Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi. Since the first application of DNA-delivered RNA interference (RNAi), the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of double stranded RNA precursors that are processed by the RNAi pathway has lead to potent gene-specific knockdown. Importantly, such strategies can permit the long-term delivery of shRNAs to overcome the limitation of transient suppression by small interfering RNAs (siRNAs). Building upon the early experimental success of expressed shRNAs, the delivery of multiple RNAi effectors, known as combinatorial RNAi (co-RNAi), can offer considerable advantages over the use of single molecule knockdown strategies . Co-RNAi is particularly important for evolving targets that require long-term treatment such as highly mutable RNA viruses like human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Recent studies have shown that the replication of these viruses can be suppressed for periods as long as 75 days by the expression of two of more shRNAs simultaneously -5. In adThere are currently three main methods to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and microRNA-embedded shRNAs. The expression of multiple shRNAs from a single construct encoding several separate promoter/shRNA cassettes offers the potential for relatively straightforward vector construction as previously validated RNAi cassettes can be simply assembled as tandem repeats. Recent studies have included the use of shRNA cassettes in combinations of two , three ,7,8, fouThe use of lhRNA or extended shRNAs (e-shRNAs) represent a likely progression from single site targeting as long dsRNA are naturally processed as part of the RNAi pathway, and such molecules have been shown not to induce interferon mediated responses -16. A nuThe insertion of shRNA sequences into naturally occurring microRNA (miRNA) precursor sequences represents a potentially favourable strategy as effector molecules should be processed and exported by the same cellular pathways as endogenous miRNAs. Moreover, it has been found that miRNA mimic shRNAs can abolish competition of siRNAs and shRNAs for transport and incorporation into RISC . The insPrevious studies have clearly demonstrated that each co-RNAi strategy can be used to achieve highly effective gene suppression. However, these experiments generally focus on the use of one chosen co-RNAi approach, often involving substantial optimisation of that system. To provide an overall picture of the strengths and weaknesses of each method of co-RNAi, we took a set of active siRNAs and delivered them using validated and proven expression systems without extensive method-specific optimisation. By directly comparing the ability of these shRNAs to initiate knockdown of the same gene targets, this study provides practical information for researchers looking to express multiple validated siRNA sequences using an existing co-RNAi platform.All siRNA and shRNA sequences and their target reporter plasmids used in this study are listed in Table Sequences of DNA oligonucleotides used to construct the miRNA-embedded shRNA plasmids are listed in Additional File shRNA, lhRNA and miRNA-embedded RNAi cassettes were cloned into the retrovirus RCASBP-(A)-CN-EGFP  using thRenilla luciferase gene  by PCR amplifying target sequences and inserting these into the NotI and XhoI site downstream of the e Figure . The fol-4 Table .2 at 39°C and used for reporter assays and for retrovirus growth. Plasmid DNA and siRNA transfections were carried out in 96-well plates using 125 ng of each plasmid DNA or 50 nM siRNAs using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. For retrovirus growth, 2 μg of RCAS-U6/shRNA plasmid DNA was transfected into DF-1 in 6-well plates and passaged 6 days post-transfection into T75 flasks for RNA isolation or 96-well plates for reporter plasmid transfection.The DF-1 cell line derived from line 0 chicken embryonic fibroblasts  were maiRenilla luciferase was determined by taking the normalised levels compared to background Firefly luciferase for each sample transfected in four replicates ± standard error and is representative of at least two independent experiments.The reporter vector psiCHECK™-2 carrying the sequences of the various shRNA target transcripts were assayed for luciferase expression using the Dual Glo Luciferase Assay System (Promega) following the manufacturer's instructions. The relative expression of target specific 32P] ATP (Amersham) and T4 polynucleotide kinase (New England Biolabs) to generate high-specific-activity probes. Hybridization, washing, and autoradiography were carried out as previously described [Total RNA was extracted from cultured DF-1 cells infected with retroviral vectors using TRIzol reagent (Invitrogen) according to standard methods described by the manufacturer. Samples of 30 μg total RNA were resolved using a 15% polyacrylamide-1 × Tris-borate-EDTA-8 M urea gel and blotted to a GeneScreen Plus membrane (Perkin-Elmer). DNA oligonucleotides with sequences complementary to the shRNAs were end labelled with [γ-escribed .Experimental data was analysed for statistical significance using two-tailed unpaired T-tests, where P values of less than 0.05 were considered significant. Statistical analysis was carried out using GraphPad Prism version 5.0b.The simultaneous expression of several RNAi effectors can be achieved using various approaches, including multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and microRNA-embedded shRNAs Figure . For theTo test if single-gene targeting dual-U6 promoter constructs could provide additive gene suppression and to assess the impact of promoter orientation, the activity of plasmids with one U6/shRNA cassette in a forward orientation and a second cassette in either orientation were compared Figure . Co-tranRenilla expression, although this was only equal to the single U6/shRNA constructs. Despite these lhRNAs not providing an additive affect, these data further confirm that this lhRNA configuration is effective and robust, as no optimisation of shRNA or spacer sequences was required. It has been shown that shRNA efficacy can be considerably improved by the use of naturally occurring loop sequences, such as the miR-30 loop, instead of arbitrary sequences [Renilla for both reporters, although the suppression by sh-2 when in the second position was considerably reduced. The most efficient lhRNA, U6/lh-2-1a, was less effective compared to the single shRNA controls, but reduced both target genes by nearly 80%. Given that sh-1a was most efficient in the first position, and sh-2 was most efficient in the second position, these data suggest that siRNA location in the lhRNA does not determine efficacy alone, but the individual properties of each siRNA is also an important determinant.Several parameters that determine effective processing of lhRNAs have been tested and have resulted in effective gene silencing and inhibition of HIV-1 replication ,19,20. Bequences ,31. To tTo validate the activity of shRNAs embedded into a miRNA context, dual shRNA expression constructs were generated using the vector pRFPRNAi . This plTo determine which of the three co-RNAi methods represents the most effective and robust strategy for gene suppression, we directly compared the most efficient single and double-gene knockdown vectors for their ability to suppress reporter gene activity. To avoid the inconsistencies associated with co-transfection of RNAi and reporter plasmids, and to provide a background that is more applicable to experimental gene knockdown studies, we inserted the most effective of each dual RNAi cassette into the retroviral vector RCASBP(A)-CN-EGFPm5. However, Considering that the recombination and deletion of repeated U6 sequences in viral vectors has been seen before ,41, prioStable cell lines were produced by incubating transfected DF-1 cells for 6 days until at least 95% of were EGFP positive and were passaged into a larger vessel for RNA isolation or into 96-well plates for transfection with reporter plasmids. The expression of sh-1a was examined by Northern blot hybridization using a probe for sh-1a Figure . The posThe limitations of lhRNA-mediated RNAi are evident both in previous studies and by the data presented in the current study. It has been shown that the siRNA activity diminishes with distance from the lhRNA base -21, and To further investigate the versatility of the miRNA-embedded co-RNAi approach, we tested the effect of having various siRNA sequences in either of the 2 miRNA loci of pRFPRNAi. To test if the shRNAs expressed from pRFPRNAi were effective in the absence of the surrounding miRNA sequences, we constructed "miRNA mimic" shRNAs based on the miR-30 structure that encoded siRNA stems extended by 3-nt and a single mismatched nucleotide at the 5' end Figure . Co-tranIn the current study, it was shown that cassette location and orientation in the dual-U6/shRNA vectors did not markedly affect shRNA activity. However, a previous report found that the abundance of individual shRNAs expressed from similar constructs decreased with when four shRNA cassettes were present , althougCo-RNAi has been used to achieve potent gene silencing of single and multiple-gene targets by means of a number of different techniques. Although all of these methods can result in efficient gene suppression, such studies often involve the detailed optimisation of the chosen system and experimental findings are not reported in the context of the other delivery options. Since the initial use of pol III expressed shRNAs in mammalian cells ,44 this Following the identification of effective siRNA molecules for target genes of interest, the selection of a delivery strategy for co-RNAi presents several options, each of which appear to offer reliable gene knockdown. To determine which of these methods is the most effective, predictable and robust, we took a set of active siRNAs and directly compared their ability to suppress gene expression when delivered by each co-RNAi platform. Since the goal of this study was also to determine the ease at which each technique could be applied to any given set of siRNAs, we used standard 19-nt siRNAs rather than molecules that have been specifically optimised and selected for each individual expression system. This study therefore analyses the adaptability of standard siRNA sequences that would be generated by publicly available algorithms or as custom designed