The Endocrine Disruptor Screening Program (EDSP), mandated by the United States Congress in the Food Quality Protection Act of 1996, attempts to protect public health from adverse endocrine effects of synthetic chemical compounds by establishing a new testing regime. But the complexities and uncertainties of endocrine disruption and its broader regulatory and social context all but ensure the failure of this policy. This article addresses the issues facing EDSP comprehensively and in detail, in order to move beyond the current regulatory paradigm and foster discourse on a positive role for scientists in support of EDSP's end goal: to protect public health. The United States Environmental Protection Agency (EPA) created the Endocrine Disruptor Screening Program (EDSP) to regulate endocrine-disrupting chemicals (EDCs) as mandated in the Food Quality Protection Act of 1996 (FQPA) and the Safe Drinking Water Amendments Act of 1996 (SDWAA). Unlike the more easily appreciated effects of toxic chemicals, EDCs interact with the human body indirectly by mimicking, blocking, or otherwise disrupting the normal function of hormones. The prolific study of endocrine disruption has since uncovered many unconventional and worrisome mechanisms, exposures, and effects [[D]evelop a screening program, using appropriate validated test systems and other scientifically relevant  information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or such other endocrine effects as the [EPA] Administrator may designate [esignate .If such an effect is discovered, "the [EPA] Administrator shall, as appropriate, take action under such statutory authority as is available...as is necessary to ensure the protection of public health" .Unfortunately, due to four complicating factors, EDSP cannot protect public health. The first complication is practical considerations. EPA estimates the universe of potential EDCs numbers more than 87,000 items. Testing this many chemicals would take an unreasonable investment of time and resources, but even scientifically prioritizing among them is highly problematic. The second complication is hazard complexity. Establishing relationships between EDCs and health hazards proves very difficult if not impossible. Endocrine-disrupting action breaks all the rules and assumptions that have guided toxicology through the era of modern chemical regulation. Without these simplifying assumptions, science cannot establish causation efficiently or with sufficient certainty for regulation. The third complication is exposure complexity. Determining exposure levels becomes more important and more difficult because the low-dose effects of many EDCs means that low-dose and transient exposure can be just as or more dangerous than high-dose and prolonged exposure. Assessments typically discount these ill-defined exposures, but we can no longer assume them insignificant. The final complication is regulatory deficiencies. Although FQPA and SDWAA provided new authority to test for endocrine disruption, they provided no new authority for the regulation of EDCs. As a result, multiple government agencies must manage future test-positive EDCs under their jurisdiction using fragmentary and incomplete statutory authorities and different regulatory standards. This introduces significant confusion to the institutional and decision-making aspects of the EDSP regulatory framework.and legal standards, and an ethical position that prioritizes profit over human health by placing the burden of proof on public and environmental health advocates. These assumptions remain all but unchallenged in the U.S. context, and thus comprise a paradigm. While this paradigm has faced some critique in the context of carcinogenic pesticides and toxic industrial chemicals, questions of its efficacy remain unresolved. Because EDCs present new and fundamental difficulties for the science underlying the regulatory paradigm, a critical analysis of EDSP provides a more compelling case that the current chemical regulatory paradigm is in need of radical revision.The EDSP policy design represents revision at the margins of U.S. chemical regulatory policy, not a radical revision. EDSP employs the same basic strategy used to regulate carcinogenic pesticides or toxic industrial chemicals – scientifically proving harm prior to regulating a chemical. Two important aspects of this strategy include an epistemological assumption that science has the capacity to 'prove' harm under the relevant scientific This study investigates the policy design of EDSP and its broader context. The above four complications play varying roles in each stage of the EDSP policy design as discussed below. See figure The following discussion provides a comprehensive empirical basis for considering alternatives to the status quo. This study aims to integrate the many factors conditioning the failure of EDSP for the purpose of fostering constructive discussion on U.S. regulatory policy concerning EDCs and chemicals more generally. The author does not possess a unique answer to the many and complicated issues surrounding endocrine disruption and the U.S. chemical regulatory paradigm. Given that no simple, well-developed alternatives exist that merit immediate consideration by decision-makers, it stands to reason that more creative and open discussions of EDCs, the chemical regulatory paradigm, and possible roles for the scientific community may provide long-term payoffs in public and environmental health protection well worth our attentions today.The design of EDSP consists of three main stages: priority setting, screening and testing, and a risk analysis leading to potential regulation. The complications shown in figure Politics section below). Politics and policy design play significant roles in the modern chemical regulatory regime. Hence, a comprehensive analysis of EDC regulation must take politics and policy design as well as science into account. The appropriate standard by which to judge these disparate policy elements is Congress's mandated end goal: to protect public health. We will return to this issue.In other words, there is reason and precedent to doubt our ability to accomplish this feat. More importantly, despite our best efforts to mobilize science in support of this difficult task, policy mechanisms allow chemical use and abuse to go forward regardless of scientific results or lack thereof. "Pesticides are registered for use while important health and safety data are still being generated; they may continue to be used after evidence of their hazards is given to EPA; they may be registered through alternative processes that bypass important tests; and they may never be required to be tested for certain kinds of hazards"  and quantitative structure activity relationships (QSAR) both discussed below, have attracted attention and resources due to a general recognition of this problem. But more important than our current lack of data, EDCs operate with a high degree of complexity. Because of system complexity, some uncertainty about endocrine disruption probably cannot be resolved – resulting in some abiding doubt about the significance of a chemical as a potential endocrine disruptor. The scientific community and EPA seem quite cognizant of this complexity, but its relevance for policy and for the end goal of protecting public health deserves careful attention.EPA has not realized their ideal of priority setting based on hazard and exposure information because of the catch-22 mentioned above. As a result, "EPA's proposed approach focuses on human exposure-related factors rather than on a combination of exposure- and [hazard]-related factors" . While tScreening and testing section below) implies that exposure to some EDCs at extremely dilute doses may have a greater effect than massive exposure to that same chemical. Transient or low-concentration EDCs may also pose a greater risk than other high-exposure chemicals. Low-dose effects and other exposure complexities make exposure alone a poor proxy for setting priorities. Since different vulnerabilities and sometimes different health effects manifest at different developmental stages, any exposure-only judgment will run into significant difficulties defining spatial and temporal boundaries for exposure determinations. Some short-lived chemicals may have important endocrine- disrupting effects, but may not show up in EPA's most robust exposure data sources: biological sampling and environmental monitoring [To set priorities based on exposure factors alone, one must assume greater exposure to a chemical implies greater potential hazard (or some other arbitrary assumption). For EDCs, this assumption is scientifically insupportable. The complexity of low-dose effects  in the first place. While these limitations may or may not be overcome in time, at present the methodology is not useful for setting priorities. It is instructive, however, to consider the justifications for the development of QSAR. "Systematic toxicity testing, using conventional toxicology methodologies, of single chemicals and chemical mixtures is highly impractical because of the immense numbers of chemicals and chemical mixtures involved and the limited scientific resources" . QSAR waTo amass the evidence necessary for regulation, EPA designed two tiers of scientific assays. Tier 1 screening involves short-term assays to detect potential chemical interaction with the endocrine system. Tier 2 testing involves long-term assays to establish such interactions, explore more complicated endpoints, and establish dose-response relationships. If enough data exists, a chemical can go straight to Tier 2 testing. Otherwise chemicals are assigned to Tier 1, where chemicals are prioritized and screened, with all positive results forwarded for Tier 2 testing , whiThe monotonic assumption allows for statistically significant results using smaller sample sizes exposed to higher doses for shorter periods of time. Linearly scaling these results down to typical exposure levels presumably yields approximate quantitative rates of, for example, disease or cancer. Abandoning this assumption decreases testing efficiency and multiplies the time and other resources necessary to understand the potential hazard posed by a chemical. A quote from University of Washington, Seattle toxicologist David Eaton, states the issue simply: "It's just too expensive ... you'll never be able to characterize [a low-dose effect] to the point where people think it's real" . Non-monOther factors complicate a scientific determination of hazard. Two chemicals can interact in ways that alter their effects. Some chemicals together inhibit their individual effects, reducing or preventing an adverse effect where one is expected. Others simply add their effects together, and yet others interact synergistically, magnifying the effect either or both would normally have. "Synergistic interactions are the most problematic, because they