The role of amino acids as substrates for protein synthesis is well documented. However, a function for amino acids in modulating the signal transduction pathways that regulate mRNA translation has only recently been described. Interesting, some of the signaling pathways regulated by amino acids overlap with those classically associated with the cellular response to hormones such as insulin and insulin-like growth factors. The focus of this review is on the signaling pathways regulated by amino acids, with a particular emphasis on the branched-chain amino acid leucine, and the steps in mRNA translation controlled by the signaling pathways. The response of translation initiation to a change in amino acid and/or hormone availability can be general, i.e., affecting the translation of most if not all mRNAs, and/or specific, i.e., affecting the translation of a single class or subset of mRNAs. Both the general and specific responses can be mediated through regulation of either the met-tRNAi and/or mRNA binding steps. The specific response may also involve an additional regulatory site, i.e., the phosphorylation status of ribosomal protein rpS6, one of the proteins composing the 40S ribosomal subunit. Learning how the cell recognizes a sufficiency of amino acids is presently the objective of intense research. Present evidence, however, suggests multiple recognition sites and multiple signaling pathways. Below, we summarize our current knowledge of the signaling pathways known to respond to changes in amino acid availability. In addition, the translation initiation factors and mRNA structural elements that are involved in changes in both global and specific modulation of mRNA translation are discussed.Recent advances in biomedical research reveal a key role for amino acids as nutritional signals in the regulation of a number of cellular processes. Studies employing a variety of cell types and different tissues demonstrate that one such process affected is the regulation of gene expression through modulation of the translation of messenger RNA (mRNA). The studies show that cells recognize changes in amino acid availability and generate alterations in signal transduction pathways that are also regulated by hormones and growth factors. The cells then respond to the integrated signaling input by either upregulating or downregulating translation initiation, i.e., the process during which initiator methionyl-tRNA  and internal ribosome entry sites (IRESs). Phosphorylation of eIF2α is mediated by any of four known eIF2α kinases in mammalian cells: the mammalian ortholog of the yeast general control non-derepressing kinase-2 (mGCN2), the heme-regulated inhibitor (HRI), the protein kinase dsRNA-activated (PKR), and the PKR-like endoplasmic reticulum kinase  and livresponse . Howeverresponse . A recenresponse . Thus, iHRI was first identified in rabbit reticulocytes and shown to be activated in response to hyperoxia and iron and heme deficiency . SubsequPKR is a ubiquitously expressed serine-threonine protein kinase that is activated by double-stranded RNA and is induced by interferon [reviewed in ]. PKR isi essentially halts cell growth and triggers apoptosis . Morrsk . TheAlthough eIF2α phosphorylation is one mechanism for enhancing the translation of mRNAs containing an IRES element(s), it is not unique. For example, during apoptosis or infection by certain types of viruses, eIF4G is cleaved. The normal function of eIF4G is to assemble the translation initiation factors eIF4A and eIF4E and the poly(A) binding protein into a complex that mediates the binding of mRNA to the 40S ribosomal subunit. Cleavage of eIF4G during apoptosis or viral infection separates the binding domain for PABP and the mRNA cap binding protein, eIF4E, from the domains that bind eIF4A and allow ribosome attachment (referred to as the middle fragment of eIF4G or M-FAG). A recent study reported that M-FAG generated in etoposide-treated cells M-FAG promotes the preferential translation of certain, but not all, IRES-containing mRNAs including Apaf-1 and death-associated protein (DAP)-5 . MoreoveAnother structural element within the 5'-UTR of some mRNAs that is involved in selective mRNA translation is an oligopyrimidine tract, referred to as a TOP sequence, immediately downstream of the 5'-cap structure ,69. MessMost mRNAs that are efficiently translated, e.g. GAPDH and β-actin, have 5'-UTRs that are short (<200 nt), have a low content of G and C residues, and are relatively unstructured . In contSecondary structure may also function as a cis-acting regulatory element through the binding of specific trans-acting factors. A well-characterized example of such regulation is the modulation of ferritin and δ-aminolevulinate (ALA) synthase mRNA translation in response to changes in iron availability . Both thNutrients, and in particular certain amino acids, play important roles in the control of gene expression through their ability to modulate the initiation phase of mRNA translation. All essential amino acids have the potential to globally regulate mRNA translation through the eIF2α kinase mGCN2. In addition, changes in eIF2α phosphorylation can selectively modulate the translation of mRNAs encoding particular proteins if the 5'UTR of the mRNA contains uORFs and/or IRES elements. Selective control of mRNA translation can also occur through changes in signaling through mTOR. Activation of S6K1 by mTOR leads to phosphorylation of rpS6 and eIF4B which is thought to promote preferential translation of TOP mRNAs and mRNAs with highly structured 5'-UTRs, respectively. In addition, mTOR phosphorylates the eIF4E binding proteins leading to enhanced assembly of the eIF4F complex. In combination with eIF4B phosphorylation, enhanced eIF4F assembly leads to preferential translation of mRNAs with highly structured 5'-UTRs. Although other amino acids have been shown to increase signaling through mTOR, leucine is arguably the most potent of the amino acids in activating the pathway.Both authors contributed equally to the writing of this manuscript.None declared.


In this paper I will describe some of the sentinel events in Aboriginal and Torres Strait Islander health policy and strategy during 2003 and the early part of 2004. This will involve discussion on the:• National Strategic Framework in Aboriginal and Torres Strait Islander Health• National Strategic Framework for Aboriginal and Torres Strait Islander Peoples Mental Health and Social and Emotional Well Being 2004–2009• National Aboriginal and Torres Strait Islander Health Performance Framework• The roll-out of the Primary Health Care Access Program• The National Aboriginal and Torres Strait Islander Social Survey and the National Indigenous Health SurveyThese developments are consistent with a policy agenda that has evolved, in general terms, since the release of the National Aboriginal Health Strategy in 1989. However, I will also consider significant developments in the broader context for Aboriginal and Torres Strait Islander affairs, particularly the decision made in early 2004 by the Howard government to abolish the Aboriginal and Torres Strait Islander Commission (ATSIC). While the key events and developments that are reported in this paper elaborate on an agenda that has been developing for more than a decade, the decision to abolish ATSIC is likely to have a revolutionary impact on the future development of Aboriginal health strategy. Following the lead of the National Aboriginal Health Strategy (NAHS) , nationa• Framework Agreements in Aboriginal and Torres Strait Islander Health ;• Joint Planning Forums .st of July 2003, the "National Strategic Framework for Aboriginal and Torres Strait Islander Health" . Agreement has also been recently brokered that details strategies for Indigenous social and emotional well-being, which is one of the Key Result Areas for the "National Framework". Significant progress has also been made in the development of a national performance management framework for Aboriginal and Torres Strait Islander health that aligns with the "National Framework".The NAHS has been the guiding framework for action in this field since it was endorsed in 1989. Consequently, it was significant that the Australian Health Ministers Conference endorsed its successor on the 31The agenda in Aboriginal and Torres Strait Islander health strategy that was adopted by the health portfolio post 1995 had focussed on reform priorities focussed on the development of :• The capacity of primary health services to respond to Aboriginal and Torres Strait Islander health need (with a particular focus on financing and workforce);• disease and risk strategies that aimed to improve Aboriginal and Torres Strait Islander health outcomes;• the evidence base for policy and practice in this sector .With respect to primary care capacity, the roll-out of the Primary Health Care Access Program (PHCAP) continues to be one of the central planks of this agenda and I will provide an overview of recent progress. Significant progress has also been made over the last couple of years in the development of the Australian Bureau of Statistics Indigenous Survey program, which promises to enhance the information available to assist decision-making within the sector. I will provide a report on the recent developments in the roll-out of this program.While the recent developments in Aboriginal and Torres Strait Islander health policy and strategy represent an evolution of a health reform agenda that has been developing for more than a decade, the abolition of ATSIC points to a much more revolutionary change in the broader institutional and programmatic context for Aboriginal affairs. ATSIC had play a critical role in integrating Australian government programs in Indigenous affairs and providing an institutional structure that facilitated Aboriginal and Torres Strait Islander input into policy and program development. ATSIC, for instance, continued to play a role in health strategy following the transfer of specific health program responsibilities in 1995. It retained, for instance, responsibility for the delivery of environmental health service. A memorandum of understanding was developed between the Department of Health and Human Services and ATSIC to support collaboration between the sectors . ConsequThe National Aboriginal and Torres Strait Islander Health Council (NATSIHC) oversaw the development of the "National Framework". However, this process was stalled by political conflict between the key stakeholders. In December 2000, Council members representing the National Aboriginal Community Controlled Health Organisation (NACCHO) resigned in protest over a consultation draft of the 'National Framework'. NACCHO, which is the peak body representing the Aboriginal community controlled health sector, was concerned with :The way the Draft is written distances Aboriginal and Torres Strait Islander people, undermines the concept of Aboriginal community control of primary health care service delivery and diminished structures which NACCHO believe are still useful. The document's tone and language is wrong in a number of ways...Following further negotiation, NACCHO withdrew its resignation, and the NATISHC was reconstituted with revised membership and Terms of Reference . DespiteThe agreed "National Framework" consists of two documents:• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Framework for action by Governments", which sets out a five- to ten-year reform agenda in 9 key result areas .• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Context", which outlines the rationale for the Framework and its context .There are nine Key Result Areas set out in the Framework including :• Community controlled primary health care: building community capacity so that individuals and communities can better address and manage their own health needs.• Health system delivery framework: improving the responsiveness of the mainstream health system to Indigenous Australians and developing stronger partnerships between mainstream and Indigenous-specific services.• A competent health workforce: improving the training, supply, recruitment and retention of appropriately skilled health professionals, health service managers and policy officers in both mainstream and Indigenous-specific health services.• Emotional and social well-being: improving outcomes with respect to mental health, suicide, family violence, substance misuse and male health .• Environmental health: improving the delivery of safe housing, water, sewerage and waste disposal.• Wider strategies that impact on health: undertaking action in portfolios outside the health sector and implementing health gain strategies in the areas of education, employment transport, food and nutrition, custodial health, aged and disability services, recreation and exercise.• Data, research and evidence: aiming to improve the quality of information about how well the health sector is meeting the needs of Indigenous Australians.• Resources and finance: aiming to provide an optimal level of resources for Indigenous health commensurate with levels of need, costs of delivering services and community capacity to deliver health outcomes.