siRNAs to be used in each of the delivery platforms described. Prior to testing the siRNAs in the co-RNAi vectors, the expression of these sequences as regular 19-nt shRNAs resulted in equivalent gene suppression Figure , illustrOur data showed that both dual-U6/shRNA and miRNA-embedded expression plasmids increased gene suppression of single-gene targets compared to equivalent to single shRNAs. In contrast, the single-gene targeting lhRNAs only suppressed gene expression to levels equivalent to the first siRNA, which is not surprising considering the reported gradient-effect in which they are produced. For the double-gene targeting plasmids, both lhRNA and miRNA-embedded shRNAs were more variable and showed slightly decreased gene knockdown compared to the dual-U6/shRNA plasmids, which performed equally well as single shRNAs. Using retroviral-delivered co-RNAi, each expression platform was directly compared showing that although the single-gene targeting miRNA-embedded vector was reasonably effective, it was only the dual-U6/shRNA vectors that achieved a similar level of both single and double reporter gene knockdown to the control shRNAs. However, considering that these vectors were not able to provide enhanced gene knockdown (as seen in the co-transfections in Figure By comparing the efficacy of different siRNA molecules expressed as lhRNAs or miRNAs, it appeared that both the location and individual properties of each siRNA could affect silencing activity. Perhaps qualities such as sequence and local structure of different molecules can directly impact upon efficient processing and production of siRNAs, possibly through changes in their thermodynamic properties. For example, when expressed as an lhRNA, sh-1a was most effective in the first position, whereas sh-2 was more effectual in the second position. Furthermore, when expressed as miRNA-embedded shRNAs, sh-1a was most efficient in position 2, whereas sh-2 which was most effective in position 1. To further explore this, we examined the individual efficacy of three different shRNAs expressed from each of the locations as embedded-miRNAs and found enormous variation ranging from increased to completely abolished activity. This suggests that the individual sequence of each shRNA can have a marked impact on the processing and subsequent activity of shRNAs expresses from each loci of this vector. Unlike lhRNAs and miRNA-embedded shRNAs, U6-transcribed regular 19-nt shRNAs do not appear to be affected by their surrounding sequences. However, since reduced levels of individual shRNAs is caused by increasing numbers of RNAi cassettes , we testThe parameters that determine shRNA efficacy are now very well defined; the same cannot be said however, for constructs that express multiple RNAi effectors. By directly comparing the same set of siRNAs expressed using three separate methods of co-RNAi we have identified some of the strengths and pitfalls associated with each technique. Overall, the use of multiple U6/shRNA cassettes offered the most reliable and predictable gene knockdown of both single and multiple-gene targets, an effect that was not inhibited by having up to five separate shRNA cassettes. It remains to be seen however, the impact of having multiple pol III promoters on cellular RNAi machinery, endogenous miRNA processing and the expression of native pol III transcripts. In such a case, the use of RNAi effectors that resemble naturally occurring molecules such as miRNAs and long dsRNAs, may best avoid unwanted cellular effects, especially considering that miRNA mimic shRNAs can abolish competition of siRNAs and shRNAs for transport and incorporation into RISC . In any LSL constructed the U6/shRNA, lhRNA, reporter vectors, and retroviral vectors, carried out all RNAi experiments, and drafted the manuscript. NJVH constructed the miRNA-embedded constructs and helped draft the manuscript. SAW developed the pRFPRNAi vectors and helped with the manuscript. VN helped conceive the study, participated in its design and coordination, and assisted with writing the manuscript. All authors read and approved the final manuscriptList of oligonucleotides used to construct co-RNAi vectors used in this study. Additional Table S1 - DNA oligonucleotides used to construct shRNA vectors. Additional Table S2 - DNA oligonucleotides used to construct lhRNA vectors. Additional Table S3 - DNA oligonucleotides used to construct miRNA-embedded shRNA vectors.Click here for fileSchematic depiction of the construction of multiple U6/shRNA vectors encoding up to five shRNA cassettes. Schematic depiction of the construction of multiple U6/shRNA vectors encoding up to five shRNA cassettes. Using a series of PCR and cloning steps, consecutive U6/shRNA cassettes were inserted in a step-wise method. For each of the three steps, one additional U6/shRNA was added to the previous vector, starting with the dual-U6/shRNA construct dU6-F1a-F2. PCR fragments encoded the U6 promoter, an shRNA sequence and added unique enzyme sites. The term sh3 refers to the third U6/shRNA cassette, sh4 refers to the fourth U6/shRNA cassette and sh5 refers to the fifth U6/shRNA cassette. The name of each resultant vector is shown to left of each construct.Click here for file


The relationship between presence of diabetes and adverse neighborhood and housing conditions and their effect on functional decline is unclear. We examined the association of adverse neighborhood (block face) and housing conditions with incidence of lower-body functional limitations among persons with and those without diabetes using a prospective population-based cohort study of 563 African Americans 49-65 years of age at their 2000-2001 baseline interviews.Participants were randomly sampled African Americans living in the St. Louis area (response rate: 76%). Physician-diagnosed diabetes was self reported at baseline interview. Lower-body functional limitations were self reported based on the Nagi physical performance scale at baseline and the three-year follow-up interviews. The external appearance of the block the respondent lived on and five housing conditions were rated by study interviewers. All analyses were done using propensity score methods to control for confounders.109 (19.4%) of subjects experienced incident lower-body functional limitations at three-year follow-up. In adjusted analysis, persons with diabetes who lived on block faces rated as fair-poor on each of the five conditions had higher odds  of developing lower-body functional limitations than the referent group of persons without diabetes who lived on block faces rated as good-excellent. At least 80 percent of incident lower-body functional limitations was attributable to the interaction between block face conditions and diabetes status.Adverse neighborhood conditions appear to exacerbate the detrimental effects on lower-body functioning associated with diabetes. Diabetes has many adverse health effects, but one of the most important with serious consequences for living independently in the community is the impact of diabetes on lower-body function . Poor loNeighborhood conditions have been shown to predict incident lower-body functional limitations ,8 and adThe sampling design of the African American Health cohort has been described elsewhere . BrieflyAll subjects received in-home, baseline evaluations (average = 2.5 hours) between September 2000 and July 2001. Baseline response was 76% (998/1320). All procedures were approved by the Institutional Review Boards of Saint Louis University and the University of Michigan.Follow-up in-home interviews averaging 1.5 hours were conducted 36 months after baseline assessments. Of the 998 persons who participated at baseline, 853 were successfully interviewed at follow-up. Since 51 persons had died between baseline and follow-up, the response for surviving subjects was 90.1% (853/947). No attrition bias during waves 1 through 4 was evident for any of the major variables involved in the current analysis. A total of 290 persons (weighted) had two or more lower body functional limitations at baseline and were excluded from further analysis. Thus, 563 persons (weighted) had one or fewer lower-body functional limitations at baseline and comprised the study sample.Five items (0 = no difficulties to 1 = difficulty) from the Nagi physical performance scale assessed lower-body functional limitations, which were summed to form the outcome measure (ranging from 0 to 5) . Items iAssessment of neighborhood conditions was comprised of interviewer observations of the block face on which the respondent lived and participants' self-reported neighborhood desirability. An "objective" assessment of the external appearance of the block face (neighborhood) in front of the homes where the participants resided was done by the survey team using a previously published assessment tool  during hWe also obtained a subjective measure of neighborhood conditions from respondents at baseline using a four-item scale of the neighborhood as a place to live, general feelings about the neighborhood, attachment to the neighborhood, and neighborhood safety from crime . ParticiAssessment of housing conditions was an observed five-item scale based on the interviewer's ratings at the baseline interview of the cleanliness inside the building; physical condition of the interior; condition of furnishings; condition of the exterior of the building; and a global rating . The test-retest reliability was at least 0.68 for each condition . In the The baseline interview asked respondents about the presence of physician-diagnosed diabetes  .Baseline covariates included in the analysis were patterned after other African American Health cohort research . Socio-dHealth at baseline was measured by the self-rated health status question of the Short-form 36, depressive symptoms , a count of the number of self-reported physician-diagnosed severe chronic conditions ever experienced . Also assessed at baseline were body mass index, current smoking status, risk of alcohol abuse (CAGE), the Yale physical activity instrument, and grip strength.The conceptual model of the relationship among the various types of variables is displayed in Figure First, we calculated unadjusted measures of association  and interaction between diabetes and block face/housing conditions for the risk of incident lower-body functional limitation at 3-year follow-up. For each block face and housing condition, a