indicate that the effects of multiple chemicals together can be significantly more powerful than might be predicted simply by adding up their effects one at a time. Regulatory science rarely incorporates any interactions; it is incapable, at present, of coping with synergies" . RegardlAnother serious complication involves the selection of testing endpoints, or dysfunctions possibly caused by EDCs. Some of the dysfunctions already identified through animal studies include cancer susceptibility and birth defects, but also more subtle endpoints like immunological dysfunction, suppression of secondary sex characteristics, decreased fertility, increased aggression, decreased mental capacity and focus, disrupted brain development, etc ,35. WhilA risk analysis concludes the EDSP policy design. EPA claims it will use its standard human health risk assessment process for EDCs . Simply Priority setting section and the hazard complexities treated in the Screening and testing section. These complexities include: HAZARD – low-dose effects, mixtures and synergies, and uncertain endpoints; and EXPOSURE – transient and low-concentration exposure to EDCs, maternal metabolism of bioaccumulated EDCs, varying vulnerability and response by developmental stage, poorly understood exposure sources, vulnerable subpopulations, and cultural, social, and economic patterns and spatial and temporal bounds of chemical release and exposure. More science cannot resolve all of these complexities and uncertainties.Because EPA's risk analysis demands an explicit integration of hazard and exposure data, the risk assessment itself is vulnerable to the exposure complexities treated in the in utero might easily be a chaotic or stochastic system. Ambiguity refers to a situation in which existing scientific data support equally valid but competing interpretations of risk (see [But these complexities translate into an even more pernicious difficulty, as decisions assumed answerable by science must be made under conditions of scientific ambiguity. Ambiguity is related to complexity as follows: Complexity refers to properties of the system under study, not the study itself. Such properties include interactive effects (like synergies), feedback loops, temporal delays between cause and effect, chaotic or stochastic system behavior, large numbers of intervening variables, and inter-individual variations. Uncertainty refers to limitations of the human analysis of a complex system. While science is often employed 'to reduce uncertainty,' the complexity of a system sets bounds on the prospective certainty of informed scientific judgment. For example, further science can never remove the chaos from a chaotic system or the randomness from a stochastic one. And to illustrate the point, exposure of a fetus to environmental EDC contamination isk , the Federal Food, Drug, and Cosmetic Act (FFDCA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), or the Safe Drinking Water Act (SDWA). The agencies with regulatory jurisdiction for EPA's list of 87,000 chemicals are: the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) in the U.S. Department of Health and Human Services, and the Food Safety and Inspection Service (FSIS) in the U.S. Department of Agriculture. EDSTAC recommended and EPA adopted the following list of chemicals for endocrine disruptor testing: 75,500 commercial chemicals listed under TSCA, 900 pesticide active ingredients, 2,500 pesticide inert ingredients, 5,000 cosmetic ingredients, 3,000 food additives, an unspecified number of nutritional substances, and an unspecified number of natural hormonally active plant residues .Testing and enforcement authority for this universe of chemicals is fragmentary. For example, EPA has authority under FIFRA and FFDCA to set tolerances for pesticides on food, but enforcement authority falls to FSIS for meat and poultry products, and FDA for other food items. The authority is also incomplete. For example, FDA has authority over the estimated 5,000 cosmetic chemicals, but FDA has no authority to require any information from the manufacturer or to mandate product safety testing. FDA's regulatory authority over cosmetics begins only after a product (possibly without any safety information) enters the market. Additionally, the standards by which to regulate differ. Under TSCA and SDWA the economic costs of regulation must be balanced against the public health threat, but under FIFRA and FFDCA, economics can be considered in only narrowly crafted situations – the standard is largely health-only based. FFDCA and FIFRA as amended by FQPA use the "reasonable certainty of no harm" standard. This standard translates into a 95% certainty that fewer than one in a million additional cancer deaths will occur due to the expected exposure over a lifetime. No translation specific to EDCs of this standard is yet available. TSCA, on the other hand, must prevent "unreasonable risk" of injury to health or the environment. A risk is "unreasonable" if the risks exceed the benefits associated with that activity, including economic benefits. SDWA explicitly requires a consideration of the cost of compliance to state, local, and other water systems when setting safety standards.The estimated 2,500 pesticide inert ingredients may defy regulation due to trade secret norms, poor EPA data quality, and historic government neglect ,40. AddiThe complexity of the institutional and legal landscape (multiple interacting agencies with multiple overlapping mandates and authority) creates substantial regulatory confusion. In this situation, the powers and responsibilities of different government agencies might be interpreted differently by other agencies or by affected parties. Such confusion leaves room for interpretation that may require long delays and intensive court battles to resolve. The history of TSCA indicates that such confusion  as well as the menace of legal action can lead to crippling regulatory inaction . By requFinally, the role of politics in regulatory decision-making deserves note. The conventional ideal of regulation under the current paradigm is that good science leads to an informed decision-maker who can then remove or limit a proven hazardous chemical from commerce (or, rarely, prevent its introduction). The complexities of endocrine disruption science, practical considerations, and the regulatory deficiencies discussed above impose limitations on this conventional ideal. Neglecting the role of politics in regulatory decision-making, however, is perhaps this ideal's most significant omission. A variety of academic and government research as well as environmental and public interest group analysis points to the failure of testing regimes to produce significant regulation or protect public health . Hazard complexities, including low-dose effects, mixtures and synergies, and uncertain endpoints, contribute further obstacles to an unambiguous scientific determination of harm (see Screening and testing section). Under current policy mechanisms, these complications of endocrine-disruption science will prevent any meaningful regulatory action. Essentially, endocrine disruption is too complex and our science too uncertain; most scientific information regarding EDCs will remain ambiguous, with the available information supporting quite different judgments of risk.The internal validity of 'science leads to regulation' presumes the capacity of science to prove harm with sufficient certainty to regulate. Exposure complexities, including transient and low-concentration exposure to EDCs, maternal metabolism of bioaccumulated EDCs, varying vulnerability and response by developmental stage, poorly understood exposure sources, vulnerable subpopulations, and cultural, social, and economic patterns and spatial and temporal bounds of chemical release and exposure, place the ability of science to make solid exposure determinations in significant doubt  came to the conclusion that the failure of testing in the past means we need to test better in the future ,41. ThouThe relevance of science for regulatory decision-making lies predominantly outside the current trend of increasingly detailed mechanistic investigation of endocrine disruption. While endocrine disruption science can contribute much to a decision-maker, it cannot provide unambiguous information that 'objectively' determines the correct decision. But science could help guide decision-making under conditions of ambiguity; uncertainty does not entail 'anything goes.' On the contrary, the uncertainty itself might guide decision-making better than any other factual information. The 2001 Intergovernmental Panel on Climate Change reports provide a precedent in the explicit treatment of uncertainty – the reports indicate the relevance of scientific information for decision makers by ranking scientific conclusions by Bayesian confidence estimates  . This emA franker treatment of uncertainty can improve the relevance of science to decision-making and promote realistic expectations of science and the scientific community. Such progress might turn the spotlight on more significant impediments to regulatory decision-making under conditions of ambiguity, such as the difficulty of testing 87,000 chemicals and the role of politics and values in making regulatory decisions. Scientists can take action within their own communities to support the policy goal of protecting public health by exploiting and improving the role scientific information plays in chemical regulation. The possibility that scientists can empower decision makers and the public by developing community standards and norms explicitly addressing uncertainty is one creative idea, but more are needed. The key to progress is taking an active and creative role in support of the protection of public health. Waiting for a solution in the form of national legislation has failed to inspire significant change over the last several decades. But explicit action within the scientific community that discourages unrealistic expectations of science will support long-term progress on chemical regulatory policy and the protection of public health.DDT – dichlorodiphenyltrichloroethaneEDC(s) – endocrine disrupting chemical(s)EDSP – (U.S.) Endocrine Disruptor Screening ProgramEDSTAC – Endocrine Disruptor Screening and Testing Advisory CommitteeEPA – (U.S.) Environmental Protection AgencyFDA – (U.S.) Food and Drug AdministrationFFDCA – (U.S.) Federal Food, Drug, and Cosmetic ActFIFRA – (U.S.) Federal Insecticide, Fungicide, and Rodenticide ActFSIS – (U.S.) Food Safety and Inspection ServiceFQPA – (U.S.) Food Quality Protection ActHPV – high production volumeHTPS – high-throughput pre-screeningPCBs – polychlorinated biphenylsQSAR – quantitative structure activity relationshipsSDWA – (U.S.) Safe Drinking Water ActSDWAA – (U.S.) Safe Drinking Water Amendments ActTSCA – (U.S.) Toxic Substances Control ActThe author(s) declare that they have no competing interests.


Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity. TGFβ family members and their receptors are involved in setting up the left-right body axis early in development. This article clarifies the role of Lefty and elucidates the molecular basis for signaling diversity between the family members The analysis of whole-genome sequences has revealed that most signaling systems consist of multiple ligands that converge on a relatively small set of receptors and pathway-specific transcription factors. In the case of human transforming growth factor-β (TGFβ) signaling, 42 TGFβs converge on seven type I receptors, five type II receptors, and two classes of Smad signal transducers (reviewed in oep) lack all endoderm and most mesoderm, similar to the double mutants for the zebrafish Nodal-related genes cyclops and squint (sqt)  . Moreove mutants . During  mutants .The EGF-CFC protein Cripto is highly overexpressed in human epithelial cancers, such as breast and colon carcinomas , or zebrafish Vg1 (a chimeric molecule containing the Xenopus ActivinβB prodomain fused to the mature domain of zebrafish Vg1) in zebrafish embryos (goosecoid (gsc). ActivinβB, ActivinβA, Sqt, and Vg1-induced ectopic gsc expression in wild-type embryos  expression was only weakly induced , receptors (mouse ActRIIB[KR]/Myc and human Alk4[KR]/Flag), and a coreceptor (mouse Cripto/Flag) in  embryos . Similar embryos , Sqt for embryos A. In con embryos A. Since  embryos , Lefty s embryos B. Since  embryos , these t embryos C. To detep zebrafish embryos and analyzed the ectopic induction of the downstream genes ntl and gsc. Sqt, Vg1, and Activins induced these genes in wild-type embryos, allowing us to determine which chimeric ligands were active. Activins, but not Sqt or Vg1, were active in MZoep mutants, allowing us to test which sequences conferred EGF-CFC coreceptor dependence or independence.The finding that TGFβ ligands that activate Activin receptors can be grouped into a EGF-CFC coreceptor-dependent class that is susceptible to inhibition by Lefty  and a class that is independent of EGF-CFC proteins and resistant to Lefty (Activin) prompted us to examine which sequences underlie this ligand diversity. We therefore generated chimeras between Activins (EGF-CFC-independent) and Sqt or Vg1 (EGF-CFC-dependent) Figures  and 5. Aoep embryos  were sufficient to confer EGF-CFC independence. Further dissection of this region into loop alone (SqtActβB[loop]) or β8 alone (SqtActβB[β8]) yielded no or much weaker activity in MZoep mutants as compared with wild-type embryos  and Vg1ActβB[loopβ8] (Vg1 with an ActivinβB loop-β8 region) both induced gsc expression in MZoep mutants with similar efficiencies as in wild-type embryos  and Vg1. Sqt embryos D. These  embryos E and sugActβB[loopβ8] can signal in an EGF-CFC-independent manner in vivo, suggesting that this chimeric protein might bind to ActRIIB and Alk4 receptors in the absence of EGF-CFC coreceptors. To test this idea, we coexpressed and immunoprecipitated epitope-tagged ligands (ActivinβB/HA, Sqt/HA, SqtActβB[loopβ8]/HA), receptors (ActRIIB[KR]/Myc and Alk4[KR]/Flag), and a coreceptor (Cripto/Flag) in Xenopus embryos  in Sqt did not enhance activity in MZoep mutants. These results suggest that multiple residues contribute to coreceptor independence, with the type II receptor-binding interface being an essential determinant.An alignment of the loop-β8 region of EGF-CFC-dependent and EGF-CFC-independent TGFβs A revealson assay . All conoep mutants. Chimeras containing the Sqt Finger 2 domain . The addition of Finger 1 in SAS or ActSqt[Finger1-loopβ8] relieved the inhibitory effect of the loop-β8 region of Sqt in wild-type embryos , previous studies have suggested that heterologous Nodal, Lefty, and EGF-CFC proteins have similar activities in zebrafish . For exaLefty1 and left-side specific Lefty2 (ΔASELefty2) mutants have been thought to be caused by inappropriate spread of Nodal signaling  fused to an HA-epitope/XhoI fragment (YPYDVPDYALE) and followed by the mature chimeric ligand into pcDNA3 vector. S denotes Sqt; A denotes ActivinβB. The boundaries for Sqt mature ligand domains are as indicated: Finger 1 (Asn263 to Cys325), Heel (Pro326 to Cys358), and Finger 2 . The boundaries for Xenopus ActivinβB mature ligand domains are as indicated: Finger 1 (Cys215 to Cys299), Heel (Pro300 to Cys335), and Finger 2 (Ile336 to Ala370). Full-length chimeras were generated by incorporating the indicated regions into Sqt, ActivinβB, or zebrafish Vg1, which were then subcloned into the pT7TS vector , loop (Phe348 to Ile354), β8 , and β9 (Met357 to Ala370). The Xenopus ActivinβA loop-β8 region sequence is FDRNNNVLKTDIAD . The zebrafish Vg1 loop-β8 region is from Try332 to Asp345. pcDNA3-zebrafish Vg1/HA, pcDNA3-Squint/HA, pCS2-Alk4(KR)/Flag (a kinase-defective mutant of human Alk4 with Lys234 to Arg234 substitution), pCS2-ActRIIB(KR)/Myc (a kinase-defective mutant of mouse ActRIIB with Lys217 to Arg217 substitution), and pCS2-Cripto/Flag have been described elsewhere  Sqt/ActivinβB chimeras were generated by subcloning the prodomain of S vector . The boulsewhere .Xenopus embryos at the one- to two-cell stage were used for injections into the animal pole.Plasmids were linearized and sense strand-capped mRNA was synthesized using the mMESSAGE mMACHINE system (Ambion, Austin, Texas, United States). Zebrafish embryos were dechorinated by pronase treatment and injected between the one- and four-cell stage. gsc and ntl (Zebrafish embryos at 24 h were mounted in 2% methylcellulose and photographed using a Zeiss (Oberkochen, Germany) M2Bio dissecting microscope. In situ hybrization was performed as described previously , using R and ntl .LacZ mRNA. The A3-luc reporter DNA construct (25 pg)   was alsoXenopus embryos were harvested at stage 10. For chemical cross-linking of proteins, animal halves were incubated in PBS with 10 mM 3,3′-dithiobis(sulfo-succinimidyl propionate) (DTSSP) (Pierce Biotechnology, Rockford, Illinois, United States) and incubated for 2 h on ice. Coimmunoprecipitation was performed as described previously (eviously . Purifieeviously . Sampleshttp://www.ncbi.nlm.nih.gov/entrez/query.fc-gi?db=Nucleotide) accession numbers for the sequences discussed in this paper are ActivinβA (Q9W6I6), ActivinβB (Q91350), human ActRIIB (P08476), mouse ActRIIB (P27040), rat ActRIIB (P38445), Alk4 (Z22536), BMP7 (P23359), Cripto (P51865), Cryptic (P97766), cyclops (P87358), GDF1 (P20863), goosecoid (P53544), Lefty1 (Q9W6I6), Lefty2 (P57785), mLefty1 (Q64280), Nodal (P43021), no tail (Q07998), one-eyed pinhead (O57516), Pitx2 (P97474), squint (O13144), VEGF-D (P97946), and Vg1 (P09534).The GenBank (


Drosophila melanogaster for two consecutive years; susceptibility to three organophosphates tended to increase in the fall. To examine the hypothesis that variation in fitness among resistant and susceptible genotypes could trigger the change of genetic constitution within the fall population, we investigated density-independent population projection trajectories starting from single adult females with characteristics of chromosome-substituted lines resistant and susceptible to the three organophosphates.Seasonal fluctuations in susceptibility to organophosphate insecticides were observed in the Katsunuma population of Density-independent population projection trajectories, expressed as the ratios of the number of each chromosome-substituted line to that of line SSS, for which all chromosomes were derived from the susceptible line, showed significant declines in numbers with time for all the resistant chromosome-substituted lines.The declining tendency in the density-independent population projection trajectories of the resistant chromosome-substituted lines could explain the simultaneous decline in the levels of resistance to the three organophosphates, observed in the Katsunuma population in the fall. The development of insecticide resistance in insect pest populations is a population genetic process, in which insecticides select for initially rare resistant mutants within a population . AlthougR factors that, in every respect except insecticide resistance, are deleterious from a survival standpoint.""Since the genes causing insecticide resistance were at low frequency in the population before the insecticide began to be applied, it must ordinarily be true that they are to some extent disadvantageous; otherwise they would have been common. Therefore the selection for resistance should ordinarily involve the replacement of the original genes with Under this theory, it could be expected that there is variation in fitness among resistant and susceptible genotypes, and that resistant genotypes have lower fitness than susceptible genotypes, which could result in the change of frequencies of resistance factors within a population.Drosophila melanogaster is one of the well-established natural populations in Japan [Drosophila flies drastically increase in number on them [The Katsunuma population (Yamanashi Prefecture, Japan) of in Japan . Katsunu on them ,5. In th on them ,7. LowerIn the Katsunuma population, the drastic change in food and breeding environments occurs from summer to fall. Almost density-independent conditions are suddenly created by masses of squeezed grapes. However, the period of these conditions might not be long enough for the population to attain a stable age-structure; rapid expansion and contraction occur during a rather short period of time. In fact, the Katsunuma population rapidly decreased in mid-November . Therefo7 on Day 50 and 3.6 × 1013 on Day 100, starting from a single adult female . The frFollowing the previous study, which introduced calculating the intrinsic rates of increase for the resistant and susceptible chromosome-substituted lines, this study sheds some more light on the genetic basis of the seasonal fluctuations in susceptibility to three organophosphate insecticides, observed within the Katsunuma population for two consecutive years . Our resVariation in fitness among resistant and susceptible genotypes could change the frequencies of the resistant genotypes in the fall Katsunuma population, where almost ampler density-independent conditions are suddenly created by masses of squeezed grapes dumped outside. Because the third chromosome of the resistant lines confers resistance to all of the three organophosphates , a signiIn this study, we calculated population projection trajectories of chromosome-substituted lines resistant and susceptible to three organophosphate insecticides. All the resistant lines showed a significant tendency toward decrease in the numbers relative to line SSS. This study strongly supports the hypothesis that there is variation in fitness among resistant and susceptible genotypes, and that resistant genotypes have lower fitnesses than susceptible ones.w; Sp/SM1; Pr Dr/TM3 [D. melanogaster population had not yet started expanding [D. melanogaster [We obtained vital statistics from chromosome-substituted lines, whose chromosomes were derived from a resistant inbred line #609-10 and a susceptible inbred line #451-4, constructed by using a balancer stock, r Dr/TM3 ,15. Bothxpanding . The resxpanding . AlthougIn this paper, each chromosome-substituted line was represented by its chromosome composition. For example, SRS indicates the chromosome-substituted line possessing the X and third chromosomes derived from the susceptible line #451-4 (S) and the second chromosome from the resistant line #609-10 (R).Density-independent population projection trajectories from a single adult female were obtained for each chromosome-substituted line, after constructing a 19 × 19 Leslie matrix for each line ), age-specific survival probabilities ), and zero [P(x) values lie between 0 and 1, whereas f(x) values are necessarily more than or equal to zero [f(x), some values are zero, depending on the reproductive schedule of the organisms concerned [n(t), whose components are numbers of each age-class, the number of each age-class at the next time unit, n(t + 1), can be calculated as n(t + 1) = n(t)L [A Leslie matrix consists of elements, which are age-specific fecundity (the first column in this matrix form, and zero . P(x) va to zero . For ageoncerned . By mult = n(t)L .To construct a Leslie matrix, age-specific survival probabilities and fecundity were based on demographic data, collected for each individual adult fly cf. ,20). The. The20])In the previous study, age-specific fecundity and survival probabilities over 19 days, based on each individual, were used for calculating the intrinsic rate of increase for each replication. In this study, the age-specific fecundity and survival probabilities over 19 days were used to construct a 19 × 19 Leslie matrix Fig. , after aDensity-independent population projection trajectories were estimated as the ratios of the number of each chromosome-substituted line, totaled over all age-classes, to that of line SSS for 100 days. 95% upper confidence bounds  were seTM carried out data collection and analyses, and drafted the manuscript. BC contributed to the computer programming and provided the theoretical background. All authors read and approved the final manuscript.