• Accountability: both to communities and to governments for the delivery and effectiveness of health services.The "National Framework" was endorsed as a plan to guide all Australian governments in a coordinated, collaborative and multi-sectoral approach to achieving Aboriginal and Torres Strait Islander health gain over the next decade. It does not have a specific funding program attached to its implementation, although arguably, the roll-out of the Primary Health Care Access Program (described later) will provide additional capacity to the implementation of the "National Framework". It is also possible that the National Framework will guide the allocation of any new resources made available through the joint planning processes established under the Framework Agreements.To further these ends, it is significant that the "National Framework" was endorsed through each government's cabinet process, providing a whole-of-government commitment to its implementation in each State and Territory and at the Commonwealth level. Each jurisdiction is developing its implementation plan against which it will report annually on progress and outcomes in health portfolios and biennially on whole of government progress. The plans will identify the specific strategies and timeframes for each action area. The National Aboriginal and Torres Strait Islander Health Council will develop a plan for an independent mid-term and final evaluation.The "Social and Emotional Well Being Framework (SEWB Framework)"  is basedThe nine guiding principles for the "SEWB Framework" were been extracted from "Ways Forward" , an earlIn 2003 the Social Health Reference Group (SHRG) (established to oversee its development) conducted extensive consultations on a draft framework document. Since then the 'SEWB Framework' has endorsed by the NATSIHC and the National Mental Health Working Group in November 2003, and the Standing Committee on Aboriginal and Torres Strait Islander Health in December 2003. It was anticipated that the final 'SEWB Framework' document would be endorsed out of session by the Australian Health Ministers Advisory Council by the middle of 2004.The development of the Aboriginal and Torres Strait Islander Health Performance Framework has built on the foundations of earlier work which has established the key elements of this framework, including the:• national performance indicators in Aboriginal and Torres Strait Islander health for the Australian Health Ministers Advisory Council ;• service activity reporting for Aboriginal community controlled health services ;• Australian government health portfolio indicators ; and• the reporting against key indicators of Aboriginal and Torres Strait Islander disadvantage for the Council of Australian Governments .It is intended that the Aboriginal and Torres Strait Islander Health Performance Framework will both integrate those government performance reporting processes that have already been developed; streamline reporting processes in Indigenous health and, ensure the strategic management of policy relevant and quality information in published reports  . As star• mapping the relationship between the Key Result areas of the 'National Framework' and key domains of health performance ; and• identifying priorities for data development and improvement based on priorities of the 'National Framework'.The Primary Health Care Access Program (PHCAP) was introduced in the 1999–2000 Federal Budget to improve access to primary health care for Aboriginal and Torres Strait Islander people. PHCAP achieves this by funding increased primary health care provision, such as additional general practitioners, nurses, Aboriginal Health Workers, and through preventive and health promotional activities, such as diabetes education and management. Funds are also used for supports to service provision such as capital works and equipment. The program also aims to work with existing health services to ensure they are responsive to the needs of Aboriginal and Torres Strait Islander people.On average across Australia, PHCAP aims to bring the level of Commonwealth funding for Indigenous primary health care to three times the average MBS usage for all Australians. The key objectives of PHCAP are :• Increased availability of appropriate primary health care services where they are currently inadequate;• Local health systems that better meet the needs of Aboriginal and Torres Strait Islander people; and• Individuals and communities that are empowered to take greater responsibility for their own health.Services can be provided through a mix of arrangements, including Indigenous specific, mainstream or a combination of these. Funding can also be used to support mechanisms to assist service providers to deliver better services and enable individuals and communities to become more involved in improving their health.Up until March 2004, new and additional services have been funded in Central Australia (5 regions), Queensland (3 regions) and South Australia (4 regions) through PHCAP, as well as continued funding of services provided through the former Aboriginal Coordinated Care Trials .During 2003 a more streamlined approach to the management of PHCAP rollout was developed ,16, resuFrom these proposals, $11.8 million in funding was approved on 14 March 2004 for:• additional health professional and support staff, for example, over 20 more health professional positions in the Kimberley region of WA;• capital works for health clinic upgrades and the construction of staff housing in remote communities;• minor capital purchases such as medical equipment; and• one-off health promotional activities and health board support and training.Longer term strategies around enhancement of local service systems, to ensure they are more accessible for Aboriginal and Torres Strait Islander people, and ensuring the commitment by state/territory governments to at least maintain their funding commitments, will continue to be pursued. While the PHCAP program has provided a significant injection of resources into what is generally considered an inadequately funded primary heath care system, the amount made available through this program still does not meet its programmatic benchmarks and targets .One of the development priorities established by the heath portfolio when it took responsibility for the administration of the Aboriginal health program was to develop the evidence base to support policy reform and the development of health service capacity .The National Health Survey, undertaken by the Australian Bureau of Statistics with funding support from the Australian Department of Health and Ageing, collects information about the health status, use of health services and facilities, socio-economic status and health-related aspects of the lifestyle of Australians. The Indigenous component of this survey aims to benchmark information on a range of health issues and enable comparisons between the health characteristics of Indigenous and non-Indigenous Australians and to allow trends in the health of Indigenous Australians to be monitored over time.The Indigenous Health Survey that was run in 2001, collected data from approximately 3,500 individuals which was reportable at the national level . In 2004In parallel with the health survey program the Australian Bureau of Statistics collected data for the 2002 National Aboriginal and Torres Strait Islander Social Survey from August 2002 to April 2003 . It is pOn 20 April 2004, the Prime Minister, John Howard and the Minister for Aboriginal Affairs, Senator Amanda Vanstone, announced the intention of the Australian government to abolish the ATSIC.ATSIC had been established in 1989 when the program responsibilities of the Commonwealth Department of Aboriginal Affairs and the Aboriginal Development Corporation were merged into a structure that enable the regional allocation of resources through elected regional councils. The board of Commissioners, elected by ATSIC regional councils, was responsible for national policy development and the oversight of national programs. At the Commonwealth level, ATSIC had the lead agency responsible for the administration of a range of programs such as: community development and employment (CDEP); housing and infrastructure; cultural heritage, broadcasting services; legal services; native title, land rights and the Indigenous land fund, etc.The agency also played a critical role in co-ordinating and integrating the Aboriginal strategy across the different government program areas. ATSIC and the health portfolio had collaborated in the implementation of health infrastructure priority projects . The MemATSIC played a pivotal institutional role in the development of 'whole of government' strategies across the Australian government. It was in effect the only institutional mechanism  that enabled this. This was until the Council of Australian Governments (COAG) resolved (in 2000 and 2002) to trial, in up to 10 regions across the country, innovative administrative arrangements, developed in partnership with Indigenous communities, which aimed to provide "more flexible programs and services based on priorities agreed with communities" [From its first term in 1996, the Howard Coalition government had a conflictual relationship with the Commission. However, government confidence in the ATSIC Board deteriorated significantly under the chairmanship of Geoff Clark (first elected chairperson in 1999) to the extent that the Minister for Indigenous Affairs suspended him on the ground of misbehaviour (under section 40 of the ATSIC Act 1989) . A revieThe Federal cabinet, nevertheless, resolved to a more radical agenda than outlined in the review findings, and announced its intention to abolish ATSIC, its regional councils and the mainstreaming of the administration of all the programs for which ATSIC had been responsible. It is proposed that the elected advisory structures will be replaced by a government appointed national council. It is also proposed that Indigenous specific program dollars will be quarantined and a whole of government approach is to be developed for the delivery of Indigenous specific funding. The key elements of this reform package have been positioned within the broader context of a government commitment to reforms aimed at producing "'joined up' government and the 'seamless' delivery of programmes" . This ne• Ministerial Taskforce: which would operate as a cabinet committee, provide collaborative leadership at a government level and set strategic directions.• Secretaries group: which would support Ministerial decision-making, coordinate across government agencies, and oversight annual reporting.• National Indigenous Council: in which the Minister would appointment Indigenous leaders in health, education, employment, law and justice to provide advice and monitor performance.The proposed mechanisms and structures that would be established to deliver this 'joined-up' framework including regional partnership agreements and community shared responsibility agreements . It is also proposed to establish Indigenous co-ordination centres which will provide a single shopfront in regional and remote Australia for indigenous specific programs, lead the negotiation of regional partnerships an shared responsibility agreements but maintain line responsibility to mainstream departments.The impact of this radical reform agenda to national Aboriginal health strategy is difficult to predict. One the one hand the actual changes to the administration of the health program is insignificant . A mainstream department has administered this program since 1995. One the other hand, the implementation of this reform agenda could have potentially very significant consequences for the development of inter-sectoral strategies in Indigenous health. This depends on the success in implementing the new mechanisms, and on their effectiveness. Furthermore, the political consequences of this radical agenda on the relationship between the Australian government and Aboriginal and Torres Strait Islander peoples have yet to really become clear. Specific partnership arrangements that have been developed within the health sector are tenuous – as is evident by the politics in the development of the "National Framework". These partnerships are critical to successful implementation of strategy in Indigenous health. Consequently, deterioration in the broader relationship between Indigenous Australians and the Australian government may have significant negative consequences for the partnership processes specific to the health sector. Even though 2003 was a year in which policy and strategy in Indigenous health made no or new radical departures, it was a year of considerable tumult in relations between the Australian government and Indigenous peoples. The ramifications of this are only now beginning to unfold.ATSIC Aboriginal and Torres Strait Islander CommissionNAHS National Aboriginal Health StrategyNATSICH National Aboriginal and Torres Strait Islander Health CouncilNACCHO National Aboriginal Community Controlled Health OrganisationPHCAP Primary Health Care Access ProgramThe authors declare that they have no competing interests.