single variable with four categories was created by combining a dichotomous neighborhood/housing variable (excellent/good vs. fair/poor) with the diabetes variable as part of the additive model . MeasureTo adjust for potential confounding from covariates, we used the propensity score method to obtain adjusted estimates of the measures of association and interaction between neighborhood/housing conditions with presence of diabetes on incident lower-body functional limitations ,24. All th and 97.5th percentiles of the empirical distribution of Interaction Contrast Ratios were calculated from 1,000 data sets resampled with replacement from the original data. All statistical analyses were conducted using SAS software, version 9.1.Confidence intervals for all effect measures were calculated using the bootstrap percentile methodology. For a 95% confidence interval the 2.5Of 563 subjects with zero or one lower-body functional limitations at baseline, 109 (19.4%) experienced two or more lower-body functional limitations at the 3-year follow-up. Eighteen percent of subjects reported having diabetes at baseline and 82 percent did not. Of those with diabetes, the percentage of subjects who lived on one of the ten block face or housing conditions rated as fair or poor ranged from 10.3% to 23.3%. Of those without diabetes, the percentage of subjects who lived on one of the ten block face or housing conditions rated as fair or poor ranged from 17.2% to 20.2%. There were no statistical differences in block face or housing conditions between persons with and those without diabetes. Baseline characteristics of the study population and factors associated with incident lower-body functional limitations in univariate analysis have been described briefly in Table Of 563 subjects with zero or one lower-body functional limitations at baseline, the percentage that developed two or more lower-body functional limitations at 3-year follow-up varied according to the participant's diabetes status and each of the five block-face conditions. For example, 65% percent of persons with diabetes who lived on block faces with yards and sidewalks in fair-poor condition developed lower-body functional limitations. In contrast, 19.4 percent of persons without diabetes who lived on block faces with yards and sidewalks in fair-poor condition developed lower-body functional limitations. About 17 percent of persons who lived on block faces with good-excellent conditions (regardless of diabetes status) developed lower-body functional limitations. Similar results were observed for the other four block-face conditions. Little difference was present examining each of the five housing conditions.In unadjusted analysis, (a) persons with diabetes residing on block faces with good-excellent conditions and (b) persons without diabetes living on block faces with fair-poor conditions generally were not significantly more likely to develop lower-body functional limitations three years later than the referent group of persons without diabetes who lived on block faces rated as good or excellent , suggesting the observed interaction is specific to diabetes and neighborhood conditions.This study has demonstrated the importance of the interaction of diabetes and neighborhood conditions acting in concert on the deterioration of lower-body functional limitations in an urban African American population. This "double jeopardy" suggests that both risk factors combined increased the risk of lower-body functional limitations considerably more than diabetes or adverse block face conditions alone . In contAdverse neighborhood conditions may exacerbate the progression toward lower-body dysfunction among persons with diabetes as a result of diabetic complications , associated conditions , poor diet and low physical activity, and reduced muscle strength . We adjuStudy limitations include a study sample that involves a single race, a single city, and a restricted age range, all of which may limit generalizability. However, focusing on a single race allows the disentanglement of race and socioeconomic status. Notably, African Americans experience more diabetes and more diabetic complications than does the majority population , so the Finally, in most studies of neighborhood effects, multiple study participants are nested within their neighborhood, requiring the use of multilevel statistical techniques. In this study sample, there were 551 block faces, 363 on which only one participant resided (65.9%). Only 3.6 percent of block faces contained five or more participants. We were not able to use multilevel statistical techniques because there was not enough clustering of participants within block faces to support a robust multi-level analytic approach. In a previous study, we randomly selected one subject per block face from the block faces with more than 1 subject and showed that parameter estimates were very similar to our findings using propensity scores .In summary, there appears to be a powerful interaction between adverse block face conditions and the presence of diabetes on decline is a crucial factor for maintaining health and independence  in urban-dwelling middle-aged African Americans. Further research is needed to investigate the mediators of this powerful interaction.The authors declare that they have no competing interests.MS conceived of the study and drafted the manuscript, EMA, FDW, JPM participated in drafting of the manuscript, YY performed the statistical analysis, DKM helped conceive of the study and participated in drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/283/prepubUnadjusted measures of association  and interaction  between diabetes and block face conditions for the risk of incident lower-body functional limitation at 3-year follow-up (weighted n = 563).Click here for filePropensity score adjusted measures of association  and interaction  between diabetes and block face conditions for the risk of incident lower-body functional limitation at 3-year follow-up (weighted n = 563).*Click here for fileUnadjusted measures of association  and interaction  between diabetes and housing conditions for the risk of incident lower-body functional limitation at 3-year follow-up (weighted n = 563).Click here for filePropensity score adjusted measures of association  and interaction  between diabetes and housing conditions for the risk of incident lower-body functional limitation at 3-year follow-up (weighted n = 563).Click here for file


The Mlabri are a group of nomadic hunter-gatherers inhabiting the rural highlands of Thailand. Little is known about the origins of the Mlabri and linguistic evidence suggests that the present-day Mlabri language most likely arose from Tin, a Khmuic language in the Austro-Asiatic language family. This study aims to examine whether the genetic affinity of the Mlabri is consistent with this linguistic relationship, and to further explore the origins of this enigmatic population.We conducted a genome-wide analysis of genetic variation using more than fifty thousand single nucleotide polymorphisms (SNPs) typed in thirteen population samples from Thailand, including the Mlabri, Htin and neighboring populations of the Northern Highlands, speaking Austro-Asiatic, Tai-Kadai and Hmong-Mien languages. The Mlabri population showed higher LD and lower haplotype diversity when compared with its neighboring populations. Both model-free and Bayesian model-based clustering analyses indicated a close genetic relationship between the Mlabri and the Htin, a group speaking a Tin language.Our results strongly suggested that the Mlabri share more recent common ancestry with the Htin. We thus provided, to our knowledge, the first genetic evidence that supports the linguistic affinity of Mlabri, and this association between linguistic and genetic classifications could reflect the same past population processes. The Mlabri are a hill tribe in northern Thailand, inhabiting a dispersed area along the border with Laos ,2. TodayLittle is known about the origins of the Mlabri and most evidence comes from linguistic studies. The Mlabri language is classified as a Khmuic language, a subgroup of the Mon-Khmer language in the Austro-Asiatic language family . The avaIn this study, we analyzed populations samples from throughout northern Thailand, including the Mlabri as well as several neighboring groups, including the Htin, Hmong, Yao, and other populations speaking Austro-Asiatic and Tai-Kadai languages. Four HapMap population samples, representing Altaic, Sino-Tibetan, Indo-European and Niger-Congo language speakers, were also included in this study. We conducted a genome-wide analysis on these samples using 50K SNPs, to investigate the genetic affinity of the Mlabri, examine the concordance of genetic and linguistic affinities, and further explore probable origin(s) of this enigmatic hunter-gatherer group.Since this is the first genome-wide genetic study of this enigmatic population, we calculated several population genetic parameters, including SNP diversity, haplotype diversity and linkage disequilibrium (LD).e) were calculated based on allele frequencies of 55,561 SNPs and the results were shown in Figure e in Mlabri (0.197) is lower than that of any of other populations in which HSe is at least 0.250 (HM). The expected heterozygosity for haplotypes (HHe) were calculated based on haplotypes in 500-kb genomic regions (Methods) and the results are shown in Figure e in Mlabri (0.666) is also much lower than that of any of other populations in which HHe is at least 0.820 (TN). The HHe comparison obtained from larger size of genomic regions (1 Mb) show the similar results .Expected heterozygosity for SNPs . The number of haplotypes was estimated for two different window sizes (500-kb or 1-Mb) respectively, with adjustment for sample size difference among populations (see Methods). Again, we found that the genetic diversity was significantly lower in Mlabri than in other populations for both 500-kb segments Figure  and 1-Mb2 ≥ 0.5 , we observed this fraction to be 1.6- to 12.3-fold higher in Mlabri than in all the other Asian populations for the distance range above 10-kb to 200-kb. For those marker pairs with strong LD (r2 ≥ 0.8), the fraction in Mlabri is 2.2- to 31.3-fold higher in Mlabri than in all the other Asian populations, and 6- to 259-fold higher than in YRI. Furthermore, LD of r2 ≥ 0.8 extended more than 1 Mb in Mlabri, whereas in all the other populations, such