PLoS Biology.)Since the days of ancient Greece, mathematics has been used to describe the world in the hopes of identifying underlying laws of nature. Physicists have long relied on mathematics to understand the behavior and interaction of particles too small to observe directly. Since it's not always possible to determine the behavioral properties of a single atom or electron, physicists characterize the behavior of these particles in terms of probability and the law of averages. Likewise, it's not always easy to tell how a single protein contributes to the behavior of a cell or organism. Faced with increasingly immense datasets—from genomes, proteomes, gene expression networks, cell signaling pathways, and more—biologists are turning to the tools of higher mathematics. High-throughput technologies like genome sequencers and microarrays generate a global picture of genomic or cellular activity, but such datasets have a high noise-to-signal ratio—the details are often subject to multiple interpretations. One way computational methods can help separate the signal from the noise is by determining the likelihood of a given set of interactions and presenting a range of possible network behaviors. When sufficient information about a biological pathway is available, experimental evidence can enhance modeling approaches to help refine the nature and role of putative network behaviors.  and protein products produce the characteristic 14-segment polarity pattern. While the molecules and pathways that generate the segment polarity pattern are well known, little is known about the quantitative nature of their interactions: in what concentrations do the components (for example, mRNAs and proteins) exist and what parameters (for example, binding constants, transcription rates, and gene product life spans) govern their interactions?One model system ripe for computational analysis is the fruitfly Four years ago a group of researchers led by George von Dassow developed a model of the genetic interactions that define segment polarity, called the segment polarity network. The model used a parameter set of 48 numerical values for each computer simulation of the segment polarity pattern. Since quantitative information about the network was unavailable, the group used random values for each of the parameters, repeating the simulation for nearly 250,000 different random parameter sets. The model proved remarkably robust—the network output was largely insensitive to variation in parameter values, with a surprisingly large fraction of random parameter sets generating the desired segment polarity pattern. That so many random variables could produce the pattern means either that almost any set of parameter values can work or that only a few of the parameters are important. Now, Nicholas Ingolia reveals the mechanism accounting for this robustness and bolsters the model with recent experimental evidence.wingless (wg) and engrailed (en), within specific cells of a “prepattern” laid down early in embryogenesis is converted into the segment polarity pattern by an intercellular signaling network. Wg and en operate through positive feedback loops that activate their own expression, a process that is destined to end up with individual cells in one of two stable states of gene expression . Since each stable state is intrinsically robust—that is, resistant to changing parameters—Ingolia hypothesized that the parameters that generate the robustness of the segment polarity pattern in von Dassow's model are those that produce this bistability.To investigate the reason for the original model's robustness, Ingolia asked whether the parameters of the model could be deconstructed into the properties of individual cells. It's known, for example, that the stable expression of two genes, called wg and en expression. The overall pattern of the model, as well as its insensitivity to parameter variation, Ingolia concludes, emerges from the stable expression states of single cells. Parameters that do not produce bistability within single cells, Ingolia found, almost never generate the correct pattern, while those that do produce bistability are much more likely than randomly chosen parameters to generate the striped segment pattern. When Ingolia added new experimental variables to the model—the signaling protein produced by the sloppy-paired gene and its interactions with en—he could reduce the fraction of parameter sets that satisfied the bistability requirement but nonetheless failed to produce the segment polarity pattern, refining the model to reflect the realities of the cell.Using computational methods to simulate the behavior of individual cells, Ingolia shows that individual cells in the original model adopt three different stable states of Such computational approaches are allowing biologists to gain valuable insights into the real-world properties and behavior of staggeringly complex biological networks. It's been over 2,000 years since Pythagoras proposed that the laws of heaven and earth reflect a numerical harmony rooted in mathematical laws. Whether that notion holds for biology, bit by bit the tools of higher mathematics are peeling back the layers of complexity to identify underlying properties of living systems.


Vestibular rehabilitation (VR) is a well-accepted exercise program intended to remedy balance impairment caused by damage to the peripheral vestibular system. Alternative therapies, such as Tai Chi (TC), have recently gained popularity as a treatment for balance impairment. Although VR and TC can benefit people with vestibulopathy, the degree to which gait improvements may be related to neuromuscular adaptations of the lower extremities for the two different therapies are unknown.We examined the relationship between lower extremity neuromuscular function and trunk control in 36 older adults with vestibulopathy, randomized to 10 weeks of either VR or TC exercise. Time-distance measures (gait speed, step length, stance duration and step width), lower extremity sagittal plane mechanical energy expenditures (MEE), and trunk sagittal and frontal plane kinematics (peak and range of linear and angular velocity), were measured.Although gait time-distance measures were improved in both groups following treatment, no significant between-groups differences were observed for the MEE and trunk kinematic measures. Significant within groups changes, however, were observed. The TC group significantly increased ankle MEE contribution and decreased hip MEE contribution to total leg MEE, while no significant changes were found within the VR group. The TC group exhibited a positive relationship between change in leg MEE and change in trunk velocity peak and range, while the VR group exhibited a negative relationship.Gait function improved in both groups consistent with expectations of the interventions. Differences in each group's response to therapy appear to suggest that improved gait function may be due to different neuromuscular adaptations resulting from the different interventions. The TC group's improvements were associated with reorganized lower extremity neuromuscular patterns, which appear to promote a faster gait and reduced excessive hip compensation. The VR group's improvements, however, were not the result of lower extremity neuromuscular pattern changes. Lower-extremity MEE increases corresponded to attenuated forward trunk linear and angular movement in the VR group, suggesting better control of upper body motion to minimize loss of balance. These data support a growing body of evidence that Tai Chi may be a valuable complementary treatment for vestibular disorders. Vestibulopathy decreases whole body dynamic postural control and causes functional limitations -4. Limitmechanisms underlying such improvements should differ substantially. A better understanding of how balance dysfunction interventions affect lower extremity neuromuscular function during ADL may be useful for developing gait training exercises, and for providing a fuller understanding of the link between motor function and balance.Alternative therapies, such as Tai Chi (TC), have recently gained popularity as a treatment paradigm for a variety of human ailments, including balance impairment -13. TC eIn this report, we present preliminary data from a blinded randomized clinical trial comparing the effects of VR and TC on gait function, joint kinetics and trunk kinematics in older adults. While the overall aim of this study was to determine the effects of balance rehabilitation on gait characteristics, we directed our efforts in this paper to better understand the relationship between mechanical energy transfers along the lower extremity kinematic chain (ankle-knee-hip), and forward and side-to-side velocity of the trunk.Our general hypothesis was that adults with balance impairment from vestibulopathy who receive the VR or TC intervention will improve gait function as indicated by time-distance measures. Our specific hypotheses are based on the following rationale: Recent studies in healthy older adults -21, withBased on the above rationale, and the specific treatment programs described in the following sections, we hypothesized: 1) TC treatment will improve lower extremity motor control by increasing ankle mechanical energy expenditure (MEE) contribution, and decreasing hip MEE contributions, to total energy of the leg, more than VR; and 2) that improved trunk control following TC will be positively correlated with improvements in lower extremity motor control, while improvements in trunk control following VR will not be correlated with improvements in lower extremity motor control. The latter may indirectly implicate other mechanisms, most likely improvements in VOR/gaze stability .Fifty-three patients with balance impairment due to vestibular hypofunction were recruited and randomized into two treatment groups: VR, a group vestibular rehabilitation intervention, and TC, a group Tai Chi exercise. Of the 53 patients admitted, 15 dropped out or were excluded prior to completing the intervention. The majority of drop outs were due to a new medical condition unrelated to the study preventing participation (e.g. fractured foot, acute back pain) or due to the sudden need to care for an ill family member. Two subjects were eliminated because of lack of force plate data to use in the data analysis . Of the 36 subjects remaining for analysis, 17 subjects were randomized into the VR treatment group  and 19 subjects in the TC group .