This paper reports on a study investigating the epidemiology of sporadic cryptosporidiosis in the North West of England and Wales using a Geographical Information System (GIS) to map location of residence of cases. Some 747 reports of cases were made to CDSC North West of which 649 reports were suitable for analysis. Cases were plotted on the maps of water supply zone and water quality area boundaries, provided by the two main water utilities.It was notable that there were major spatial variations in attack rate across the North West and Wales. The most dramatic example was the large difference between the Greater Manchester conurbation with many reports and Liverpool with none. Given the distribution of previously detected waterborne outbreaks in the region it was initially thought that drinking water source may be an explanation. However, an analysis of the distribution of cases in the Greater Manchester area showed no correlation with any of five water supplies that serve the conurbation.Our study has shown a dramatic variation in the incidence of laboratory confirmed cryptosporidiosis within two regions of the United Kingdom. Further analysis has not been able to prove drinking water as a likely explanation of this variation which so far remains unexplained. Cryptosporidium. Most infections in the UK are due either to C. parvum (previously known as C. parvum genotype 2 or bovine strain) or C. hominis (previously known as C. parvum genotype 1 or human strain) [Cryptosporidium spp. are protozoan parasites. In otherwise healthy individuals they tend to cause a self-limiting form of gastroenteritis which can last for several days and, sometimes, weeks. In patients with certain forms of immune deficiency, most notable the Acquired Immune Deficiency Syndrome (AIDS), the infection can cause a severe and prolonged diarrhoeal illness which, prior to the widespread use of at highly effective antiretroviral therapy was often fatal [Cryptosporidiosis is infection with species of the genus Cryptosporidiosis has now become the most commonly identified protozoal cause of gastroenteritis in the United Kingdom. Most of the epidemiological data to-date has been related to reports of outbreaks. Between the years 1983 to 1997 there were 80 outbreaks cryptosporidiosis in England and Wales affecting 4649 individuals . OutbreaThe epidemiology of sporadic (non-outbreak-related) cases is largely unknown. Of three large case-control studies reported in the past few years only one found an association with drinking mains water -9. The sThe question remains what proportion of sporadic cryptosporidiosis infections may be due to the consumption of mains drinking water. This paper reports a study using GIS to investigate the epidemiology of sporadic cryptosporidiosis and particularly address the issues of whether sporadic cases are also associated with consumption of drinking water.Table It can be seen that there is substantial spatial variation in the distribution of reported cases. In part, this variation can be explained by variation in population density. However, much of the variation is unexplained. For example, reports from Liverpool are very uncommon, whilst reports from Greater Manchester are very common.It was decided to investigate the excess case reporting from Greater Manchester in further detail to look for any possible association with water supplies. Water to the Greater Manchester area comes from five main water treatment works; Lostock (derived from Thirlmere in the Lake District and chlorinated but not filtered), Woodgate Hill , Arnfield-Godley , Buckton Castle  and Wybersley .In order to determine whether there was any relationship between attack rate and water supply, all water supply zones in the North West that received any water from one or more of these five supplies were identified Figure . For eacAs already mentioned, care should be taken in the interpretation of this analysis. It is notable that the proportion of reports that could not be allocated a correct postcode varied from one health authority to another to some extent. Also variation in attack rate between water supply zones or water quality areas was as likely to be due to differences in population size as to differences in reported cases. This was most obvious in zones/areas with relatively small population sizes where random effects could have a particularly important affect. However, there are a number of obvious features.The most obvious is the large number of cases from the Greater Manchester conurbation. This covered the Bury and Rochdale, Manchester, Salford, Stockport, West Pennine, and Wigan and Bolton Health Authorities. This excess of cases in Manchester is even more remarkable when compared with the virtual absence of cases from the Liverpool conurbation . The reason for the excess of cases in Greater Manchester is unclear. Although different reporting habits could play a part, we doubt that it could explain more than a small part of the difference. Reporting practices are not that greatly different across the North West . A sero-Cryptosporidium [An alternate explanation could be that the increase represents different water supplies. Salford, and Wigan and Bolton Health Authorities get much of their water supply from Thirlmere, a supply known to be prone to contamination by poridium , none ofporidium . NeverthA further explanation could be that the Manchester population experience other risk factors more commonly than the Liverpool population. Possible explanations include contact with contact with animals, visiting swimming pools and overseas travel. We do not have access to data to show whether or not people from Manchester are more exposed to these factors than people from Liverpool. However, Manchester is closer to a major National Park, The Peak District. If people from Manchester use their proximity to the Peak District to spend more time in the countryside and so are more likely to come into contact with farm animals, this could explain the difference. This would be an interesting hypothesis to test in a further study.In addition to Greater Manchester, there are also areas of increased reporting from North Wales and from North West Lancashire. These hotspots also remain unexplained. North West Lancashire, however, receives much of its water from Thirlmere and a water source cannot be excluded. However, many cases were reported from the Fylde peninsula which only receives a small proportion of its water from Thirlmere.et al. [The use of GIS to study the spatial distribution of cases has been useful in identifying geographical variation, but not necessarily for identifying the reasons for this variation. However, initial analysis does not support the hypothesis that differences in drinking water source is the major reason for this variation. We agree with Dangendorf et al.  that GISConsultants in Communicable Disease Control in the North West Region of England and in Wales were asked to forward details of cryptosporidium cases upon notification from the laboratory. A data collection form was completed for each case, giving the following details: name, address, postcode, date of birth, GP name, GP address and date of notification. The form was faxed or e-mailed to Communicable Disease Surveillance Centre (CDSC) – North West as soon as possible.Enhanced surveillance for the North West of England and Wales were set up separately, North West England in mid December 2000 and Wales in February 2001. Both ran until February 2002. To check for accuracy, the data were audited every 2 months. Each CCDC was sent a list of the cases they had notified to CDSC North West in the preceding 2 months. Any cases that had not been notified were forwarded to CDSC.The first stage in the geographical analysis was to check the 747 records for possible duplicate records. These were selected on the criteria of 2 individuals with identical names, dates of birth and postcodes being present in the database. Given that a postcode contains on average only 15 addresses the chances of these being legitimate is highly unlikely. Through this procedure 10 records were deleted from the database. Consequently 737 cases of cryptosporidiosis were identified during the period of enhanced surveillance.The next step was to assign a grid reference to each postcode and this was achieved using the Royal Mail Postcode Address File. Eighty eight records were excluded as either, an incomplete postcode was entered into the cryptosporidiosis database or a match could not be found in the postcode address file. Therefore, in total the database was reduced to 649 cryptosporidiosis cases. These were plotted as points against a backdrop of the water supply zones for the two main water utilities. The water supply zone and water quality area boundaries were provided by the two main water utilities (United Utilities and Welsh Water). A "water supply zone" is an area designated by a water undertaker providing water to the residences of not more than 50,000 people. In general the source is consistent across a particular zone.Using the GIS each case was also assigned its corresponding water supply zone and the number of cases in each WSZ was divided by the population, based upon data supplied by the two water utilities, to produce the attack rate maps. The analysis was undertaken in ArcGIS 8.1 using point in polygon techniques .PRH was lead on study design, did the statistical analyses and co-wrote the paper.SH co-designed the study and co-wrote the paper and undertook most of the data collection.QS co-designed the study and co-wrote the paper.SW co-designed the study and co-wrote the paper.IL undertook the geographical analyses and co-wrote the paper.KO co-designed the study, co-wrote the paper and obtained data on the water distribution.RC co-designed the study and co-wrote the paper.