strong LD extended only up to 200 kb.The significantly reduced genetic diversity in Mlabri was also reflected by its extent of linkage disequilibrium (LD). We assessed the extent of LD among markers with minor allele frequency (MAF) ≥ 0.05 Figure  and ≥ 0.5 Figure  or r2 ≥ 8 Figure . For marThe genetic characteristics obtained from above analysis, such as significantly increased LD and extremely reduced haplotype diversity are both consistent with the view from a previous study  that theWe first studied the clustering relationships among 446 individuals representing 13 populations in Thailand and the CHB and JPT from the HapMap project . We used an allele sharing distance (ASD)  as the gThe above clustering relationships among individuals were also confirmed by principal components analysis (PCA) at the individual level . As showSince the closer relationship between Mlabri and Htin could be due to recent gene flow from Htin to Mlabri or vice versa, we further performed Bayesian cluster analysis as implemented in the STRUCTURE algorithm  to examiBecause the analyses discussed above were all consistent in showing that individuals from the same population cluster together, it is meaningful to evaluate the genetic relationships among populations. A maximum likelihood tree of populations , based oHowever, the Htin showed signs of admixture in both STRUCTURE and frappe analyses Figure . This raIn this study, we analyzed genome-wide SNP data on the Mlabri, as well as several neighboring populations and HapMap population samples. The Mlabri population shows several substantial differences from the other populations: significantly increased LD, extremely reduced haplotype diversity and small effective population size (29), all of which are consistent with the view that the Mlabri were recently founded from a very small number of individuals of an agricultural group but subsequently adopted their current hunting-gathering lifestyle, as proposed by a recent study based primarily on mtDNA and Y chromosome data . AlthougBoth model-free and model-based clustering analyses strongly suggest that the Mlabri share a degree of common ancestry with the Htin, a group speaking Tin language. In this case -- as is the general rule in many human populations -- the genetic affinity of these populations is consistent with its linguistic affinity. This result, to our knowledge, is the first genetic evidence supporting the linguistic affinity of the Mlabri and Tin languages. Cavalli-Sforza and colleagues showed an apparent congruence between linguistic phyla and genetic clusters, and they proposed that this congruence indicates "considerable parallelism between genetic and linguistic evolution" . SubsequHunting and gathering was presumably the subsistence strategy employed by human societies for more than two million years, until the end of the Mesolithic period. Contemporary hunter-gatherer groups are often thought to serve as models of an ancient lifestyle that was typical of human populations prior to the development of agriculture. However, there has been complex interaction between hunter-gatherers and non-hunter-gatherers for millennia. There are contemporary hunter-gatherer peoples who, after contact with other societies, continue their ways of life with very little external influence. There are also contemporary groups usually identified as hunter-gatherers do not have a continuous history of hunting and gathering, and in many cases their ancestors were agriculturalists and/or pastoralists who were pushed into marginal areas as a result of migrations, economic exploitation, and/or violent conflict . Our curIn summary, our results strongly suggested that the Mlabri share more recent common ancestry with the Htin, a group speaking a Tin language. This result, to our knowledge, is the first genetic evidence supporting the linguistic affinity of the Mlabri and Tin languages. We proposed that Htin is most likely the source population from which the Mlabri genetically originated in case cultural reversion occurred in the history of Mlabri.Samples from the Mlabri as well as other 12 populations were collected in Thailand. The sample information, including sample size, ethnic and linguistic information is shown in Table Genotype data of 13 Thailand population samples generated using Affymetrix Genechip Human Mapping 50K Xba array were obtained from the Pan-Asian SNP Initiative . DetaileHaplotypes of 22 autosomes were inferred for each individual from its genotypes with fastPHASE  version e) was calculated based on allele frequencies.Heterozygosity for each SNP (HSe), the genome was divided into 500-kb regions, with each region having roughly 14 SNPs. HHe were calculated for each region using haplotype frequencies [e were averaged over all windows.To calculate heterozygosity for haplotypes  without replacement in each population. Note that Mlabri has the smallest sample size in all the populations studied. For a population with sample size larger than 36 chromosomes, the sampling was repeated 100 times for each segment and the average of the number of haplotypes of all replications was taken as the number of haplotypes.The cumulative proportion given a number of haplotypes was obtained by estimating the proportion of the sliding-windows across the genome carrying equal or less haplotypes.2 following Hill and Weir [Linkage disequilibrium (LD) between SNPs were measured using rand Weir  and calcPrincipal component analysis (PCA) was performed at individual level using EIGENSOFT version 2.0 .We used an allele sharing distance (ASD) ,43 as a The tree of individuals was reconstructed based on ASD distance and using Neighbor-Joining algorithm  with theAncestry of each person was inferred using a Bayesian cluster analysis as implemented in the STRUCTURE program ,46. We rThe program frappe  implemenSX, DK, MS, and LJ conceived and designed the study. JK contributed to the Tai-Kadai sample collection. MS and MS performed the technical work on SNP genotyping and QC procedures for all population samples from Thailand. SX collected data and performed the analysis. SX and LJ wrote the paper, to which all authors contributed. All authors read and approved the final manuscript.e) in 17 populations.Contains Figure S1 - Haplotype heterozygosity  shown on the abscissa. A: showing maximal probability estimation of ten runs at each K (from K = 2 to K = 9); B: showing probability estimation at K = 2 to K = 18 in all ten runs.Click here for fileContains Figure S2 - Geographical distribution of Thailand population samples. Red dots on the map indicated sampling locations. Information of population IDs can be found in Table Click here for file


Pyrococcus furiosus wereseparated into membrane and cytoplasmic fractions and each was analyzed by 2D-gelelectrophoresis. A total of 66 proteins were identified, 32 in the membrane fraction and 34in the cytoplasmic fraction. Six prediction programs were used to predict the subcellularlocations of these proteins. Three were based on signal-peptides  and three on transmembrane-spanning α-helices . A consensus of the six programs predicted that 23 of the 32 proteins(72%) from the membrane fraction should be in the membrane and that all of the proteinsfrom the cytoplasmic fraction should be in the cytoplasm. Two membrane-associatedproteins predicted to be cytoplasmic by the programs are also predicted to consistprimarily of transmembrane-spanning β-sheets using porin protein models, suggesting thatthey are, in fact, membrane components. An ATPase subunit homolog found in themembrane fraction, although predicted to be cytoplasmic, is most likely complexed withother ATPase subunits in the membrane fraction. An additional three proteins predicted tobe cytoplasmic but found in the membrane fraction, may be cytoplasmic contaminants.These include a chaperone homolog that may have attached to denatured membraneproteins during cell fractionation. Omitting these three proteins would boost themembrane-protein predictability of the models to near 80%. A consensus prediction usingall six programs for all 2242 ORFs in the P. furiosus genome estimates that 24% of theORF products are found in the membrane. However, this is likely to be a minimum valuedue to the programs’ inability to recognize certain membrane-related proteins, such assubunits associated with membrane complexes and porin-type proteins.Cell-free extracts from the hyperthermophilic archaeon


Genomic data has revealed that homing endonucleases are extremely widespread in T-even-like phage, as evidenced by the astounding fact that ~11% of the T4 genome encodes homing endonuclease genes, with most of them located outside of self-splicing introns. Detailed studies of the mobile td intron and its encoded endonuclease, I-TevI, have laid the foundation for genetic, biochemical and structural aspects that regulate the mobility process, and more recently have provided insights into regulation of homing endonuclease function. Here, we summarize the current state of knowledge regarding T4-encoded homing endonucleases, with particular emphasis on the td/I-TevI model system. We also discuss recent progress in the biology of free-standing endonucleases, and present areas of future research for this fascinating class of mobile genetic elements.Mobile genetic elements are common inhabitants of virtually every genome where they can exert profound influences on genome structure and function in addition to promoting their own spread within and between genomes. Phage T4 and related phage have long served as a model system for understanding the molecular mechanisms by which a certain class of mobile DNA, homing endonucleases, promote their spread. Homing endonucleases are site-specific DNA endonucleases that initiate mobility by introducing double-strand breaks at defined positions in genomes lacking the endonuclease gene, stimulating repair and recombination pathways that mobilize the endonuclease coding region. In phage T4, homing endonucleases were first discovered as encoded within the self-splicing  T. T10/11-atalysis . In consatalysis .The monomeric I-TevI interacts with two regions of its 38-bp homing site . The DNAI-TevI uses both sequence and distance determinants in selecting its CS . AlthougThe zinc finger imparts another layer of regulation to I-TevI cleavage. In addition to the structural diversification that zinc fingers provide, an interesting aspect of these modules is