-1 for the sum of right and left ear caloric stimulation at 27 and 44°C warm water stimulation of both ears and ≤8°·sec-1 slow phase velocity for the sum of 35 cc of ice water stimulation in each ear) and decreased VOR gains during passive rotational testing at up to 50°·sec-1 . Patients with unilateral vestibular hypofunction had damage only on one side, including at least 30% unilaterally reduced caloric response, positional nystagmus while lying with the damaged ear down, and/or confirmatory abnormalities on rotational testing . Patients with bilateral deficits are typically more disabled than are those with unilateral deficits. The average time post-onset of vestibulopathy for the 36 subjects included in the analysis was 3.05 years (range 0.58 – 12 years). All subjects were community dwelling and reported varying degrees of limitations in locomotor activity. Twenty of the 36 subjects were female, and the 36 subjects were 59.5 ± 11.5 years old (range, 41–81), 1.70 ± .11 m tall and 83.6 ± 16.5 kg in weight (breakdown by treatment group is shown in the Results section). All subjects had at least one course of VR since the time of onset of their vestibular symptoms. Inclusion criteria required that each subject did not have VR for >6 months from study enrollment. The testing protocol was approved by MGH institutional review board, and all subjects provided written informed consent according to institutional guidelines on human research.Unilateral or bilateral vestibular hypofunction (UVH or BVH) diagnoses were obtained as previously described . BrieflyThe VR and TC treatment interventions were provided in a total of six (3 VR and 3 TC groups) small groups with an average of 8 subjects per group. Each intervention program met once weekly, on separate weekdays, for 10 weeks in the same exercise room. The weekly sessions for both intervention groups lasted approximately 70 minutes. Each treatment program was lead by the same instructor for the three treatment cohorts. The instructors were blinded to the exercises provided to the other treatment program. One or two assistants were available for each session for all treatment groups to insure participants' safety. All treatment sessions included time to: 1) review material introduced in prior sessions; 2) introduce new material; 3) ask questions and share personal experiences or concerns regarding the practices; and 4) cool down and rest.The TC intervention incorporated three objectives outlined in a balance-related TC program developed by Wolf and colleagues ,17. FirsThe VR intervention used in this study was a comprehensive exercise program designed to improve the problems specifically associated with damage to the peripheral vestibular system ,6,10,29.Subjects performed two-to-four gait trials along a 10 m level walkway at baseline testing, and at testing following the intervention program, at their freely selected pace upon the command "Please walk as you normally would, as if taking a brisk walk in the park". Body segment kinematics were acquired at 150 Hz with a four-camera Selspot optoelectric tracking system (Selective Electronics, Partille, Sweden), used to acquire position and orientation data of 11 segments (both feet, shanks, thighs and arms, and the pelvis, trunk and head). Collection of kinematic data and processing is described in more detail elsewhere . KineticParameters selected for data analysis consisted of dynamic gait function (time-distance measures), lower extremity neuromuscular control , and trunk stability .Gait function was assessed with standard time-distance measures ,32, incl(+), the amount of concentric mechanical energy expended with segment-to-segment energy transfer), eccentric transfer (MEE(-), the amount of eccentric mechanical energy expended with segment-to-segment energy transfer), and no-transfer (MEE(o), the amount of concentric and eccentric energy expended without segment-to-segment energy transfer) conditions. The total joint MEE is simply the sum of these components (MEE(t) = MEE(+) + MEE(-) + MEE(o)). Leg MEE is the sum of joint MEE (ankle, knee and hip) for different conditions, or totals. Percentage contribution of joint MEE  to leg MEE  was then calculated.Neuromuscular function of the lower extremities was assessed using mechanical energy expenditure (MEE) of the ankle, knee and hip, relative to the total MEE of the leg, and were computed as described previously ,33. BrieTrunk stability was assessed using kinematics of the trunk center of mass , and con(+), MEE(-) and MEE(o) contributions) required significance at α = .017 for at least one comparison, α = .025 for the second comparison, and α = .050 for the third comparison. Families of four members  required significance at α = .013 for at least one comparison, α = .017 for the second comparison, and so on. All p-values given will be unadjusted, but the adjusted α is given for each comparison where appropriate. SPSS for Windows (v10, SPSS Inc. Chicago, IL) was used for all statistical analyses.One-way ANCOVA was used to compare change scores between the two groups, using the baseline values as covariates. Paired samples t-tests compared the change in each variable for each group from baseline to post-intervention testing. Pearson correlations were used to examine associations between change scores in lower extremity MEE and change scores in trunk velocities, for each treatment group. Due to the large number of comparisons in this exploratory study, a Ryan-Holm step down Bonferonni approach was used to control for type I errors , using ap = .223), height  or weight . There was no significant difference in proportion of UVH and BVH in the treatment groups (Chi-square, p = .429), or proportion of men and women in the treatment groups (Chi-square, p = .709).The two groups were not different in age  for any of the variables examined. There were, however, significant changes pre- and post-treatment within each group. These latter results appear to suggest that clinically important differences in each group's response to the therapies exist. Thus, the remainder of the results presented will focus on the within-groups comparisons.p = .044) and step length (p = .045), but not in gait speed (p = .060) or step width (p = .390); the TC group improved in gait speed (p = .009) and step length (p = .010), but not in stance duration (p = .055) or step width (p = .313).Both groups improved (unadjusted α = .05) following intervention in time-distance measures (see Table (t) for each joint, and the sum of all the joints (leg). Although the total leg MEE(t) change was similar, the distribution of joint MEE(t) were quite different for the two treatment groups. Comparison of the change in percent contribution of MEE for each transfer condition for each joint to leg MEE showed that only the TC group had significantly reduced (p < .001, adjusted α = .017) relative hip concentric MEE(+) and increased (p = .019, adjusted α = .025) relative ankle concentric MEE(+), following training. These data are shown in Figure Figure p = .009, adjusted α = .013) peak trunk forward velocity during stance phase of gait following treatment, while the VR group's increase was similar though not statistically significant (p = .018, adjusted α = .013). There were no significant changes in forward velocity range, nor were there significant changes in peak or range of lateral trunk velocity for either group. The VR group, however, did show a significant increase in peak trunk angular velocity (p = .007, adjusted α = .017) and range of trunk angular velocity (p < .001, adjusted α = .013) in the frontal plane. There were no significant changes in trunk angular velocity in the frontal plane for the TC group, and neither group showed significant changes in peak and range of trunk angular velocity in the sagittal plane. These data are summarized in Figure TC group had significantly increased . For the TC group, however, changes in range and peak of forward velocity of the trunk was positively correlated with changes in leg MEE . Figure Correlation analysis between changes scores in leg MEE and trunk kinematics revealed significant relationships for both treatment groups. Most striking was the consistent directional relationship between trunk velocity and leg MEE within each of the treatment groups. For the VR group, changes in range and peak of forward velocity of the trunk was negatively correlated with changes leg MEE  for both treatment groups was observed, confirming our general hypothesis, our data suggest that the mechanisms underlying those improvements differ, and appear to be linked to differences in neuromuscular responses of the lower extremities to the treatment programs. Specifically, our data suggest that changes in the relative contribution of individual joints to total leg mechanical energy expenditure (MEE), and the relationship between changes in lower extremity mechanical energy expenditure and changes in upper body kinematics, are different between TC and VR interventions Figure . Further(t)  for ankle, knee and hip, and total leg MEE(t), for each group. The increases in leg MEE(t), which were similar for both groups, were apparently achieved by different neuromuscular adaptations of the individual joints. Where the VR group appear to increase total MEE(t) of each joint to gain a total MEE increase of the leg, the TC group show a distinctive pattern of substituting ankle plantar-flexor contribution for hip extensor/flexor contribution. Prior studies on the relative roles of ankle and hip kinetics in gait [We found that TC patients significantly increased the contribution of ankle MEE to total leg MEE and decreased contribution of hip MEE to total leg MEE following the treatment program, while the VR group showed no significant change in ankle or hip MEE contributions following intervention Figure . Althoug in gait ,20,24 su(+) for the changes in concentric MEE(+) of individual joints. While both groups decreased the relative contribution of concentric hip MEE(+), only the TC group increased the contribution of ankle concentric MEE(+), while the VR group increased the contribution of knee concentric MEE(+). Concentric energy transfer represents the energy expended by muscles in concentric contraction when energy is being transferred between segments. Because concentric contraction represents work being done by the muscles (as opposed to eccentric contraction, which is work being done on the muscles), we can interpret the above finding as meaning that, for the TC group, a greater proportion in the change in concentric work done by the leg muscles is attributed to the change in concentric work of ankle plantar-dorsiflexors, while for the VR group, this contribution decreases.Figure One possible reason concentric ankle MEE contribution may have increased significantly in the TC group, but not the VR group, is because the TC and the warm-up exercises improved ankle flexibility. Tight ankles (limited range of motion, ROM) may preclude