ST estimates, Nei's DA distance and phylogenetic relationships.Approximately 800,000 primarily feral dogs live on the small island of Bali. To analyze the genetic diversity in this population, forty samples were collected at random from dogs in the Denpasar, Bali region and tested using 31 polymorphic microsatellites. Australian dingoes and 28 American Kennel Club breeds were compared to the Bali Street Dog (BSD) for allelic diversity, heterozygosities, F-statistics, GST of 0.088 and also with high bootstrap support to the Australian dingo and Akita in the phylogenetic analysis.The BSD proved to be the most heterogeneous, exhibiting 239 of the 366 total alleles observed across all groups and breeds and had an observed heterozygosity of 0.692. Thirteen private alleles were observed in the BSD with an additional three alleles observed only in the BSD and the Australian dingo. The BSD was related most closely to the Chow Chow with a FThis preliminary study into the diversity and relationship of the BSD to other domestic and feral dog populations shows the BSD to be highly heterogeneous and related to populations of East Asian origin. These results indicate that a viable and diverse population of dogs existed on the island of Bali prior to its geographic isolation approximately 12,000 years ago and has been little influenced by domesticated European dogs since that time. Bali, a province of the Republic of Indonesia, is an island just 87 km from north to south and 142 km from east to west and home to more than 2.9 million people [More than 90% of the residents of Bali are Hindu  with mytAs a result of overpopulation, many BSDs suffer from chronic skin diseases, internal parasites, parvo- and distemper-virus infections, and malnutrition. In an effort to reduce the dog population and to care for their medical needs, the Bali Street Dog Foundation (Yayasan Yudisthira Swarga) was founded in 1998 . They prThe BSD population is of interest for both its genetic diversity and historical relationships. It is also a population that has bred more or less randomly for thousands of years with limited genetic influx, due mainly to geographic barriers and a strict rabies control program in effect since 1926. The present study is concerned with the genetic diversity of this unique canine population and its relationship to other canine subpopulations in Asia and throughout the world. Data presented herein was derived from the DNA testing of 40 BSD samples from the Denpasar city region of Bali with 31 polymorphic microsatellite loci. The genetic diversity of the BSD was compared to that of the Australian dingo and 28 American Kennel Club (AKC) breeds.T values greater than 0.700. Average HS was 0.577 for the 30 subpopulations, with all but three loci having HS values greater than 0.500. The HS and HT values were closest for C23.123 and farthest for C22.279 and C10.404. HWE analysis revealed that all but one locus had at least one population out of equilibrium for the 30 populations sampled. C01.424, C31.646 and CPH16 had 7 populations out of HWE and AHT130 did not have any populations with p values below 0.05. The level of locus diversity attributable to subpopulation structure was evaluated with two statistics – RST and FST. Both statistics gave similar average values at 0.230 and 0.236 respectively. However, RST ranged from 0.098 to 0.486 while FST ranged from 0.179 to 0.328.Analysis of locus diversity across all 30 subpopulations revealed that the number of observed alleles ranged from six to 20 with a total of 366 for all loci Table . OverallE) and observed (HO) heterozygosities  and a 28.4% higher HE than the average AKC breed (0.573). HO was also highest in the BSD at 0.692, versus 0.426 in the Australian dingo and 0.563 in the average AKC breed.Overall, the BSD was the most genetically diverse population surveyed here, displaying 239 total alleles out of the 366 seen in all 30 subpopulations or 65.3% of the total observed alleles .IS estimates were calculated to assess the level of inbreeding for each subpopulation . Appearing at the highest frequency was allele 201 of locus CPH08 in the BSD, Australian dingo and Chow Chow at frequencies of 21.3%, 10% and 20%, respectively. Further, the BSD, Australian dingo, Chow Chow and Akita share allele 113 of locus C22.279 at frequencies of 23.8%, 67.5%, 15% and 5%, respectively. Results for locus PEZ08 demonstrate a lack of influence of European alleles where a high frequency of deviations from n+4 alleles were observed in the AKC breeds sampled yet no deviation from n+4 alleles was observed in the BSD or the Australian dingo.ST analysis was performed for each locus between the BSD and the two closest subpopulations: the Australian dingo and Chow Chow  and the Australian dingo , and least closely with the Airedale Terrier .Further distance analysis was performed for all 31 loci between all 30 subpopulations using both Nei's DA distance and pairwise Fes Table . Across Genetic distance relationships amongst the five Asian subpopulations were further explored using neighbor-joining dendograms with four non-Asian subpopulations for comparison Fig. . The BSDO was high in three feral dog subpopulations of Korea, Sakhalin and Taiwan, ranging from 0.539 in the Taiwanese to 0.717 in the Korean dogs. Given that the loci used in that study had an average allele number of 7.75, these values are similar to the HO of 0.692 observed in the BSD. Wilton et al. [O of 0.387 using microsatellites with an average allele number of 6.93, similar to the HO of 0.426 for the Australian dingoes reported herein with an average allele number of 11.8.Microsatellites have been previously used to assess genetic diversity and relationships in feral dog subpopulations ,7. Kim eIS. Even after adjusting for sample size, the BSD maintains their status as the most heterogeneous population in the study. Unlike the Australian dingo which exhibits a much lower level of diversity, the BSD findings suggest either a large founding population on Bali and/or a consistent genetic influx since the geographic isolation of ~12,000 years ago. This data also supports that the BSD appears to approximate a randomly breeding population with little selection pressure.Given the size of the island of Bali, it is extraordinary that 800,000 feral dogs can thrive and maintain such high levels of genetic diversity. Of all the subpopulations surveyed here, the BSD has the highest number of observed alleles, the highest heterozygosity, the fewest number of loci out of Hardy-Weinberg equilibrium and the lowest FE, such as the Boxer with a HE of 0.320, breeds like the Jack Russell Terrier have a high HE of 0.713 and overall their HE is higher than that of the dingo. Of first note, the selection of the dogs that contribute to a breed composition mostly occurs prior to official breed recognition primarily by genetic drift due to geographic isolation and selection for particular working or physical characteristics. After official breed recognition future breeding choices are based primarily on the availability of sires and dams that approximate the breed standard. As a result, there is a founding population that proceeds to breed mostly by convenience. Also, many breed standards have changed considerably over the years resulting in retention of a certain level of diversity within each breed, some breeds retaining much more than others. Finally, dogs comprising the comparison AKC breeds were sampled from across the United States, removing any geographical bias of the genotypes observed and slightly elevating the heterozygosities.When comparing the heterogeneity of the BSD to that observed within the AKC breeds some caveats should be addressed. One may initially expect long established, well-defined dog breeds to be much less heterogeneous than reported here. While some breeds do have a low HT is 0.577. The average values for the 11 subpopulations surveyed in the Kim et al [The average allelic diversity of the loci used in the present study was 11.8 alleles per locus, versus 7.75 in the Kim et al work . Howeverim et al  work werST and FST values were nearly identical across all subpopulations and all loci, indicating that approximately 23% of the differences observed in allele frequencies can be attributed to differences between subpopulations. FST provides an unbiased estimate of genetic drift between subpopulations by comparing alleles identical by state. RST takes advantage of the stepwise mutation model, which assumes that mutations most often occur as whole repeat unit losses or gains from the original allele size. As a result, the number of mutations provides an estimate of time from divergence. It is interesting, therefore, to compare RST and FST values by locus. Eighteen of the 31 loci studied have an RST to FST ratio greater than 1.1  over that of other study groups. Blood samples from the Australian dingo were taken from captive animals in Australia. Australian dingoes were known to be unrelated by at least one generation.Dogs from 28 American Kennel Club (AKC) breeds, equally representing the AKC group designations, were sampled with buccal swabs for a previous study . Twenty Thirty-one of the 100 microsatellites multiplexed into 12 PCRs by the Veterinary Genetics Laboratory  had beenForward primers were synthesized and dye labeled with either Fam, Hex or Vic, or Tamra or Ned , Foster City, CA). Reverse primers were synthesized by Operon . Primer sequences and concentrations for all markers are available as BSD and AKC breed DNA was derived from buccal cells harvested from the inside of the cheek with nylon bristle cytology brushes . Samples were collected by owners or field volunteers and submitted directly to the laboratory. DNA was extracted by heating a single swab for 10 min at 95°C in 400 μl 50 mM NaOH and then neutralized with 140 μl 1 M Tris-HCl, pH 8.0. Australian dingo DNA was extracted from blood using a standard sodium hydroxide digest.2, 200 μM of each dNTP , 0.6 unit AmpliTaq (ABI), and 2% DMSO (Sigma) then covered with 15 ul Chill-out™ Liquid Wax  to prevent evaporation. One of five thermal cycler programs was used for each primer mix ranging from 56° to 64° degrees for the annealing temperature. All PCR work was done in polycarbonate 96-well v-bottom microtiter plates  on MJ Research PTC-100 thermal cyclers . Protocols are also available in A 2 μl aliquot of extract was used in each PCR which equates to approximately 50 ng DNA. All markers and DNAs were amplified with a PCR reagent mix of 1X PCR buffer (ABI), 4.17 mM MgClOne μl aliquots of PCR product were mixed with 2 μl Fluorescent Ladder (CXR) 60–400 (Promega 400) or Internal Lane Standard 600 (Promega 600)  fluorescent size standard, denatured on MJ Research PTC-100 thermal cyclers for three minutes at 95°C, then held at 5°C or placed on ice for at least one minute before gel loading. Two μl aliquots were then loaded onto a 6% denaturing polyacrylamide gel and run on an ABI 377 Automated Sequencer using ABI 10" × 7 1/8" short plates (12 cm). Gels were run at 1.10 kV (constant) voltage, 60.0 mA current, 200 W power, 51°C and 40.0 mW (constant) laser power for up to 2 hours when using Promega 400, and up to 3 hours using Promega 600. DNA fragment analysis was performed with in-house designed STRand software , which rO) were