their ability to be regulated by oxidation and reduction (redox) reactions . Indeed,uvsX gene, consistent with its similarity to intron-encoded GIY-YIG endonucleases, which also generate 2-nt 3' extension [seg endonucleases  [segA, segE, mobB, and mobC. This arrangement of embedded promoters favors retention of the endonuclease in the phage genome, because any deletion event that removed the essential host promoter would be detrimental to the phage.The regulatory elements that govern expression of the three T4 intron-encoded endonucleases I-TevI, I-TevII, and I-TevIII were deduced soon after the discovery of the introns and endonucleases themselves . Primer s Figure . In contly 32.1) , with sisegD) are all transcribed from promoters that lie in the non-coding regions upstream of the endonuclease ORF , or in the 3' region of the gene immediately upstream of the endonuclease . Interestingly, these "endonuclease-specific" promoters are either middle (5 instances) or late (2 instances), with no occurrences of early promoters, suggesting that there is some advantage to expression of endonucleases >5 min post infection.Seven free-standing endonuclease genes  or one of two middle promoters upstream of or internal to 24.2 . Yet, transcripts initiated from these promoters would have to read through intrinsic transcriptional terminators up- and downstream of segD. Given the antisense orientation of segD and extensive transcriptional terminator in this region of the T4 genome, it is not unreasonable to assume that segD transcript levels are vanishingly low.All of the homing endonuclease genes in T4 are present in the same transcriptional orientation as the surrounding genes, with one notable exception. The uncharacterized free-standing GIY-YIG endonuclease y Figure . This arlacZ fused to the I-TevI late promoter during phage infection. Although not experimentally demonstrated for I-TevII or I-TevIII, it is likely that each endonuclease also functions like I-TevI to autoregulate its own expression.An added layer of transcriptional regulation was recently discovered for I-TevI . In examtd intron and I-TevI in the phage population  tract immediately downstream of the stem structure [mobE and segF, possess intrinsic transcriptional terminators in the 5' end of their coding regions [mobE endonuclease is inserted in the nrdA/nrdB intergenic region of a number of T-even phage, and its expression is dependent on promoters upstream of nrdA. The terminator internal to mobE was predicted to be weak based on the length of the poly(U) tract [mobE terminator [nrdB gene downstream of mobE is not affected by the presence of the terminator in mobE, because a middle promoter is located in the intergenic space separating the 3' end of mobE and 5' end of nrdB [mobE terminator is to limit read-through transcription from the nrdA promoter, modulating transcript levels of nrdB to coordinate synthesis of NrdB  with that of NrdA (the large subunit) [mobE expression, reducing the amount of mobE-containing transcripts.Transcriptional termination mediated by intrinsic or rho-independent terminators plays a key role in regulating the expression of T4 genes, and many intrinsic terminators have been computationally identified . T4 termtructure -52. Inte regions ,53. The U) tract , and RNarminator . Howeversubunit) . More spsegB and segG by the host enzyme RNase E may be an adaptation to reduce endonuclease transcript levels [mobE transcript in the T-even-like phage Aeh1 that infects Aeromonas hydrophila [segB and Aeh1 mobE, the extent and timing of RNase E processing is unknown, while for segG, RNase E processing has been shown to increase the stability of the downstream gene 32, facilitating translation [segG or segB to act as mobile elements, as both endonucleases are inherited at high frequency in the progeny of T4 × T2 co-infections [Similarily, post-transcriptional processing of T4 t levels ,56. An Rdrophila . For T4 nslation . It shoufections ,39.td, preventing translation of I-TevI at early and middle times during infection. A late promoter, immediately upstream of I-TevI, is positioned such that late transcripts do not include sufficient sequence to form the inhibitory RNA structure, freeing the RBS and facilitating translation of I-TevI at late times [segB, and for mobE in phage Aeh1 , in Aeromonas phage 25 [seg43(25), but the hairpin does not sequester the RBS that is immediately downstream from the base of the stem  that are a reasonable match to the T4 consensus can be identified upstream of only six of the twelve endonuclease genes . However, the RBSs are not positioned at the optimal distance of 6-9 nucleotides from the AUG codon. For instance, the predicted segD RBS is 2 nucleotides upstream of the AUG codon, whereas the mobC RBS is 27 nucleotodies upstream, questioning whether or not these sequences represent bone-fide translation start sites.For the remaining free-standing homing endonucleases in phage T4, the translational control mechanisms are not obvious, and have yet to be addressed experimentally. The free-standing endonuclease ORFs start with an AUG initiation codon, the lone exception being mobE, the AUG iniation codon of mobE overlaps by one nucleotide with the first of two termination codons of the upstream nrdA gene, creating the following arrangement UAAUGA. This arrangement of overlapping initiation and termination codons is suggestive of translational coupling, a mechanism where termination of the upstream protein is linked to initiation of translation the downstream protein [nrdA/mobE overlap may represent a mechanism to limit translation of MobE endonuclease, in addition to the aforementioned transcriptional termination in mobE.Six of the twelve free-standing endonucleases have no discernable TIRs, with five of the endonuclease ORFs overlapping the upstream ORFs. In the case of  protein . Such ar protein -67. In msegC (20-bp overlap), segF (11-bp overlap), mobA (23-bp overlap), and mobC (24-bp overlap). Here, the mechanism of translational control is likely to be ribosome 'scanning', whereby the ribosome does not dissociate and diffuses along the mRNA within a seven-codon window up- or down-stream of the termination codon [In other cases, the extent of overlap with the upstream ORF is more extreme, as evidenced for the homing endonucleases on codon . If an AmobE in phage Aeh1, are subject to multiple-layers of regulation, whereas other homing endonucleases are not as tightly regulated. Whether the intron-encoded endonucleases and mobE are more 'toxic' and require multi-layered regulation is an interesting possibility that requires experimental confirmation. Intriguingly, Kruezer and co-workers generated a T4 phage where the I-TevI operator site was deleted and replaced by a middle promoter, with no noticeable effect on phage viability [Clearly, all of the mechanisms described above are negative regulatory mechanisms that function to downregulate the levels of homing endonucleases in T4-infected cells, suggesting that unregulated expression of endonucleases would be detrimental to T4. One obvious rationale for downregulating endonuclease function relates to the sequence-tolerant binding ability of homing endonucleases, and the potential for introducing (presumably) deleterious nicks and DSBs at ectopic sites throughout the T4 genome. Curiously, certain endonucleases, namely the intron-encoded endonucleases and iability . Similariability .td intron and translation of full-length thymidylate synthase [td intron, the 3' end of the ORF extends into the structured region of the intron and contributes key nucleotides that form critical secondary structures [td intron splicing, an accumulation of unspliced td pre-mRNA, and a thymidine-dependent phenotype [However, recent work suggests that the stringent regulation of I-TevI is required to facilitate efficient splicing of the synthase . An inteructures . This obructures . Indeed,henotype . These ohenotype ,73.One interesting commonality among all the regulatory mechanisms is the restriction of endonuclease function to middle or late times in the phage infective cycle ,74. We pin vitro and in vivo characterization of endonuclease-mobility pathways that rely on extremely efficient DNA repair and recombination pathways to promote dissemination of homing endonucleases through populations of phage lacking them. Our understanding of factors that influence endonuclease mobility and retention in phage genomes is still in its infancy, but studies with eukaryotic homing endonuclease systems may provide some clues. In particular, studies on intron-encoded yeast homing endonucleases have elucidated a cyclical life cycle that allows homing endonuclease genes to escape degeneration and deletion due to lack of intron-less alleles for homing [Phage T4 is an oddity among T-even phage, encoding 15 homing endonucleases. We pondered the significance of this observation in 2000, suggesting that the number of completely sequenced T-even phage genomes was too few to say anything definitive about endonuclease distribution . In the r homing ,82. Relar homing ,83. Morer homing .nrdB gene encoding the small subunit of aerobic ribonucleotide reductase is cleaved by both mobE, an HNH endonuclease of phage T4 [hef (homing endonuclease-like function), encoded in phage U5 [nrdD and nrdG genes, are targeted by seg and mob homing endonucleases [One unifying characteristic of phage-encoded homing endonucleases is the observation that most endonuclease genes are inserted within or near phage genes that are functionally critical, such as DNA polymerases and ribonucleotide reductases. Targeting of functionally critical phage genes by homing endonucleases is an evolutionary strategy to maximize spread because very similar genes and target sites will be present in related genomes , and to phage T4 , and by phage U5 . Similarucleases . Given ttrans homing pathway involves a free-standing homing endonuclase, mobE, mobilizing the defunct I-TevIII endonuclease and nrdB intron in phage T4 [The insertion sites of many self-splicing group I introns also correspond to functionally critical sequences in phage genomes. Insertion of the intron into a functionally critical region is thought to prevent deletion of the element from the phage genome, as only a precise deletion of the intron or intein will restore a functional host gene sequence, whereas an imprecise deletion would likely be lethal. The propensity for homing endonucleases