the optimal structural alignment to coordinate mechanical energy sufficiently to increase propulsion , perhapsAs shown in Figure Because the VR exercise program may increase subjects' awareness of eye and head movement strategies that cause dizziness and instability ,47, a moAlthough the preliminary results presented here suggest the lower extremities may play an important role in the ability of vestibulopathic patients to improve gait function, several limitations of the present study may prevent broad generalization of the results. Our small sample size was perhaps the most important limitation. It is probable that the lack of between-group significant changes, particularly in light of many significant within group differences (pre-post intervention), was due to high variances obscuring group mean differences. Indeed, a larger sample size for controlling type II errors (increasing power), and better control of type I errors for multiple statistical tests, is warranted for future full-scale studies. The large age range within groups may have also contributed to high variability, but note that more heterogeneous samples in fact enhance external validity, including generalizability, of the results. We also did not include a no-treatment control group in the experimental design of this preliminary study. Because vestibulopathic patients may learn to compensate spontaneously, a no-treatment or sham-treatment group would be necessary to determine if changes in gait function are truly a result of the interventions. This is unlikely, however, given the inclusion criteria that all subjects must have had stable symptoms for 6 months and were on average 3 year post-onset of vestibulopathy. Given that both groups improved gait function in our randomized design comparing substantially different interventions suggests that the effects observed were not spurious. It must be recognized, however, that assumed improvements in function, via increased gait speed for example, may be limited ,49. As wWe conclude that VR and TC can successfully improve gait function, as determined by common time-distance measures, in patients with vestibulopathy. We further conclude, however, that TC improves lower extremity motor control more than VR, by selective redistribution of joint energetics, which appears to engender a more vigorous gait and better trunk control. TC, as a complementary treatment to VR, may allow for better control of the trunk through reorganization of lower-extremity motor patterns, elicited from the flowing, controlled TC exercises.The author(s) declare that they have no competing interests.All authors participated in the overall study design, contributed to the interpretation of data and writing/editing of the manuscript, and have read and approved the final manuscript. CAM conceived the hypotheses for this manuscript and carried out the data analysis; DEK was the principal investigator of the project; SWP was the neurologist associated with the project; DMS conducted the patient testing and assisted in development of the vestibular rehabilitation program; PMW conducted the Tai Chi intervention; and SLW was the project consultant.The pre-publication history for this paper can be accessed here:


In the emerging field of environmental genomics, direct cloning and sequencing of genomic fragments from complex microbial communities has proven to be a valuable source of new enzymes, expanding the knowledge of basic biological processes. The central problem of this so called metagenome-approach is that the cloned fragments often lack suitable phylogenetic marker genes, rendering the identification of clones that are likely to originate from the same genome difficult or impossible. In such cases, the analysis of intrinsic DNA-signatures like tetranucleotide frequencies can provide valuable hints on fragment affiliation. With this application in mind, the TETRA web-service and the TETRA stand-alone program have been developed, both of which automate the task of comparative tetranucleotide frequency analysis.Availability: TETRA provides a statistical analysis of tetranucleotide usage patterns in genomic fragments, either via a web-service or a stand-alone program. With respect to discriminatory power, such an analysis outperforms the assignment of genomic fragments based on the (G+C)-content, which is a widely-used sequence-based measure for assessing fragment relatedness. While the web-service is restricted to the calculation of correlation coefficients between tetranucleotide usage patterns of submitted DNA sequences, the stand-alone program generates a much more detailed output, comprising all raw data and graphical plots. The stand-alone program is controlled via a graphical user interface and can batch-process a multitude of sequences. Furthermore, it comes with pre-computed tetranucleotide usage patterns for 166 prokaryote chromosomes, providing a useful reference dataset and source for data-mining.Up to now, the analysis of skewed oligonucleotide distributions within DNA sequences is not a commonly used tool within metagenomics. With the TETRA web-service and stand-alone program, the method is now accessible in an easy to use manner for a broad audience. This will hopefully facilitate the interrelation of genomic fragments from metagenome libraries, ultimately leading to new insights into the genetic potentials of yet uncultured microorganisms. At present, a majority of the microbes from natural microbial communities cannot be transferred into pure cultures, either because proper cultivation conditions have yet to be found, or due to currently unidentified fundamental obstacles . For decGammaproteobacteria [The metagenome-approach allows for the first time to circumvent the restrictions imposed by the limited culturability of environmental microorganisms . Now, DNbacteria -6, and tbacteria , respectHowever, the metagenome-approach is not without its limitations and problems. A major constraint is that especially small genomic fragments, as are obtained from libraries that have been constructed with fosmids or cosmids as cloning vectors, often lack suitable phylogenetic marker genes. This leads to the problem that fragments belonging to the same organism cannot be reliably identified as such unless they overlap. In order to nonetheless interrelate such genomic fragments, measures such as the (G+C)-content or BLAST hits and codon usage of the fragment's coding regions are commonly used to assess whether two unlinked fragments from a metagenome library belong to the same organism. These measures, however, can produce ambiguous or even misleading results, and should be supplemented by additional tools that assess the relatedness of the genomic fragments .Since numerous studies have shown that oligonucleotide frequencies within DNA sequences exhibit species-specific patterns -18, compIn order to automate and facilitate such an analysis, the TETRA software suite was developed, comprising both, a web-service and a stand-alone program.et al. (2004) and the TETRA online manual.The algorithms that are used within TETRA have been described elsewhere . In brieThe TETRA web-service  has been REAL Software Inc., Austin, Texas) and is available for Mac OS X. Versions for Linux and Windows are also available, but differ in details regarding their implementation and features. The counting of the tetranucleotides in the current version of TETRA stand-alone program is done by ocount – a self-written C program that has been integrated into the program.The TETRA stand-alone program can be downloaded for free from the TETRA website . The curThe TETRA web-service computes correlation coefficients between tetranucleotide usage patterns of DNA sequences, which can be used as an indicator of sequence relatedness. Details on the in- and output formats is available in the comprehensive online documentation .The stand-alone version of TETRA has many additional features that are not available via the TETRA web-service. Firstly, it comes with pre-computed tetranucleotide usage patterns of all 166 prokaryote chromosomes that were publicly available by June 2004  is computed in less than 10 minutes on a dual 1.8 GHz G5 (IBM PPC 970) computer. Newly computed tetranucleotide usage patterns are displayed within the 'Navigator' window, which is the central place for data management, access to the raw data and the calculation of plots and correlation coefficients Figure . Raw datA detailed documentation of the TETRA stand-alone program and its functions is available via the program's online help system.As has been demonstrated in a previous study , the anaEscherichia sp., Shigella sp., Yersinia sp. or Mesorhizobium sp., Sinorhizobium sp., Bradyrhizobium sp.). However, more distantly related genera or even species with larger evolutionary distances are often not correctly clustered (e.g. Prochlorococcus sp.).While the method works quite well for sequences in the range of 40 kb, it is certainly not suited for the analysis of single-read end-sequences, which are usually shorter than 1 kb. Since the phylogenetic signal within tetranucleotide usage patterns is faint, the method performs weakly for whole genome phylogenetic tree reconstructions. In a whole-genome tree calculated from the pre-computed 166 prokaryotic chromosomes (data not shown), organisms are mostly grouped at the species level and at the level of genera, when these are closely related : Platform independent (web-service); Mac OS X • Programming language: REALbasic• Other requirements: none• License: none• Any restrictions to use by non-academics: noneBLAST – basic local alignment search toolmegx – marine environmental genomicsTETRA was implemented by HT and JW, TL contributed to the TETRA web-service, MB and FOG contributed important ideas regarding implementation, features and tested the programs.