determined by direct counting for each of the 30 subpopulations. Hardy-Weinberg equilibrium (HWE) tests were performed using Genepop version 3.4 [ST estimates and subpopulation expected heterozygosities (HE) for the 30 breeds or dog groups were performed using Genepop version 3.4 [IS estimates (inbreeding coefficient of each subpopulation) for each allele following Weir and Cockerham [Allelic diversity and observed heterozygosities  and its associated parameters, HS (average heterozygosity among subpopulations) and GST (coefficient of genetic differentiation), were calculated across all loci using the public domain software, DISPAN [ST [ST [Gene diversity or total population heterozygosity (H, DISPAN . Two addSPAN [ST  and RST  [ST [ST ,28 were Allele frequencies were used to compute a matrix of genetic distances , which wBSD: Bali Street DogIS, FST RST, GST: F-statistics indicesFS, HT, HE, HO: Heterozygosity indicesHHWE: Hardy-Weinberg equilibriumA: Number of allelesNAES: American Eskimo DogAS: Australian ShepherdAT: Airedale TerrierBCO: Border CollieBLT: Bull TerrierBMD: Bernese Mountain DogBS: Brittany SpanielBT: Belgian TervurenBU: BulldogBX: BoxerBZ: BorzoiChow: Chow ChowDingo: Australian dingoDP: Doberman PinscherGH: GreyhoundMBT: Miniature Bull TerrierPG: PugRR: Rhodesian RidgebackGR: Golden RetrieverJRT: Jack Russell TerrierKE: KeeshondLR: Labrador RetrieverNE: Norwegian ElkhoundPG: PugPM: PomeranianPPN: PapillonPWC: Pembroke Welsh CorgiRR: Rhodesian RidgebackWM: WeimaranerYT: Yorkshire TerrierDNI performed the majority of data acquisition and analysis, wrote first draft of the manuscript and prepared the final draft for submission. ALS performed the majority of sample processing, assisted in data acquisition and the writing of the subsequent drafts of the manuscript as well as final draft preparation. SG sampled the dogs tested, provided background for the manuscript and assisted in the final draft preparation. ANW provided the Australian dingo data for comparison and assisted in the subsequent drafts of the manuscript. NCP directed the research and assisted in the writing of the manuscript. All authors read and approved the final manuscript."MS 2784776144318916 Supplement1.xls" and contains the primer sequences, expected size range, primer concentration used and annealing temperatures used. In a separate sheet within the same file the protocols for each PCR reaction are listed.Click here for file"MS 2784776144318916 Supplement2.xls" and contains the individual genotype data for each animal used to derive the statistics and phylogenetic results presented herein.Click here for file


Many different things combine to cause epidemics of disease. Among these factors are the characteristics of the infecting organism, the resistance of the host, and, as is increasingly realized, climatic conditions.El Niño, the best known climatic disturbance, is caused by a warming of the Pacific Ocean, which then affects the climate globally. Previous work has suggested that this recurring phenomenon can have a profound effect on the incidence of many diseases, including dengue, malaria, and diarrheal diseases.PLoS Medicine, Sultan and colleagues from a climate research institute and an infectious diseases center in France looked at the relation between climate and meningitis outbreaks in Mali in West Africa, a region that every year between February and May sees devastating epidemics of meningococcal meningitis affecting up to 200,000 people. The most important recurring climatic event in this region is a dry wind, known as the Harmattan, that blows throughout the winter, causing a drop in humidity and the production of vast quantities of dust.In a paper in this month's What the authors found was that over the years 1994–2002, the week of the onset of the yearly meningitis epidemic came at around the same time as the peak of one measure of the wind—the sixth week of the year. As Pascual and Dobson say in their Perspective article on this study, “Sultan and colleagues' study is exceptional in that it illustrates a clear relationship between an external environmental variable and the initiation of disease outbreaks.”How do climatic changes influence disease? In some cases, such as the role of flooding in spreading a waterborne disease, the causes are perhaps obvious, but why should a dry wind affect disease incidence? Previous works have suggested that the climate can work in a number of ways, by influencing the life cycle of both disease vectors and the disease-causing organism, and, as here perhaps, by affecting the resistance of the host. Sultan and colleagues speculate that the drying effects of the wind on the mucous membranes could increase the chances of the organism getting established in the human host. Whatever the causes, one very useful feature of climate is that, once the patterns are understood, they can often be predicted.A way of predicting these meningitis epidemics could be enormously useful. Sultan and colleagues looked at only a few years, but if these findings are confirmed over a longer time period, they could make preparing for an epidemic much more efficient.


Nutrition & Metabolism (N&M) will publish articles that integrate nutrition with biochemistry and molecular biology. The open access process is chosen to provide rapid and accessible dissemination of new results and perspectives in a field that is of great current interest. Manuscripts in all areas of nutritional biochemistry will be considered but three areas of particular interest are lipoprotein metabolism, amino acids as metabolic signals, and the effect of macronutrient composition of diet on health. The need for the journal is identified in the epidemic of obesity, diabetes, dyslipidemias and related diseases, and a sudden increase in popular diets, as well as renewed interest in intermediary metabolism.A new Open Access journal,  Nutrition & Metabolism (N&M) include 1) an awareness of an epidemic of obesity, diabetes, dyslipidemias and related diseases, 2) a sudden increase in the popularity of diets, such as low carbohydrate diets, to achieve weight loss and combat diabetes, and 3) a renewed interest in intermediary metabolism accompanied by the development of new tools and techniques for genomic and metabolic analysis.Recent events that provide the rationale for a new Open Access journal, ) and similar motions exist in the US congress [N&M feel that, at this point, the burden of proof is on proponents of perpetuating the current system. We are, however, not doctrinaire on this point and believe one should pay for a service if it is valuable. Beyond information, printed collections provide convenience and we intend to offer bound copies of articles on individual topics as the journal proceeds.With the considerable activity shown in these areas, rapid and easily accessible dissemination of new information is clearly valuable. Whereas articles in existing journals do discuss intermediary metabolism in a nutritional context, there is a need for a unique and explicit focus for this discipline. In addition, it is precisely because publications in nutritional biochemistry are spread over such a large number of existing journals, few libraries and almost no individual can subscribe to all. It is in areas like this that free, open access becomes important. There is a large published debate on open access . Most rcongress ,3. The eN&M has its own strengths and interests as indicated by the board of editors . Three areas of particular interest are lipoprotein metabolism, amino acids as metabolic signals, and the effect of macronutrient composition of diet on health. This is reflected in our opening research articles by Darimont, et al. on the control of obesity and lipid structure by adrenergic systems, and by Volek, et al. on the effectiveness of low carbohydrate diets, and differential effects on fat and lean mass.Nutrition and metabolism is a broad field and we welcome submissions from all areas of nutrition and related biochemistry. Like any journal, however, , while not recommending any particular diet, highlighted many of the relevant issues in macronutrient control of metabolism. In our initial publications, contributors to the conference will provide reviews of the various topics covered. In the first posting, Klaas Westerterp summarizes the importance of macronutrient composition in thermogenesis, and Stephen Phinney discusses the impact of ketogenic diets on physical performance. Kimball and Jefferson review the regulation of mRNA translation in general. The article provides a nice overview of various mechanisms involved in the control of protein synthesis when amino acids become limiting. Perhaps the most important from a practical standpoint, Nuttall and Gannon summarize potential benefits of higher protein diets in diabetes.The sudden popularity of low carbohydrate diets is one of the most remarkable phenomena in nutrition today. A recent editorial by Walter Willett points out how important it is that we understand them . SimilarN&M is free. Articles are included in PubMed and archived in PubMed Central. Online submissions can be made at .Nutrition & Metabolism welcomes contributions in all areas of research in which nutrition interacts with biochemistry and molecular biology. Emphasis will be on the molecular, biochemical, and physiologic understanding of various metabolic pathways. The journal will publish Original Research, Reviews, Commentaries and Perspectives, Brief Communications, Methods and Book Reviews. Access to all articles in


E. Coli genes is affected by changes in the level of chromosome supercoiling, suggesting that supercoiling transmits regulatory signals from the environment to many cellular pathways.Microarray analysis shows that transcription of 306  Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.The chromosome of We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions.The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell. Escherichia coli is a circular double-stranded DNA molecule that is maintained in a negatively supercoiled state. Supercoiling induces torsional tension in the DNA, and thus can influence processes that involve the opening of the double helix, such as replication initiation [The chromosome of itiation , DNA looitiation  and tranitiation . A numbeitiation ), suggesE. coli, supercoiling is maintained at a precise range during log phase growth by the topoisomerases DNA gyrase, topoisomerase I (topo I), and topoisomerase IV (topo IV) [gyrA and gyrB), and for topo I (topA). Relaxation of the chromosome upregulates gyrA and gyrB and downregulates topA as a form of feedback control [fis , ilvG (an amino-acid synthase subunit) and cydAB (an oxidase involved in aerobic respiration), has been found to be sensitive to supercoiling [In topo IV) -7. DNA gtopo IV) -10, whertopo IV) . Togethetopo IV) ,13. The  control -16. Thisrcoiling -19, suggrcoiling . If supercoiling ,17,18.E. coli K-12 genome to systematically identify those genes that respond to relaxation of the chromosome during log-phase growth. We used antibiotics and mutations in the topoisomerase genes to change supercoiling levels by independent mechanisms and thus discerned the general effects of chromosome relaxation. We classify supercoiling-sensitive genes, or SSGs, according to