and introns to target conserved sequences forms the core of a recently proposed evolutionary scenario termed collaborative homing, for the origin of mobile introns by recombination between an endonuclease-lacking intron and a free-standing endonuclease that is "pre-adapted" to target the intron insertion site of the endonuclease-lacking intron, creating a highly efficient composite mobile genetic element ,88. The phage T4 .Bacillus subtilis [Because homing endonucleases utilize DNA repair and recombination pathways to promote mobility, significant co-conversion of sequence flanking the endonuclease's cleavage site sequence is associated with endonuclease-mediated mobility ,18,39. Tsubtilis ,90. ThusmobE insertion into nrdA large subunit gene of aerobic ribonucleotide reductase of Aeromonas hydrophila phage Aeh1 [mobA insertion into the topoisomerase large subunit gene 60 of phage T4 [seg43(25) insertion associated with gene 43, encoding a B-type DNA polymerase of Aeromonas phages 25 [mobE and mobA cases, the homing endonuclease has inserted into a functionally critical region of the host gene, splitting the gene into separate coding regions as compared to related phage. For the seg-like insertion of phage 25, it is difficult to ascertain whether the split gene 43 structure arose by insertion of the seg homing endonuclease, because the related phage 44RR possess a different genetic arrangement, consisting of an intercistronic untranslated sequence (IC-UTS) that splits gene 43 into 43A and 43B [seg endonuclease invaded an already split gene created by the insertion of the IC-UTS.More dramatic effects on phage gene structure and function arise from what appear to be homing endonuclease transposition events, whereby an endonuclease gene has inserted into a site that is different from the insertion site of analogous homing endonucleases in related phage genomes. Such transposition-like insertions include the age Aeh1 , the mobphage T4 , and a shages 25 . In the  and 43B . Thus itnrdA gene of phage Aeh1 encodes a fully functional aerobic ribonucleotide reductase with activity similar to canonical enzymes that consist of a single NrdA polypeptide [43A and 43B genes of phage 25 co-purify when overexpressed, and possess DNA polymerase activity [mobA insertion has not been studied in detail, phage T4 topoisomerase has long served as a model enzyme and possesses an unusual subunit structure with respect to other phage-encoded and bacterial topoisomerases [Regardless of their origins, each insertion splits a contiguous coding region into two distinct polypeptides that must somehow reassemble to form a functional enzyme. Recent work has shown that the split ypeptide . Similaractivity . Althougomerases , consistmob endonucleases spread between genomes by nicking their target sites rather than introducing a double-strand break is an intriguing area of future research. From a genomic perspective, the relatively small size of phage genomes coupled with extraordinary advances in sequencing technology has revealed that homing endonuclease genes are widespread, but not as abundant as predicted based on laboratory experiments. It remains to be determined if more phage genome sequences can offer insight into evolutionary processes that regulate homing endonuclease distribution, as it already clear from existing sequences that complex regulatory mechanisms have evolved to control the expression of homing endonucleases. Clearly, there are interesting evolutionary forces at work, and experimentally manipulating regulatory controls will likely be required to understand the impact of homing endonuclease activity on phage genome structure and function.T-even phage have proven to be an attractive and tractable model system for studying the biology of homing endonucleases in the last 20 years, and we have learned much about the molecular details of mobility pathways and regulatory mechanisms. Many of these details are applicable to homing endonucleases in eukaryotic systems, and also have provided insight into the mobility pathways of other mobile elements such as inteins and group II introns. From a mechanistic perspective, how the The authors declare that they have no competing interests.DRE, EAG and MB wrote the manuscript. All authors read and approved the final manuscript.


The regulation of gene expression is complex and occurs at many levels, including transcriptional and post-transcriptional, in metazoans. Transcriptional regulation is mainly determined by sequence elements within the promoter regions of genes while sequence elements within the 3' untranslated regions of mRNAs play important roles in post-transcriptional regulation such as mRNA stability and translation efficiency. Identifying cis-regulatory elements, or motifs, in multicellular eukaryotes is more difficult compared to unicellular eukaryotes due to the larger intergenic sequence space and the increased complexity in regulation. Experimental techniques for discovering functional elements are often time consuming and not easily applied on a genome level. Consequently, computational methods are advantageous for genome-wide cis-regulatory motif detection. To decrease the search space in metazoans, many algorithms use cross-species alignment, although studies have demonstrated that a large portion of the binding sites for the same trans-acting factor do not reside in alignable regions. Therefore, a computational algorithm should account for both conserved and nonconserved cis-regulatory elements in metazoans.We present CompMoby (Comparative MobyDick), software developed to identify cis-regulatory binding sites at both the transcriptional and post-transcriptional levels in metazoans without prior knowledge of the trans-acting factors. The CompMoby algorithm was previously shown to identify cis-regulatory binding sites in upstream regions of genes co-regulated in embryonic stem cells. In this paper, we extend the software to identify putative cis-regulatory motifs in 3' UTR sequences and verify our results using experimentally validated data sets in mouse and human. We also detail the implementation of CompMoby into a user-friendly tool that includes a web interface to a streamlined analysis. Our software allows detection of motifs in the following three categories: one, those that are alignable and conserved; two, those that are conserved but not alignable; three, those that are species specific. One of the output files from CompMoby gives the user the option to decide what category of cis-regulatory element to experimentally pursue based on their biological problem. Using experimentally validated biological datasets, we demonstrate that CompMoby is successful in detecting cis-regulatory target sites of known and novel trans-acting factors at the transcriptional and post-transcriptional levels.de novo discovery of evolutionarily conserved and nonconserved cis-regulatory sequences involved in transcriptional or post-transcriptional regulation in metazoans. This software is freely available to users at .CompMoby is a powerful software tool for systematic  The increasing number of sequenced genomes of multicellular eukaryotes, including human, along with high-throughput methods such as whole genome microarray expression data, allows for systematic characterization of the cis-regulatory elements that control gene expression. Regulation of gene expression occurs at multiple levels in metazoans, including transcriptional and post-transcriptional. Transcriptional regulation involves binding of transcription factors (TFs) to short cis-regulatory elements, or transcription factor binding sites (TFBSs), that are generally 5–15 basepairs (bp) long. TFs bind to specific TFBSs on DNA, which leads to activation or repression of gene transcription ,3. The ade novo motifs in the noncoding regions (NCRs) on a genome-wide scale without prior knowledge of the trans-acting factor and the labor costs of experimental techniques. NCRs are defined as any DNA sequence residing outside the translational start and end site of all known genes of a genome. In general, motifs are more difficult to detect in metazoans because the overall genomes are much larger than yeast, including the NCR . Furthermore, regulatory elements can reside far upstream, downstream, in introns   de novo  introns  for revi introns . Many of introns ,10.Algorithms for detecting motifs in metazoans tend to leverage evolutionary conservation to reduce the background noise ,10. TherWe previously introduced the CompMoby (Comparative MobyDick) algorithm for the study of transcriptional regulation in metazoans, which was successfully applied to embryonic stem (ES) cells . We havede novo motif discovery, which may lead to identification of a new TF, miRNA, or RBP. Currently, we restrict analysis of miRNA and RBP target sites to the 3' UTR sequences since studies have shown that most known metazoan miRNA targets reside in the 3' UTR [The CompMoby software integrates species specific and evolutionary conservation information as input into the MobyDick algorithm ,17 and fe 3' UTR ,3.CompMoby has the advantage of being comprehensive and flexible because the software does not only rely on alignments, and cis-regulatory motifs that are alignable do not have to be 100% conserved. CompMoby can also capture degenerate cis-regulatory sites in its clustering step, where similar motifs that are not necessarily exact matches are grouped together. This flexibility is a useful feature because cis-regulatory sites for both TFs and miRNAs are often degenerate in animals . Our algde novo cis-regulatory elements functioning at the transcriptional as well as the post-transcriptional level in metazoans. The nature of the algorithm allows the biologist to systematically identify the exact positions of putative cis-regulatory elements that are conserved and/or species specific on a genome-wide scale for experimental follow-up, which may provide insight into further understanding the complex regulatory networks of metazoans.The CompMoby software provides the user with a tool to identify 10 p-values; the second contains only motif clusters that pass a user defined Bonferroni corrected -log10 p-value cutoff and identifies the sequence source  of the motifs within the cluster . Depending on the user specified options, there are three different types of output files that can be produced from the alignment wrapper script: a file containing all NCRs in