Feline leukemia virus (FeLV) induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR) comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation.Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication.Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in the FeLV-945 LTR. c-Myb binding and CBP recruitment to the LTR positively regulated virus production, and thus may be responsible for the replicative advantage conferred by the 21-bp triplication. Considering that CBP is present in hematopoietic cells in limiting amounts, we hypothesize that FeLV-945 replication in bone marrow may influence CBP availability and thereby alter the regulation of CBP-responsive genes, thus contributing to altered hematopoiesis and consequent hematologic disease. The variable clinical outcome of FeLV infection is thought to reflect this genetic diversity [et al., manuscript submitted].Feline leukemia virus (FeLV) is a simple gammaretrovirus that induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. Like other natural retroviruses, FeLV is not a single genomic species but is a genetically complex family of closely related viruses subject to selective pressures in the host. Variant genomes are generated during virus replication as a result of both error-prone reverse transcription and recombination. The consequence of this variation is a diverse population that is continuously shaped iversity ,2. FeLV-iversity ,4, ChandThe 21-bp triplication was previously shown to provide transcriptional enhancer function to the LTR that contains it, and to function preferentially in primitive hematopoietic cells . In K-56Regarding the molecular mechanism by which the 21-bp triplication functions in the context of the LTR, at least two possibilities have been considered. One possibility is that the 21-bp triplication functions to maintain the appropriate spacing in the LTR between the enhancer and the promoter. A spacer function might be particularly relevant in an LTR like FeLV-945 in which the enhancer is not tandemly repeated. Substitution of the 21-bp repeat element with unrelated sequence of the same length, however, was observed to ablate the replicative advantage, thus indicating that the 21-bp triplication does not perform solely a spacer function . An alteAs described above, previous studies suggested that preserving the protein binding sites may confer a selective advantage that accounts for the precise sequence conservation of the 21-bp triplication in the FeLV-945 LTR . In the To evaluate whether c-Myb binding to the 21-bp triplication regulates LTR function, reporter plasmids were constructed in which expression of the firefly luciferase gene was driven by the U3 region of an FeLV LTR containing one, two or three copies of the 21-bp element. Reporter gene constructs were introduced by lipid-mediated transfection into feline embryonic fibroblasts (FEA) along with increasing amounts of a c-Myb expression vector. Fibroblasts were selected because the level of endogenous c-Myb expression in those cells is low or absent ,15. The Previous studies had shown that the 21-bp triplication contributes enhancer function to the LTR in a cell type-specific manner, and that it is significantly more active in K-562 cells as compared to a fibroblast line ,15. WhenSupershift EMSA was used to evaluate whether c-Myb and CBP might be present in the specific complex that forms on the 21-bp triplication. Specific complex formation was observed using a radiolabeled probe representing the 21-bp triplication in the presence of nuclear extracts from either K-562 cells or feline 3201 T-cells. In both cases, mobility of the complex was reduced (supershifted) when reacted with antibody to c-Myb. When the same reaction was performed in the presence of an antibody to CBP, an identical supershift was observed Figure . No compet al., manuscript submitted]. The 21-bp triplication was shown to enhance transcription from the FeLV LTR and to confer a replicative advantage to the virus, at least in part through the specific binding of unidentified nuclear proteins to the repeat motif [bcl-2 promoter was shown to be effectively competed by 50-fold molar excess of a cold oligonucleotide carrying a high affinity c-Myb binding site [The natural FeLV isolate, FeLV-945, was originally identified from lymphoid and other hematopoietic disorders in a geographic and temporal cohort. A unique 21-bp repeat motif in the FeLV-945 LTR was observed to be precisely conserved among animals in the cohort that exhibited malignant, proliferative or degenerative hematopoietic diseases of non-T-cell origin [,4, Chandat motif . In the at motif ,11), theat motif ,19. Indeing site . A possiet al., manuscript submitted], it is likely that the tropism of FeLV-945 in vivo included the hematopoietic progenitors in which c-Myb is expressed. Considering this possibility, we hypothesized that c-Myb may act as a transcriptional regulator of FeLV-945. In support of this hypothesis, reporter gene assays showed that an LTR containing the triplication was responsive to c-Myb in a dose-dependent manner  and was maintained in RPMI 1640 medium with 10% FBS. The FEA cell line, a continuous line of feline embryonic fibroblasts, was obtained from Dr. Jennifer Rojko and was grown in Eagle minimal essential culture medium supplemented with 10% fetal bovine serum (FBS), 0.1 mM non essential amino acids and 50 μg/ml gentamicin reagent solution (Invitrogen, Carlsbad, CA,). 3201 is an FeLV-negative thymic lymphoma cell line of feline origin [me sites . The FeLme sites .TAACGGTTCCGTAGTGA) or a CREB consensus binding site (5'-AGAGATTGCCTGACGTCAGAGAGCTAG). Protein-DNA complexes were resolved by 6% polyacrylamide gel electrophoresis in 0.25X TBE buffer (1X TBE buffer is 89 mM Tris base, 89 mM Boric acid and 2 mM EDTA). Gels were then dried at 80°C and exposed to radiographic film for varying periods of time. In some reactions, monoclonal antibody (4 μg) to either c-Myb or CBP, or isotype control antibody, was added after the 30-minute incubation period and incubated overnight at 4°C. Complexes were then resolved by 6% polyacrylamide gel electrophoresis as described above. The mouse monoclonal IgG1 antibody to c-Myb was raised against a recombinant protein corresponding to amino acids 500–640 of the human protein (Santa Cruz Biotechnology, Santa Cruz, CA). The mouse monoclonal IgG1 antibody to CBP was raised against a peptide corresponding to amino acids 2422–2441 of CBP of human origin (Santa Cruz Biotechnology, Santa Cruz, CA).A double-stranded oligonucleotide probe containing the 21-bp triplication and 40 bp of flanking sequence from the FeLV-945 LTR was radiolabeled using the synthetic oligonucleotide GS945 as template (5'- GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGTCTCCAGGCTCCCCAGTTGAC -3'), the filling primer (5'-CTGGTCAACTGGGGAGCCT-3') and the Klenow fragment of DNA polymerase (Invitrogen, Carlsbad, CA) to complete the duplex. A homologous probe containing only a single copy of the 21-bp element was similarly synthesized using the oligonucleotide GS61E as template (5'-GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTCTCCAGG CTCCCCAGTTGAC-3'). Nuclear extract from K-562 cells was obtained from Active Motif (Carlsbad, CA). Nuclear extracts from FEA and 3201 cells were prepared using the Nuclear Extract Kit from Active Motif (Carlsbad, CA) according to manufacturer specifications. Nuclear extracts were also prepared from FEA cells following the lipid-mediated transfection  of FL-Myb, a c-Myb expression vector in which full length murine c-Myb cDNA was inserted into the multiple cloning site of pcDNA3.1 . DNA-protein binding reactions included 5 μg of nuclear extract and 2.4 ng of radiolabeled probe in a 20 μl reaction containing 1 mM Tris pH 7.5, 7.5 mM NaCl, 1 mM EDTA, 0.7% glycerol, 0.1 mM DTT and 2 μg poly(dI-dC). Reactions containing nuclear extracts from K-562 cells were incubated at 4°C for 30 minutes. Reactions containing nuclear extracts from 3201 or FEA cells were incubated at 30°C for 30 minutes. In some reactions, unlabeled probe was added to the reaction as a specific competitor, or HindIII/HaeIII-digested bacteriophage lambda DNA was included as non-specific competitor. Some reactions included as competitor a double-stranded oligonucleotide containing a known high-affinity c-Myb consensus binding site  and +/- (5'-GCTGAAACAGCAGAAGTTTCAAGGCCACTGCCAGCAGTTTCAAGGCCACTGCCAGCAGATTCAAGGCCACTGCCAGCAGTCTCCAGGCTCCCCAGTTGAC-3') that contained a point mutation in the first or second binding site, respectively (mutated base indicated by boldface and underline). The indicated point mutation had previously been shown to ablate c-Myb binding [AluI/HincII and substituted into the LTR of FeLV-A/61E. The KpnI/PstI fragment of the resulting recombinant LTR was then excised and cloned into the pGL2-Basic luciferase reporter plasmid. Finally, a luciferase reporter plasmid was developed that contained the isolated 21-bp triplication cloned upstream of the SV40 promoter in pGL2-Promoter (Promega Corp., Madison, WI). A double-stranded DNA fragment containing the 21-bp triplication was generated by PCR amplification using the oligonucleotide GS945 (described above) as template and primers fw945-kpn1 (5'- GCTCGGTACCAGCTGAAACAGCAGAAGTTTC) and rv945-sac1 (5'- ATGCTGAGCTCAACTGGGGAGCCTGGAGACT). The resulting amplification product was digested with KpnI/SstI and inserted into the multiple cloning site upstream of the SV40 promoter in the reporter plasmid.Luciferase reporter plasmids were constructed to contain the U3 region of an FeLV LTR containing one, two or three copies of the 21-bp element. The U3 region of the FeLV-A/61E LTR, containing one copy of the 21-bp element, was cloned into the firefly luciferase reporter plasmid pGL2-Basic (Promega Corp., Madison, WI). The LTR was then substituted between  binding . A doubl5 cells were seeded in triplicate into 6-well tissue culture plates. The next day, reporter plasmids (500 ng) were introduced into cultured cells by lipid-mediated transfection  in the presence of pRL-TK (5 ng) in a 100:1 ratio. pRL-TK encodes Renilla luciferase and was used as an internal control for transfection efficiency. Firefly and Renilla luciferase activities were quantified 24 hours later using the Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI). Data from triplicate wells were analyzed statistically using one-way ANOVA and Bonferroni post test. Statistical significance was considered as p < 0.05. In some assays, the c-Myb expression vector FL-Myb (described above) was added to the transfection in increasing amounts (50 ng – 500 ng). The 5XMRE plasmid was used in reporter gene assays as a positive control. This plasmid contains five tandem c-Myb binding sites cloned upstream of a luciferase gene .For reporter gene assays, 2 × 105 K-562 cells, uninfected or chronically infected with recombinant FeLVs GA-945L and GA-61EL (described above) were seeded in triplicate into 24-well tissue culture plates. A full-length CBP expression vector  was introduced into the cells by lipid mediated transfection using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Culture supernatants were collected three days later and reverse transcriptase activity was quantified as previously described [5 × 10escribed . Data frNone declared.SLF developed reporter gene constructs and performed binding assays, gene expression and virus replication assays. SP identified and initially demonstrated c-Myb binding sites. KRR developed the reporter gene assays. LSL directed the experimental design, implementation and interpretation of data. All authors read and approved the final manuscript.