their response to DNA relaxation. Therefore, we call 'relaxation-induced genes' those genes whose expression is increased upon DNA relaxation, and 'relaxation-repressed genes' those whose expression is repressed by DNA relaxation.In this study, we used cDNA microarrays ,22 repregyrA or topA, and their expression patterns correlated with the supercoiling level of a reporter plasmid in the cells. SSG transcripts have the same rates of RNA decay as non-SSG transcripts, and thus the changes in expression were due to a change in the rate of RNA synthesis, rather than RNA decay.An extensive statistical analysis of our experimental results revealed 200 relaxation-repressed genes and 106 relaxation-induced genes; in total, around 7% of all genes in the genome were found to be significantly affected by supercoiling changes. Many of these genes are more sensitive to supercoiling than p < 0.0001) AT-rich in their upstream sequences, and also have AT-rich coding regions. Relaxation-repressed genes had a corresponding preference for GC-rich sequences. The SSGs are dispersed throughout the chromosome, and fall into many different functional classes. We propose that the large number and functional diversity of the SSGs reflects the role of supercoiling as a second messenger that responds to environmental changes and feeds into different regulatory networks.We discovered that the sequences of the relaxation-induced genes are significantly (tetA gene in pBR322 anchors this plasmid to the membrane [We sought to determine the genes that are activated or repressed by relaxation of the (-) supercoils in the chromosome. To isolate the expression changes due to the loss of supercoiling from those due to the experimental approach, we used three different methods to relax the chromosome. In two of the methods we inhibited gyrase and topoIV with either quinolone or coumarin antibiotics, and in the third we used a temperature-sensitive strain in which gyrase is inhibited at 42°C. Because it is technically difficult to quantify the supercoiling state of the bacterial chromosome, we used a plasmid, pBR322, in the strains as a reference. Co-transcriptional translation of the membrane , and thumembrane . The supgyrA and gyrB) or topo IV (parC and parE) as controls, to separate expression changes due to undiscovered drug targets from those directly due to changes in supercoiling. When we inhibited gyrase by treating gyrA+ parCR cells with 15 μg/ml norfloxacin, the reporter plasmid in the cells was rapidly relaxed (Table gyrAR parCR strain remained (-) supercoiled. After 30 minutes, there was a 103-fold drop in viability in the sensitive strain, but only a 17% drop in the resistant strain. A norfloxacin concentration of 50 μg/ml fully inhibited both gyrase and topoisomerase IV in the wild-type strain (data not shown), while the resistant strain retained wild type levels of (-) supercoiling and showed only a slight drop (15%) in viability, indicating that we did not overcome the drug resistance. At bacteriocidal concentrations similar to these, quinolones cause a decrease in the sedimentation coefficient of the nucleoid, indicating relaxation of the chromosomal supercoils [t = 0) and was labeled with Cy3 (green). RNA samples taken 2, 5, 10, 20 and 30 minutes after drug addition were labeled with Cy5 (red). The labeled experimental and reference samples were mixed in equal amounts before hybridization to a microarray.The quinolone antibiotic norfloxacin selectively and immediately inhibits gyrase and topo IV -26. We upercoils . The refInhibition of topoisomerases by quinolones leads to double-strand breaks in the chromosome . Thus, nWe also relaxed the chromosome using novobiocin, a coumarin antibiotic that inhibits gyrase, and at a higher concentration, topo IV ,31. NovogyrB234 cells upon shift to the restrictive temperature and subsequent relaxation of the DNA  to determine which genes change significantly during an experiment can bias expression analysis towards genes with very low or variable expression levels . We usedet al. . Brieflyrocedure . The corp-value represents the robustness of the response to relaxation, whereas correlation with plasmid supercoiling may represent sensitivity to changes in supercoiling levels. For example, a gene that is always completely repressed in response to relaxation will have a low p-value, but may show little correlation with intermediate levels of supercoiling. Similarly, a gene with more variable expression may have a higher p-value, but may also have a higher sensitivity to intermediate supercoiling levels. Taken together, these metrics provide a detailed account of supercoiling sensitivity.As an independent metric of supercoiling sensitivity, we measured how closely gene expression followed the level of DNA supercoiling, by calculating the correlation of the expression of each gene across all of the experiments with the level of supercoiling in the reporter plasmid. Relaxation-induced genes showed a positive correlation with plasmid linking number  supercoiling is lost, both linking number and gene expression increase), up to a maximum observed Pearson correlation coefficient of 0.91. Relaxation-repressed genes showed a negative correlation with plasmid linking number to a minimum Pearson coefficient of -0.88. The majority of genes  showed no strong correlation with plasmid supercoiling, resulting in Pearson coefficients between 0.5 and -0.5. The p-values for all of the genes versus their correlation to plasmid supercoiling are plotted in Figure p-values and little correlation with plasmid supercoiling. Those genes with the lowest p-values  tended to be more strongly correlated (or anticorrelated) to plasmid supercoiling. The data for all genes can be found in Additional data file 2. Among all genes there is a continuous variation in both p-value and correlation to plasmid supercoiling. We found a total of 306 genes at p < 0.05, which we define as SSGs. Of these, 106 genes were induced by DNA relaxation and have a positive correlation with plasmid linking number, while 200 genes were repressed by relaxation and these have a negative correlation with plasmid linking number. The correlations of the SSGs with plasmid supercoiling are shown in Figure p-value. Just over half of the SSGs have high significance, p < 0.005. The high redundancy of our data  minimized the influence of any single array measurement. Thus we can be confident that the genes we classed as SSGs have a reproducible response to supercoiling changes.The p-value (from the top). Each colored entry in the diagram corresponds to one spot on one array . Conversely, these relaxation-repressed genes should have low ratios (and black squares) in the control experiments 1 to 14. The significant difference in SSG expression between the controls and relaxation experiments is reflected by the sharp contrast between the mostly black controls and the bright green relaxation experiments. At the top we have shown a model expression profile representing the supercoiling of the reporter plasmid in each experiment  are listed, along with their correlations to plasmid supercoiling levels.Figure p-values, correlations to supercoiling, and expression levels in each experiment can be found in Additional data file 3.The 106 genes that are induced by relaxation are similarly shown in Figure gyrA+parC+ cells, plasmid supercoiling levels changed dramatically within the first minute  should respond quickly to relaxation. We used a finer time-course experiment to determine which genes had the fastest response to chromosomal relaxation. When 15 μg/ml norfloxacin was added to e Figure . Signifie Figure . We rankE. coli mRNAs have a mean half-life of 5.2 ± 0.3 minutes in LB media [The speed of the transcriptional response to relaxation, combined with the strong correlations to supercoiling of the reporter plasmid in the cells, is strong evidence that the SSGs are directly regulated by supercoiling changes. Furthermore, given that LB media , RNA synp-value with the published values of RNA half-life [We found no correlation of alf-life  and in gp = 3E-4).We searched for a basis of supercoiling sensitivity at the nucleotide sequence level by examining the AT content in and around the SSGs. We considered only the first genes in an operon. Whereas relaxation-repressed genes have a slightly depleted AT content both upstream of their promoters and within the coding sequence, relaxation-induced genes have an emphatic elevation of AT content in similar regions. The AT content of relaxation-induced genes from 800 nucleotides before to 200 nucleotides after the start codon is 54.6%, compared with 51.7% for non-SSGs. To illustrate the very low probability of selecting by chance a set of genes with such an elevated AT content, we randomly selected groups of first-in-operon non-SSGs 50,000 times and calculated AT content within the same window. We never found a set with an AT content as high as the relaxation-induced genes .This is not the only region in which the AT content of SSGs deviates from the rest of the genome. Figure Striking as these differences in AT content are for SSGs as a group, they are not sufficient to distinguish an individual SSG from a non-SSG. That is, not all genes with high or low AT content were supercoiling sensitive in our experiments. Although such genes are rare in the non-SSG population, the greater size of the pool of non-SSGs results in many genes with wide variations in AT content. Also, supercoiling sensitivity cannot solely be due to differences in AT content, as a few SSGs were highly sensitive to supercoiling changes in spite of having an AT content similar to the rest of the genome.topA [gyrA [p-values. Accordingly, although the topoisomerase genes topA and gyrA both clearly respond to supercoiling . The omission of these topoisomerase genes from our list of SSGs reflects the conservative statistical approach we used to define the list. There are probably other genes that respond to supercoiling changes in different conditions from those we tested (log-phase growth in rich media). Also, we defined SSGs by focusing on the immediate effects of relaxation, and thus considered only primary transcriptional changes, rather than downstream effects mediated by other gene products . When downstream effects are considered, changes in supercoiling are likely to affect transcription of an even greater proportion of the genome.In this analysis of supercoiling effects on transcription, we identified 306 genes that quickly and reproducibly respond to chromosomal relaxation. The comprehensive nature of our investigation, with responses of 93% of the genome  in 21 different relaxation experiments and 14 control experiments, allowed us to be more stringent than previous studies in our definition of SSGs, and to identify those genes that had statistically significant changes after the chromosome was relaxed by different methods. Genes that are sensitive to relaxation but are also affected by temperature shifts (including topA  and gyrApA [gyrA ) showed E. coli. Two early studies used either nylon membranes or two-dimensional protein gels to compare topoisomerase mutants with slightly different homeostatic levels of supercoiling, and neither study found a large number of genes [There have been several previous attempts