the entire genome; a file containing NCRs from only a subset of genes if the previous file has already been created; or both of the above files . For the 3' UTR analysis, the user has the option to analyze the entire length of the UTR sequences ; three, a quality factor  to be greater than 0.2 .From the set of three filtered dictionaries, all the motifs are grouped together by sequence similarity with the CAST clustering algorithm  to obtai10 p-values are calculated from a Poisson distribution based on the number of occurrences of the motif cluster in the input reference species sequence set compared to an expected number of occurrences derived from a random background set. The background set usually consists of the upstream or 3' UTR sequences of the same length as the input data from the entire genome of the reference species, or all other genes on the microarray from which the co-expressed genes were derived. This background sequence set can be obtained using the Ensembl Biomart feature . The user has the flexibility to upload a sequence file in FASTA format containing a background set of their choice. We recommend using a background set consisting of 5000 sequences or less and uploading a gzipped file to the CompMoby web interface due to the upload time. The -log10 p-value of each motif cluster is then Bonferroni corrected by the total number of clusters .10 p-values; the second contains only motif clusters that pass a user defined Bonferroni corrected -log10 p-value cutoff (default 0)Figure  as well 0)Figure . The web  from the CompMoby analysis of the pluripotent co-expressed gene set, showed that in mouse ES cells 56% of the tested motifs (different motifs from each of the 10 motif clusters) showed significant luciferase reporter activity compared to a control containing only a basal promoter . Interestingly, when the motifs that had significant reporter activity in mouse ES cells compared to differentiated cells were experimentally tested in human ES cells, the luciferase reporter activity was conserved. Four out of the ten motif clusters also demonstrated sequence specific binding by proteins present in ES cells using electrophorectic mobility shift assays . ExperiNanog would not have been discovered using MobyDick on the mouse sequences alone because the motif is only found in the set of sequences containing aligned blocks between mouse and human [width = 6, 8, or 10, conservation threshold = default values, and background = aligned input sequences or 1kb mouse background sequence file used for CompMoby calculation of motif enrichment. Only when CompareProspector was run with width = 8 and background = aligned input sequences was the Oct4/Sox2 binding site found.An advantage of using computational tools such as CompMoby over experimental methods is the ability to systematically predict TFBSs and their location upstream of many genes at once, thus reducing the time-consuming experimental dissection of large NCRs. In this particular case of cis-regulatory motif analysis in mammalian species, CompMoby improves upon MobyDick. The Oct4 and Sox2 binding site upstream of n Figure . Another release) discover release), width =a priori knowledge of known TFs.By analyzing both species specific and aligned sequences simultaneously, CompMoby enables a user to decide what type of motif they would like to experimentally test depending on the biological study. This feature is important for regulatory studies in model species, where the user may wish to study conserved as well as divergent regulatory pathways. Our previous study demonstrates the power of CompMoby to identify regulatory motifs that may be functional in the model organism and applicable to human, and to discover novel TFs that may have important roles in regulation by not requiring miRNAs play an important role in controlling gene expression at the post-transcriptional level and studies have shown that one miRNA may have multiple binding sites on the same target mRNA, which leads to stronger repression of the mRNA ,33. In aLim et al. generated the datasets by independently over-expressing miR-1 or miR-124 in human HeLa cells and then profiling the mRNA on whole genome microarrays. Both miR-1 and miR-124 are known for their tissue specificity in mammals, where the former is preferentially expressed in heart and skeletal muscle, while the latter is preferentially expressed in brain. Lim et al. identified 96 genes that were significantly down-regulated  at both 12 and 24 hours with miR-1 over-expression and 174 genes with miR-124 over-expression.. MobyDick analysis on the human 3' UTR sequences alone would not have been able to identify the target site of miR-1, which is derived from only the mouse sequence set  of these genes mapped to Ensembl gene identifiers (release 45) for input data into CompMoby as the reference species  or MEME (version 3.5.7) on the mouse 3' UTR sequences alone. We focused on the n-myc motif because n-myc is highly up-regulated in pluripotent cells [myc is capable of reprogramming fibroblasts to pluripotent stem cells in combination with other TFs [myc is largely interchangeable with c-myc [myc in reprogramming fibroblasts [We applied CompMoby to analyze the 3' UTR sequences of 123 co-expressed genes : Platform independent , UNIX (Perl scripts).Programming language: Perl, C.License: Accessible and free for all users.Bp: basepair; ChIP-chip: chromatin immunoprecipitation-on-chip; CompMoby: comparative MobyDick; ES cells: embryonic stem cells; miRNA: microRNA; NCR: noncoding region; RBP: RNA binding protein; TF: transcription factor; TFBS: transcription factor binding site; TSS: transcriptional start site; UTR: untranslated regionCC and HL conceived and designed the project. CC developed and coded CompMoby and web interface, and analyzed and interpreted 5' upstream and 3' UTR sequence results for all datasets. CM and RB designed, performed, and analyzed luciferase reporter experiments, and CM, RB, CC, and HL interpreted luciferase results. RY, GW, and MRS analyzed and provided list of up-regulated pluripotent cell genes for the 3' UTR analysis. CC and HL wrote the manuscript. All authors read and approved the final manuscript.Accession numbers of embryonic stem cells for upstream sequence analysis. Ensembl accession numbers of genes up-regulated in mouse embryonic stem cell compared with differentiated cells.Click here for fileAccession numbers of miR-1 over-expression. Ensembl accession numbers of genes down-regulated in human HeLa cells with miR-1 over-expression.Click here for fileAccession numbers of miR-124 over-expression. Ensembl accession numbers of genes down-regulated in human HeLa cells with miR-124 over-expression.Click here for fileAccession numbers of embryonic stem cells for 3' UTR analysis. Ensembl accession numbers of genes 1.5-fold up-regulated in all pair-wise comparisons of different types of mouse embryonic pluripotent cells to various types of differentiated cells.Click here for fileMaterials and methods for luciferase reporter assays. Materials and methods for ES cell culture and luciferase reporter assays in 3' UTR sequence analysis.Click here for file


Brain-derived ectonucleoside triphosphate diphosphohydrolases (NTPDases) have been known as plasma membrane-incorporated enzymes with their ATP-hydrolyzing domain outside of the cell. As such, these enzymes are thought to regulate purinergic intercellular signaling by hydrolyzing ATP to ADP-AMP, thus regulating the availability of specific ligands for various P2X and P2Y purinergic receptors. The role of NTPDases in the central nervous system is little understood. The two major reasons are the insufficient knowledge of the precise localization of these enzymes in neural structures, and the lack of specific inhibitors for the various NTPDases. To fill these gaps, we recently studied the presence of neuron-specific NTPDase3 in the mitochondria of hypothalamic excitatory neurons by morphological and functional methods. Results from those studies suggested that intramitochondrial regulation of ATP levels may play a permissive role in the neural regulation of physiological functions by tuning the level of ATP-carried energy that is needed for neuronal functions, such as neurotransmission and/or intracellular signaling.In the lack of highly specific inhibitors, the determination of the precise function and role of NTPDases is hardly feasable. Yet, here we attempt to find an approach to investigate a possible role for hypothalamic NTPDase3 in the initiation of the midcycle luteinizing hormone (LH) surge, as such a biological role was implied by our recent findings. Here we hypothesize that NTPDase-activity in neurons of the AN may play a permissive role in the regulation of the estrogen-induced pituitary LH-surge.We propose to test our hypothesis on ovariectomized rats, by stereotaxically injecting 17beta-estradiol and/or an NTPDase-inhibitor into the arcuate nucleus and determine the consequential levels of blood LH, mitochondrial respiration rates from arcuate nucleus synaptosomal preparations, NTPDase3-expression from arcuate nucleus tissue samples, all compared to sham and intact controls.Results from these studies may lead to the conclusion that estrogen may modulate the activity of mitochondrial, synapse-linked NTPDase3, and may show a correlation between mitochondrial NTPDase3-activity and the regulation of LH-release by estrogen. In certain tissues, nucleoside monophosphates are further hydrolyzed by 5'-ectonucleotidase to adenosine that activates P1 adenosine receptors. NTPDase4–8 have not been detected in the brain.NTPDases  have been described as an 8-member family of nucleotidase enzymes. Most NTPDases are integral membrane proteins: NTPDase1 , NTPDaseOf the NTPDases, types 1, 2 and 3 have been identified and studied in the brain. NTPDase1 was identified in neurons, glia and endothelial cells of the rat brain ,7, whereThe neuroendocrine hypothalamus is the target of a number of peripheral hormones that function as signals for the feedback-based regulation of various homeostatic systems. Estrogen, for example, targets numerous hypothalamic neurons, including those of the arcuate nucleus, to regulate the GnRH- and consequential pituitary LH-surge. We and others have previously demonstrated that in non-human primates, as well as in rats, considerable synaptic events take place in the neuroendocrine hypothalamus, termed the „estrogen-induced hypothalamic