Idd (insulin dependent diabetes) loci, which have been mapped to small chromosome intervals, for which the orthologous regions in the human genome can be identified. Here, we have conducted re-sequencing and association analysis of six orthologous genes identified in NOD Idd loci: NRAMP1/SLC11A1 (orthologous to Nramp1/Slc11a1 in Idd5.2), FRAP1 (orthologous to Frap1 in Idd9.2), 4-1BB/CD137/TNFRSF9 (orthologous to 4-1bb/Cd137/Tnrfrsf9 in Idd9.3), CD101/IGSF2 (orthologous to Cd101/Igsf2 in Idd10), B2M (orthologous to B2m in Idd13) and VAV3 (orthologous to Vav3 in Idd18).One strategy to help identify susceptibility genes for complex, multifactorial diseases is to map disease loci in a representative animal model of the disorder. The nonobese diabetic (NOD) mouse is a model for human type 1 diabetes. Linkage and congenic strain analyses have identified several NOD mouse Re-sequencing of a total of 110 kb of DNA from 32 or 96 type 1 diabetes cases yielded 220 single nucleotide polymorphisms (SNPs). Sixty-five SNPs, including 54 informative tag SNPs, and a microsatellite were selected and genotyped in up to 1,632 type 1 diabetes families and 1,709 cases and 1,829 controls.P values > 0.2), indicating that common variation in these key candidate genes does not play a major role in type 1 diabetes susceptibility in the European ancestry populations studied.None of the candidate regions showed evidence of association with type 1 diabetes ( Type 1 diabetes is a common, multifactorial disease believed to be caused in a proportion of cases by an autoimmune destruction of pancreatic β-cells by an inflammatory infiltrate comprising T lymphocytes, dendritic cells and macrophages. This process results from a complex interaction between genetic and environmental risk factors. Genetically, it is under the control of the major histocompatibility complex (MHC) [Idd regions controlling type 1 diabetes susceptibility in the NOD mouse could also determine susceptibility in humans, even though this depends on the frequency of susceptibility alleles in human populations, which affects statistical power, and that the correct candidate gene has been chosen from the Idd interval. These Idd intervals might contain many genes, including several involved in the immune response [A murine model of type 1 diabetes, the NOD mouse, spontaneously develops an autoimmune-mediated diabetes that has many similarities to the human disease. It is likely that components of the pathophysiology and genetic predisposition are conserved across species, and indeed two loci have already been shown to affect type 1 diabetes susceptibility in both species, namely the immunoregulatory MHC HLA class II and CTLA-4 genes. The other causative gene(s) in the known response . Neverthresponse ,4.Ian4/Iddm1 [Cblb (KDP rat) [CTLA4 [The central importance of T cell development and function in type 1 diabetes is evident from the susceptibility genes identified so far. The MHC class II genes are important etiologically in two rat models of type 1 diabetes, the Biobreeding (BB) and KDP strains ,6, the Nn4/Iddm1  affects KDP rat)  and CTLA) [CTLA4  (in humaIl2 [Cd101 [Nramp1/Slc11a1 [Idd5.2 [Idd9.2 [Idd9.3 [Idd10 [Idd13 [Idd18 [Nramp1/Slc11a1 from Idd5.2 [Frap1 from Idd9.2 (unpublished); 4-1bb/Cd137/Tnfrsf9 from Idd9.3 [Cd101/Igsf2 from Idd10 [B2m from Idd13 [Vav3 from Idd18 . Table In our selection of candidate genes within NOD congenic intervals, we have, therefore, biased our choice towards immune-related genes such as  [Idd9.2 , Idd9.3  [Idd9.3 , Idd10 [3 [Idd18  we chosem Idd9.3  (unpubliom Idd13  and Vav34-1BB [NRAMP1 (MIM 600266), FRAP1 (MIM 601231), 4-1BB (MIM 602250), CD101 (MIM 604516), B2M (MIM 109700) and VAV3 (MIM 605541) were re-sequenced in 32 or 96 randomly chosen UK white patients with type 1 diabetes to identify SNPs and for the selection of tag SNPs. As LD between 4-1BB SNPs was weak, eight out of nine common SNPs were genotyped  and analysed using single-locus tests.A tag SNP approach to test for association was adopted for all genes, except for 4-1BB , in orde4-1BB . Coding 4-1BB, FRAP1, or B2M were observed. A synonymous change was detected in exon 3 of NRAMP1 (MAF = 0.32) and a non-synonymous SNP (nsSNP) in exon 15 (MAF = 0.02), causing a conservative amino acid change: Asp543Asn (DIL5202/ss23142243). Interestingly, as in the case of its mouse orthologue [CD101 , VAV3β (unique exon 1 and exons 4 to 27) and VAV3.1 (unique exon 18 and exons 19 to 27) yielded six exonic SNPs  (see Table D101 see . Re-sequSNPs see . Two SNPVAV3 and a microsatellite from NRAMP1 were genotyped a priori in the whole family collection (step 1 and 2) and a single nsSNP from CD101 in step 1 families only (DIL3794/rs3754112). The nsSNP DIL3810/ss23142433 in VAV3 was not tested because it was in quite strong LD with DIL3809/ss23142432 (R2 = 0.64), so that only DIL3809/ss23142432 was genotyped. Note that in our tag approach, the two VAV3 nsSNPs (DIL1521/rs7528153 and DIL3809/ss23142432) were chosen deliberately as tag SNPs.Two common nsSNPs  from P values for association between type 1 diabetes and candidate gene tag SNPs all exceeded 0.2, as did the single-locus test P values for 4-1BB SNPs. Consequently, we did not proceed to genotype in step 2 samples for any of the candidate genes (Table VAV3 and CD101 or the microsatellite of NRAMP1 showed evidence of association (Table NRAMP1 microsatellite promoter (GT)n has previously shown linkage and association with autoimmune disease, and allele A2 with infectious disease susceptibility [In a pragmatic, phased genotyping strategy, in step 1, the multi-locus test es Table  and 4. Non Table . Allele tibility -20. The VAV3, which spans almost 400 kb, less than 10% of the genomic region of VAV3 was re-sequenced to identify SNPs. The general importance of intronic and intergenic regulatory sequences as candidates for disease susceptibility is well recognised. Hence, potential unidentified causal variants in introns or flanking regions of the genes may have been missed, and remain a target for future analyses. Despite finding no evidence of association, it remains possible that there exists a common disease variant in one or more of the six candidate genes tested, which either has an effect smaller than would be detected with this study or is in much weaker LD with the tag SNPs than any other SNP known to us [With regards to our association study in humans, intronic and potential regulatory regions were not sequenced in the candidate genes since these cover large genomic regions, which will have to wait for much more extensive polymorphism maps . For exawn to us .NRAMP (Asp543Asn, MAF = 0.02) and DIL3799/ss23142349 in CD101 . For both SNPs, P values above 0.05 were obtained (P = 0.19 for DIL5202/ss23142243 and P = 0.80 for DIL3799/ss23142349), therefore, making it less likely that these rare variants contribute to susceptibility to type 1 diabetes. Nevertheless, causal variants with MAFs less than 0.01 [Finally, the possibility of one or more rare disease variants in a locus needs to be considered . The besIdd interval may account for disease susceptibility in the NOD mouse, but the human orthologous region may lack this susceptibility variant. The scenario, in which candidate genes in the NOD Idd interval may not necessarily be harbouring a functional, causal variant in their human orthologue genes, was discussed previously [Idd interval may not be the gene causing susceptibility to disease.Taken together, these data make an association between type 1 diabetes and common variation in coding and untranslated regions of the six functional candidate genes in the investigated human-mouse orthologue regions less likely. Several possibilities may account for this. A gene  in an eviously . It is aThe tag SNP maps described here will be useful for association studies of other diseases. They will be integrated into future SNP maps encompassing the entire orthologous regions and all regulatory sequences and genes encoded within them.CD101 and NRAMP1, DIL3799/ss23142349 and DIL5202/ss23142243, a total of 3,704 type 1 diabetes cases and 3,930 controls were used.All family members were white and of European ancestral origin. The type 1 diabetes families comprised two parents and a least one affected child. The 748 type 1 diabetes families used in 'step 1' were as described previously : 472 UK NRAMP1, the 748 type 1 diabetes families described above were used in addition to 343 multiplex/simplex families from the UK, 159 Norwegian simplex families, 322 Romanian simplex families, and 60 multiplex families from the USA totalling the combined DNA sets to 1,632 type 1 diabetes families, as described previously [For 'step 2' genotyping of eviously .Nested PCR products from DNA from 96 or 32 type 1 diabetes patients were sequenced using an Applied Biosystems (ABI) 3700 capillary sequencer (Foster City, CA), and SNPs identified using the Staden Package .® assay (Third Wave Technologies, Inc. Madison WI) [NRAMP1 microsatellite was genotyped on an ABI3700 sequencer using fluorescent primers as previously described [SNPs were genotyped using the Invaderison WI)  and TaqMison WI) . The NRAescribed . Full deNRAMP1 (European Molecular Biology Laboratory [EMBL] accession numbers D50402, D50403, BC041787, L32185, BC033754), FRAP1 (UO88966), 4-1BB (UO3387), CD101 (Z33642), B2M (BC032589) and VAV3  was performed by importing Ensembl information into a temporary ACeDB database as described in Burren et al. [Annotation of n et al. .Stata statistical system and may be downloaded from our website [P > 0.05).The program for the selection of tag SNPs  and asso website . All genLMM and DJS contributed equally to this work by performing the genetic studies and writing the manuscript. AV, FP, RP, CL, JH, HF, CM, KMH, GC carried out the genetic studies and collated data. JDC performed the statistical analysis and participated in the design of the study. LJS participated in the sequence analysis. NW participated in design and collated data. KSR, CG, C I-T, DAS, DPS and LBP participated in the study design and coordination. JAT, LSW and RCT helped to draft the manuscript. All authors read and approved the final manuscript.NRAMP1. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 32 type 1 diabetes subjects. R2 values for non-typed SNPs. UTR, untranslated region.SNPs, including two deletion/insertion polymorphisms, identified in Click here for fileCD101. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 96 type 1 diabetes subjects. R2 values for non-typed SNPs. Note that DIL3969 has an allelic R2 < 0.80 due to technical difficulties with the assay. UTR, untranslated region.SNPs identified in Click here for fileVAV3. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 96 type 1 diabetes subjects. R2 values for non-typed SNPs. Note that DIL6496 and DIL6488 in block 1, and DIL1526 have allelic R2 values < 0.80, which was due to technical difficulties with those assays. UTR, untranslated region.SNPs, including four in/dels identified in Click here for fileIdd loci, the location of their human orthologous regions, and selected functional candidate gene within the Idd interval. *Estimated number of genes.Supporting published references for Table Click here for file4-1BB. Minor allele frequencies are based on the sequencing panel of 96 type 1 diabetes subjects. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Note that DIL4247/rs6694557 could not be genotyped due to assay technical difficulties. UTR, untranslated region.SNPs identified in Click here for fileFRAP1. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 32 type 1 diabetes subjects. R2 values for non-typed SNPs. UTR, untranslated region.SNPs identified in Click here for fileB2M. Novel SNPs are denoted by "ss" numbers and previously published SNPs are denoted by "rs" numbers. Minor allele frequencies are based on the sequencing panel of 32 type 1 diabetes subjects. R2 values for non-typed SNPs. UTR, untranslated region.SNPs identified in Click here for fileGenotyping counts Numbers of attempted subjects for genotyping and of subjects with genotypes.Click here for file