to measure the effects of supercoiling on gene expression in of genes ,44. ThisE. coli [ynhG, yrbL, otsB and yifE). Seven others had p < 0.1 in our relaxation experiments, and the rest had still higher p-values in this study. It is possible that these genes are only responsive to supercoiling changes in the context of osmotic stress.A more recent analysis by Church and colleagues used microarrays to gauge the osmotic stress response of E. coli . SurveyiJust as supercoiling is affected by many environmental changes, such as osmotic shock, oxygen tension, nutrient upshift and temperature change, so too do changes in supercoiling affect genes in a large number of classes. Not surprisingly, a substantial fraction (6.9%) of the SSGs encode genes involved in DNA replication, recombination, modification and repair. However, the SSGs span many other classes, and thus are well positioned to feed into many different regulatory networks. Thus, supercoiling can act as a second messenger that quickly translates environmental changes to transcriptional programs, inducing and repressing specific genes independently of protein synthesis.smtAmukBEF operon on loss of supercoiling is intriguing, given the importance of mukB, mukE and mukF in chromosome supercoiling and segregation [E. coli [Several of the SSGs warrant further inspection. For example, the repression of the regation ,46. Cons[E. coli , these gyrdD, a 'putative topoisomerase'. yrdD encodes a 19 kilodalton (kDa) protein 30-40% identical to the carboxy-terminal domain of topoisomerase I from Bacillus subtilis, Helicobacter pylori and Methanococcus jannaschii. The function of YrdD is unknown, but the repression by chromosomal relaxation provides an intriguing lead.Another relaxation-repressed gene that may be involved in chromosomal maintenance is cls (cardiolipin synthase) and ileS (isoleucine tRNA synthetase), which is consistent with the earlier discovery that these genes were involved in sensitivity to gyrase inhibitors [deoA and deoC are induced on relaxation. For these genes, DNA relaxation may be a signal of DNA damage, and their induction would allow the cell to recycle nucleotides necessary for DNA repair. Finally, the induction of rpoD, the σ70 subunit of RNA polymerase, may help the cell compensate for the increased difficulty of melting the relaxed DNA template.Chromosomal relaxation leads to the repression of hibitors . Also, wlacps and ilvGP [in vitro. The more relevant issue is promoter regulation on chromosomes in vivo, where other factors may dominate. The CRP protein increases lac operon transcription at the low to moderate superhelicities found in vivo, and the nucleoid-associated protein IHF is implicated in the supercoiling sensitivity of the ilvGMEDA operon [Salmonella leu-500 promoter by supercoiling requires that the promoter is either on the chromosome or on a plasmid anchored to the cell membrane by transcription and translation of a gene such as tetA [What is the basis of supercoiling sensitivity? Most of the well controlled analyses of supercoiling-sensitive promoters, notably of the nd ilvGP ,49-51, wA operon . Also, tA operon -55. We f as tetA . FurthergyrA and gyrB promoters, Menzel and Gellert [gyrA and gyrB. Promoter clearance has also been invoked in the mechanism of supercoiling sensitivity of some promoters in vitro [in vivo, and propose that promoter clearance is generally a key regulatory step for supercoiling sensitive transcription.It is striking that there are so many relaxation-induced genes that are relatively repressed when the chromosome is (-) supercoiled. This is surprising because (-) supercoiling should favor formation of an open promoter complex. The promoter regions of many of the genes induced by relaxation are AT rich, which will make it easier to form an open promoter complex even when the DNA is relaxed and the energy required is greater. Alternatively, the difference in AT content could reflect structural features such as curvature or flexibility. Curved sequences of DNA can influence the position of plectonemic supercoils, and thus could serve to localize a promoter sequence to the apex of a superhelical loop . We note Gellert  found thin vitro . As our The AT-rich regions of our relaxation-induced genes extend downstream of the translational start site, and thus may involve transcription elongation in addition to promoter clearance. There is growing appreciation of the regulation of transcription elongation -61. The E. coli chromosome [The SSGs are useful as topological probes of the chromosome in living cells. While the SSGs are scattered throughout the chromosome, they are not evenly spread, but rather have regions of high and low density. The SSGs are plotted on a chromosomal map in Figure romosome . The disromosome . Relaxatromosome . Here weWe have shown that supercoiling acts as a transcription factor, with positive and negative effects on specific genes while leaving the majority of the genome unchanged. Like other transcription factors such as TrpR  and ArcAE. coli K-12, strain MG1655 (Sigma-Genosys), were generously supplied by Fred Blattner (University of Wisconsin) and Carol Gross . ORFs were amplified from MG1655 genomic DNA using ExTaq polymerase (PanVera) and failed PCR reactions were attempted again using Platinum Taq (Invitrogen) or previous successful reactions as the DNA template. Ninety-six percent of the ORFs were successfully amplified. PCR conditions were set according to those supplied with the primers. DNA was precipitated with isopropanol and prepared for microarray printing as described in [tyrT, have been shown to respond to changes in supercoiling [Amino- and carboxy-terminal primers for protein-coding open reading frames (ORFs) of ribed in . We did rcoiling .Detailed instructions on slide preparation, microarray printing and processing microarrays can be found online . 384-welE. coli cells were grown with shaking in LB media to an OD600 = 0.45-0.55 at 37°C, or at 30°C for temperature-sensitive strains. Samples of cells were withdrawn at intervals and added to a 1/10 volume of either 95% ethanol plus 5% phenol or 2 M NaN3 to stop transcription. Cells were then quickly harvested by centrifugation in a microcentrifuge. The supernatant was aspirated and the pellets frozen in liquid N2. Total RNA was prepared using the Qiagen RNeasy mini kit, except that 4 mg/ml lysozyme and a 30 sec incubation was used in the first step. For each microarray, 20 μg total RNA was primed with 1-2 μg of random hexamers and labeled by reverse transcription in the presence of Cy3- and Cy5-conjugated dUTP (Amersham Biosciences). For each experiment or condition, a Cy5-labeled experimental sample was combined with a Cy3-labeled reference sample and hybridized to a processed microarray as described [escribed . After 5Scanned array images were visually inspected, and non-uniform spots were excluded from further analysis. The background was subtracted from the images that were then (median) normalized using the Scanalyze 2.0 program, v. 1.44  such that the total fluorescence in each channel was equal.E. coli genes, that could be considered for further study. Because we were interested in changes in expression levels due to variations in supercoiling rather than to drug or genetic effects, we used a two-sample comparison approach  rather than a factorial analysis approach. We tested two commonly used methods to determine differentially expressed genes in the comparison of two samples. We found that the method of Dudoit et al [et al [We tested several methods of imputation to estimate the values of spots missing due to hybridization defects (described in ), and afit et al , which cl [et al .32P-labeled DNA probes for gyrB and asnB (as a loading control) were synthesized from their respective PCR products, and radioactivity was quantified by a phosphorimager.Samples were run on formaldehyde-MOPS 1% agarose gels and blotted onto a nylon membrane . 32P-labgyrB234 mutant are in a C600 strain background, but all strains used have been described in greater detail elsewhere [ΔacrA strain that greatly slows drug efflux [0, where Lk0 for pBR322 = 4,361 bp/10.5 bp/turn = 415. Samples were run on parallel 20-cm gels containing 0, 2.8 or 10 μg/ml chloroquine for 18-26 h at 48-52 V with constant buffer recirculation, which allowed visualization of the entire distribution of topoisomers. Gels were southern blotted [32P-labeled probe made from random priming of pBR322. Radioactive blots were quantified using a phosphorimager.Plasmid DNA was extracted from cells by the alkaline lysis method  or the Qlsewhere ,35,73. Tg efflux . The supg efflux  from the blotted , and hybgyrB by both methods 5 min after addition of the gyrase inhibitor novobiocin to ΔacrA cells at 5, 20, 50 and 200 μg/ml. The microarray ratios for these concentrations were 2.3, 4.8, 4.9 and 6.3, respectively, while the ratios from northern hybridizations were 2.8, 4.7, 4.7 and 5.1. Second, as an internal control we compared the transcription of genes in 153 known polycistronic operons. We found no operons with genes that changed expression more than 1.5-fold in opposite directions (data not shown). Third, we compared two identically grown cultures with the same microarray . We used two strains that were isogenic, except that one had point mutations conferring norfloxacin resistance on gyrase and topo IV. The correlation coefficient of the gene-expression levels was 0.982, indicating the negligible variation between the cultures. In contrast, when we treated cells with the gyrase inhibitor norfloxacin , the correlation coefficient with respect to the untreated cells was only 0.391 and hundreds of genes showed large differences in expression. We conclude that gene-expression changes resulting from slight genotypic changes or experimental repeats were negligible compared with the changes resulting from topoisomerase inhibition, and that the E. coli microarrays are a reliable method for quantifying these changes.We tested the validity of our microarrays in three ways. First, we compared gene expression ratios measured with microarrays to values obtained by northern hybridization. We measured induction ratios for p-values < 0.05) over a total of 35 experiments, in which DNA gyrase was inhibited with novobiocin, norfloxacin or by a mutation that rendered gyrase temperature-sensitive. Next we determined the correlation of gene expression with the σ of a reference plasmid in the same cells. To calculate the correlation of gene expression to plasmid supercoiling, we created a model profile made up of the ratio of plasmid σ in each experiment to plasmid σ in the (supercoiled) reference for that experiment (Table We limited the list of SSGs to those whose expression difference between treatments and controls was statistically significant (nt Table . The maxThe following additional data files are available with the online version of this paper: Additional data file Data on the induction of the SOS response to DNA damageClick here for additional data fileGene-expression ratios for all genes across all experimentsClick here for additional data fileGene-expression ratios for supercoiling-sensitive genes across all experimentsClick here for additional data fileData on the reproducibility of microarray measurement of RNA levelsClick here for additional data file


Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction.We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny.The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations. We based our model on mathematical properties that alignment scores should respect; i) information theory      (22)a* and b* the random variables corresponding to the shuffled sequences of a and b respectively. The sum of the logarithms corresponds to the product of the two probabilities, an expression of the hypothesis of independence of lineage. Interestingly, equation (22) provides an expression of the symmetric effect of time on the variations that independently affected a and b.with t appears as a function of Z-score ratios. For any set of homologous proteins, it is therefore possible to measure a table of pair-wise divergence times and build phylogenetic trees using distance methods.From relation (20), , where numbrand is the number of randomizations. In the case studies presented here, we set numrand = 2000 (see Methods). By contrast, stability of MAB trees is sought by bootstrapping approaches and consensus tree reconstruction. MAB trees appear as the result of a complex learning process including possible re-adjustment of the multiple alignments after eye inspection pragmatically applied to assist the reconstruction. Alternatively, Bayesian analyses have been recently proposed for phylogenetic inference μ,+∞μ + ψ ,+∞[.In consequence, s0 is not trivial because it depends on the knowledge of the cumulative distribution function. Extensive studies provided experimental and theoretical supports for an extreme value distribution of alignment scores [i.e. the Gumbel distribution [The estimation of t scores ,43,44. Uribution , the cumθ and β (β > 0) the location and scale parameters. The probability density function g(s) is defined by dG(s) = g(s)ds. We observe with  that . Using the Taylor's polynomial formula, i.e. :with In consequence, for a Gumbel score probability distribution:ψ from a Gumbel distribution is illustrated in Figure A graphical determination of a and b is relatively high, that is s ≥ μ + ψ, then the trivial inequality s ≥ s impliesIf a pair-wise alignment score of two sequences s - μ)2)) ≥  - μ)2))     (h), we deduce that a and b, alignment was achieved with the Smith-Waterman method and the BLOSUM 62 scoring matrices, using the BIOFACET package from Gene-IT, France [z, z, z, z, with 2000 sequence shuffling. For all computations, an estimation of the Gumbel parameters θ and β was made using the computed μ and σ of any S and the formula  and θ = μ - βΓ'(1), where Γ'(1) ≈ 0.577216 is the Euler constant. In all computations, both Gumbel parameters were very close  = 35.04, SD(θ) = 0.12, mean(β) = 3.92, SD(β) = 0.08). As a consequence, the assumption Q ≈ Q was verified for any pair of sequences. We used the parameters to estimate μ = θ + βΓ '(1) , and μ + ψ ≈ μ + 10.5178 ≈ 47.85. As any pairs of computed scores are higher than this critical threshold, we used relation [To build PHYLIP trees, multiple sequence alignments were created with ClustalW . PHYLIP , France . We comprelation . Trees wCSHP, configuration space of homologous proteins, TULIP, theorem of the upper limit of a score probabilityOB conceived the main theoretical model, designed and developed the method to build phylogenetic trees and drafted the manuscript. SR and PO participated in the theoretical model refinement and in the design and development of computational methods to build TULIP trees. EM contributed to the conception of this study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Should we be considering the social and economic ramifications of a society where life-span could be limitless? Sinclair suggested, as have other experts, including his mentor Lenny Guarente and the National Institute on Aging advisory council member Elizabeth Blackburn, that Kass and other bioconservatives are creating a false alarm about life extension, because only a modest  increase in human life span is achievable by biomedical intervention, whereas Kass's apprehensions concern extreme or indefinite life extension. Kass retorted that science isn't like that: modest success tends to place the bit between our teeth and can often result in advances far exceeding our expectations.The biogerontologist David Sinclair and the bioethicist Leon Kass recently locked horns in a radio debate  of age-related molecular and cellular damage—consisting of just seven major categories of ‘rejuvenation therapy’ (I agree with these predictions in two respects: that the degree of life extension achieved by first-generation drugs of this sort may well approach the (currently unknown) amount elicitable by caloric restriction itself in humans, and that it is unlikely to be much exceeded by later drugs that work the same way. In two other ways, however, I claim they are incorrect. The first error is the assumption of proportionality: I have recently argued , from evescribed , 2004 a therapy’ —that appCoping with Methuselah and by other commentators is demographic. Life expectancy is typically defined in terms of what demographers call a period survival curve, which is a purely artificial construction derived from the proportions of those of each age at the start of a given year who die during that year. The ‘life expectancy’ of the ‘population’ thus described is that of a hypothetical population whose members live all their lives with the mortality risk at each age that the real people of that age experienced in the year of interest. The remaining life expectancy of someone aged N in that year is more than this life expectancy minus N for two reasons: one mathematical  and one biomedical . My spirits briefly rose on reading Aaron and Harris's explicit statement (p. 69) of the latter reason. Unfortunately, they didn't discuss what would happen if age-specific mortality rates fell by more than 2% per year. An interesting scenario was thus unexplored: that in which mortality rates fall so fast that people's remaining  life expectancy increases with time. Is this unimaginably fast? Not at all: it is simply the ratio of the mortality rates at consecutive ages (in the same year) in the age range where most people die, which is only about 10% per year. I term this rate of reduction of age-specific mortality risk ‘actuarial escape velocity’ (AEV), because an individual's remaining life expectancy is affected by aging and by improvements in life-extending therapy in a way qualitatively very similar to how the remaining life expectancy of someone jumping off a cliff is affected by, respectively, gravity and upward jet propulsion . The escape velocity cusp is closer than you might guess. Since we are already so long lived, even a 30% increase in healthy life span will give the first beneficiaries of rejuvenation therapies another 20 years—an eternity in science—to benefit from second-generation therapies that would give another 30%, and so on ad infinitum. Thus, if first-generation rejuvenation therapies were universally available and this progress in developing rejuvenation therapy could be indefinitely maintained, these advances would put us beyond AEV. Universal availability might be thought economically and sociopolitically implausible , so it's worth considering the same question in terms of life-span Coping with Methuselah is representative, is the most significant because of its urgency. First-generation rejuvenation therapies, whenever they arrive, will surely build on a string of prior laboratory achievements. Those achievements, it seems to me, will have progressively worn down humanity's evidently desperate determination to close its eyes to the prospect of defeating its foremost remaining scourge anytime soon. The problem  is that this wearing-down may have been completed long before the rejuvenation therapies arrive. There will come an advance—probably a single laboratory result—that breaks the camel's back and forces society to abandon that denial: to accept that the risk of getting one's hopes up and seeing them dashed is now outweighed by the risk of missing the AEV boat by inaction. What will that result be? I think a conservative guess is a trebling of the remaining life span of mice of a long-lived strain that have reached two-thirds of their normal life span before treatment begins. This would possess what I claim are the key necessary features: a big life extension, in something furry and not congenitally sick, from treatment begun in middle age.The third oversight that I observe in contemporary commentaries on life extension, among which ten years, given adequate and focused funding (perhaps $100 million per year). And nobody in Coping with Methuselah said so. This timeframe could be way off, of course, but as Wade notes (p. 57), big advances often occur much sooner than most experts expect. Even the most obvious of these lifestyle changes—greater expenditure on traditional medical care, avoidance of socially vital but risky professions—could severely destabilise the global economy; those better versed in economics and sociology than I would doubtless be even more pessimistic about our ability to negotiate this period smoothly. Overpopulation, probably the most frequently cited drawback of curing aging, could not result for many decades, but the same cannot be said for breadth of access irrespective of ability to pay: in a post-9/11 world, restricted availability of rejuvenation therapies resembling that seen today with AIDS drugs would invite violence on a scale that, shall we say, might be worth trying to avoid.It is the prospect of AEV, of course, that makes this juncture so pivotal. It seems quite certain to me that the announcement of such mice will cause huge, essentially immediate, society-wide changes in lifestyle and expenditure choices—in a word, pandemonium—resulting from the anticipation that extreme human life extension might arrive soon enough to benefit people already alive. We will probably not have effective rejuvenation therapies for humans for at least 25 years, and it could certainly be 100 years. But given the present status of the therapies listed in Coping with Methuselah (p. 143). Once AEV is achieved, there will be no going back: rejuvenation research will be intense forever thereafter and will anticipate and remedy the life-threatening degenerative changes appearing at newly achieved ages with ever-increasing efficacy and lead time. This will bring about the greatest economic change of all in society: the elimination of retirement benefits. Retirement benefits are for frail people, and there won't be any frail people. The graph just mentioned amply illustrates how much wealth will be released by this. My hope, therefore, is that once policy makers begin to realise what's coming they will factor in this eventual windfall and allocate sufficient short-term resources to make the period of limited availability of rejuvenation therapies brief enough to prevent mayhem. This will, however, be possible only if such resources begin to be set aside long enough in advance—and we don't know how long we have.Am I, then, resigned to a future in which countless millions are denied many decades of life by our studied reluctance to plan ahead today? Not quite. The way out is pointed to in