing on theories of party politics, the authors argue for five different factors that could possibly explain the share of young MPs: the age of the party leader, the age of the party, the size of party support, the party’s ideology, and the party’s formal recruitment procedure for candidates. In other words, the authors theorise that having a young network, a large outreach, and an ideology or organisational structure that attracts the young should increase a party’s share of young MPs. To test their hypotheses, the authors use data on over 6,000 Members of the European Parliament ever elected and match them with information on party characteristics. Rather surprisingly, their various statistical models show no noteworthy effect of any of the party characteristics on the representation of young MPs in the European Parliament, 1979-2019. Only parties with a more libertarian ideology, as opposed to an authoritarian ideology, are predicted to have somewhat younger MEPs. But the effects are small. This suggests that, irrespective of their individual features, there are hardly any noteworthy differences between parties’ ability to promote the election of young MPs. It follows that other parties may want to adopt other means to accomplish a better representation of the young; the authors’ suggestion of applying youth quotas within parties provides one potential avenue. In the final article by the winners of this year’s Intergenerational Justice Prize, Thomas Tozer discusses the potential of quotas in his normative contribution on the representation of the young. He makes a case for the normative desirability of “descriptive representation” of the young within political parties and trade unions. Specifically, he argues that democracy requires the promoting of substantive equality and people’s substantive interests and that descriptive representation of the young can achieve both. This is because the young have unique concerns that are significant and because their concerns might be affected by representatives’ behaviour. Quotas that require parties and trade unions to enrol a certain share of young members, Tozer argues, might be an option but not ideal because people choose to become members of such organisations. As an alternative, he proposes the creation of incentives for young people to join parties and trade unions, such as free membership. In the book review section, Emily Ford assesses Richard Katz and Peter Mair’s Democracy and the Cartelization of Political Parties (2018, OUP). The book’s central argument is that parties are developing or have already developed into cartels, driven by a desire to maintain their position in the face of declining political participation. They limit political competition between them and try to deter new party entry. Ford’s review overall is positive and she recommends the book to scholars and students as a discussion of the social pressures that parties are exposed to and how they are coping with them. In a second book review, Anna Braam writes about Ian Gough’s Heat, Greed and Human Need. Climate Change, Capitalism and Sustainable Wellbeing (2017, Edward Elgar), a recent study which suggests three steps for countries to accomplish staying below a 1.5°C rise above pre-industrial temperatures: eco-efficient production, changing patterns of consumption, and a reduction of absolute consumption. According to Braam, the book’s interdisciplinary approach – drawing on economic, ecological, political and social aspects of climate change – is convincing, and the book’s argument is credible, especially regarding the rich countries. Ann-Kristin Kölln (Guest editor, Aarhus University, Denmark) Antony Mason (IF) Maria Lenk (FRFG) Jörg Tremmel (University of Tübingen) Markus Rutsche (University of St. Gallen

Charge-state-enhanced ion sputtering of metallic gold nanoislands

Experimental results on the charge-state-dependent sputtering of metallic gold nanoislands are presented. Irradiations with slow highly charged ions of metallic targets were previously considered to show no charge state dependent effects on ion-induced material modification, since these materials possess enough free electrons to dissipate the deposited potential energy before electron-phonon coupling can set in. By reducing the size of the target material down to the nanometer regime and thus enabling a geometric energy confinement, a possibility is demonstrated to erode metallic surfaces by charge state related effects in contrast to regular kinetic sputtering

Dynamic regulation of AtDAO1 and GH3 modulates auxin homeostasis

The hormone auxin is a key regulator of plant growth and development, and great progress has been made understanding auxin transport and signaling. Here we show that auxin metabolism and homeostasis are also regulated in a complex manner. The principal auxin degradation pathways in Arabidopsis include oxidation by AtDAO1/2 and conjugation by GH3s. Metabolic profiling of dao1-1 root tissues revealed a 50% decrease in the oxidation product oxIAA, an increase in the conjugated forms IAA-Asp and IAA-Glu of 438-fold and 240-fold respectively, while auxin remains close to wild type. By fitting parameter values to a mathematical model of these metabolic pathways we show that, in addition to reduced oxidation, both auxin biosynthesis and conjugation are increased in dao1-1. We then quantified gene expression in plantae, and found that transcripts of AtDAO1 and GH3 genes are increased in response to auxin, over different time scales and concentration ranges. Including this regulation of AtDAO1 and GH3 in an extended model reveals that auxin oxidation is more important for auxin homoeostasis at lower hormone concentrations, while auxin conjugation is most significant at high auxin levels. Finally, embedding our homeostasis model in a multicellular simulation to assess the spatial effect of the dao1-1 mutant shows that auxin increases in outer root tissues, in agreement with the dao1-1 mutant root hair phenotype. We conclude that auxin homeostasis is dependent on AtDAO1, acting in concert with GH3, to maintain auxin at optimal levels for plant growth and development