synaptic plasticity“ (EISP). EISP is marked by a well-defined pattern of synaptic reorganization of both excitatory (mainly during the positive gonadotrophin feedback) and subsequently, inhibitory interneuronal contacts ,13. SincOur initial results described above lead us to the hypothesis that NTPDase-activity in neurons of the AN may play a permissive role in the regulation of the estrogen-induced pituitary LH-surge.2) 20 minutes after initial injection of saline; Group 2 will receive E2 20 minutes after initial i.c. injection of suramin (NTPDase-inhibitor); Group 3 will receive two consecutive injections of physiological saline (sham control) with a timing presented above. An additional group of animals (Group 4) not subjected to i.c. injections will be investigated as described below. Animals will be sacrificed 2-6-10-18 and 26 hours after the i.c. injections. Blood samples will be drawn from each individual and plasma LH will be determined. Brains will be quickly removed and tissue samples containing the arcuate nucleus will be obtained by punch sampling biopsy technique as described by Palkovits and Brownstein [At least five healthy, normal cycling female rats per each parameter examined (see below) will be used for the study. Animals will be ovariectomized (ovx) 7 days prior to further experimentation. Seven days after ovariectomy, animals will receive stereotaxic intracerebral (i.c) injections into the arcuate nucleus, using the stereotaxic coordinates provided by Paxinos and Watson , as follin loco treatment of the right-sided samples allows the determination and therefore the better comparison of NTPDase activities among group-members. It should be noted that since no selective NTPDase3-inhibitor is available to-date, the proposed experiments will also be performed using alternative inhibitors that are preferably less potent purinoceptor antagonists. It is also necessary that the results from the proposed experiments be confirmed once a specific inhibitor, that does not affect purinergic receptors but is highly specific for NTPDase3, becomes available.Mitochondrial oxygen consumption  will be determined from synaptosomal preparations of tissue aliquotes without further inhibition (suramin) of the sample , or with further suramin-treatment of the contralateral  sample. Please note that the initial, i.c. injection of suramin is necessary to test the effect of NTPDase-inhibition on plasma LH levels , while further The experimental design is summarized in Figure With the proposed hypothesis it is our goal to prompt further studies that investigate the relationship between the NTPDase-dependent energetic status of cells and their relevant biological functions.NTPDase3 has been previously found in the mediobasal hypothalamus . Since oWhile the function of NTPDase3 has been mostly tested by receptor antagonists, increasing efforts have been made to find selective blockers for the enzyme itself. Despite of those efforts, a fully specific inhibitor has not yet been found. Today, the lack of such specific blockers is a considerable drawback of NTPDase-research. Yet, some of the purin receptor antagonists are also capable of inhibiting NTPDase enzyme activity. One such inhibitor for NTPDases is the polyoxometalate suramine. Although suramine  cannot be considered for selective inhibition of receptors or NTPDase-activity, it can be used in some subcellular fractions of biological samples, such as synaptosomal fractions isolated from various sources. As cited above, few studies investigated the ATPase activity of brain NTPDases in synaptosomal fractions, however, none of those examined a functional link between mitochondrial respiration and NTPDase activity. We, in our most recent study, demonstrated that inhibition of NTPDase activity in synaptosomal fractions decreased ADP-dependent state 3 mitochondrial respiration, and that the hypothalamic NTPDase3 expression and the mitochondrial state 3 respiration rates are dependent on the actual estrogenic background . These rAs described in the hypothesis, we expect that inhibition of NTPDases in the AN would prevent the estrogen-induced pituitary LH release. Inhibition of AN NTPDase-activity may affect changes in intercellular purinergic signaling and may also influence intracellular energy states by the hydrolytic activity of mitochondrial NTPDase.Numerous studies reported that extracellular ATP increases the activity level of GnRH neurons and the pituitary gonadotrops -23. On tThe proposed, yet to be tested mechanism would involve the inhibition of mitochondrial NTPDase-activity in hypothalamic neurons, thereby leading to a momentary increase in mitochondrial ATP levels, but a subsequent silencing of mitochondrial respiration ; As a result, a halt in mitochondrial ATP production would occur. In this case, NTPDase-containing nerve terminals would not have sufficient amounts of ATP to fuel neurotransmission, therefore, the highly energy-demanding EISP could not be initiated, thus preventing pituitary LH-release.In any case, testing the possible influence of NTPDase-inhibition on pituitary LH-release will bring us closer to understand the relationship between the considerably versatile neuronal ATP functions and the hypothalamic regulation of pituitary LH-release.The authors declare that they have no competing interests.The authors, through their frequent and periodic scientific consultations, have taken an equal share in the formulation of the presented hypothesis article. All authors read and approved the final manuscript.


All influenza pandemic plans advocate pandemic vaccination. However, few studies have evaluated the cost-effectiveness of different vaccination strategies. This paper compares the economic outcomes of vaccination compared with treatment with antiviral agents alone, in Singapore.We analyzed the economic outcomes of pandemic vaccination (immediate vaccination and vaccine stockpiling) compared with treatment-only in Singapore using a decision-based model to perform cost-benefit and cost-effectiveness analyses. We also explored the annual insurance premium (willingness to pay) depending on the perceived risk of the next pandemic occurring.The treatment-only strategy resulted in 690 deaths, 13,950 hospitalization days, and economic cost of USD$497 million. For immediate vaccination, at vaccine effectiveness of >55%, vaccination was cost-beneficial over treatment-only. Vaccine stockpiling is not cost-effective in most scenarios even with 100% vaccine effectiveness. The annual insurance premium was highest with immediate vaccination, and was lower with increased duration to the next pandemic. The premium was also higher with higher vaccine effectiveness, attack rates, and case-fatality rates. Stockpiling with case-fatality rates of 0.4–0.6% would be cost-beneficial if vaccine effectiveness was >80%; while at case-fatality of >5% stockpiling would be cost-beneficial even if vaccine effectiveness was 20%. High-risk sub-groups warrant higher premiums than low-risk sub-groups.The actual pandemic vaccine effectiveness and lead time is unknown. Vaccine strategy should be based on perception of severity. Immediate vaccination is most cost-effective, but requires vaccines to be available when required. Vaccine stockpiling as insurance against worst-case scenarios is also cost-effective. Research and development is therefore critical to develop and stockpile cheap, readily available effective vaccines. The influenza A (H1N1-2009) pandemic has been declared a pandemic by the World Health Organization (WHO) which has led to the activation of pandemic plans worldwide. These include development of candidate pandemic vaccines and stockpiling on antiviral drugs. However, it is not possible to predict with certainty when and what strain will trigger the next influenza pandemic. The influenza virus's changing behavior, acquisition of adaptive mutations, expansion of host range, emerging transmissibility of different strains among humans, and potential for genetic re-assortment raise concerns In recent years, H5N1 vaccines have been touted as a possible pandemic vaccination strategy to protect against a potential H5N1 pandemic strain While several reports have compared the cost-effectiveness of vaccination Similar to a previous anti-viral study performed in Singapore Cost-benefit analyses were performed to compare different vaccination strategies with treatment only, and included appropriate direct and indirect economic costs such as the cost of death. Cost-effectiveness analyses were also performed based on cost per-life-saved by vaccination and treatment only to provide another analytical dimension which avoids quantifying the value of life. The annual insurance premium was also used because it is difficult to predict a vaccine's true value. The annual insurance premium was the maximum cost that the country would be willing to pay annually to avert the impact of a pandemic, and includes all costs associated with vaccination including research and development, purchase, additional stockpiles, administration, and adverse effects. For example, a country would be willing to pay 3 times the annual insurance premium for a vaccine with 3 years shelf-life including administration, warehousing, and other costs. The analyses also provide the optimal strategies and investments for vaccines depending on vaccine efficacy and other key parameters.The values for all parameters are shown in a priori (i.e. sunk costs), and is a non-recurring cost. We then evaluated if the addition of pandemic vaccines to the treatment-only strategy provided a net cost benefit. Treatment is assumed to be given to all influenza-like-illness cases, regardless of vaccination. As oseltamivir treatment is optimal when administered early within 48 hours Since several countries, including Singapore, have stockpiled oseltamivir as part of the preparedness for an influenza pandemic, we assumed that antiviral treatment is available for every resident, has been purchased As pandemic vaccination has hitherto not been deployed for the actual prevention of a pandemic, its efficacy is largely unknown. This study relied on current animal and human immunogenic studies for H5N1 candidate vaccines to determine the possible efficacies of a candidate pandemic vaccine Vaccine cost, efficacy, and cross-reactivity (as mentioned above) are 3 key variables. As a base-case scenario, we assumed the vaccination cost of USD$46.4, i.e. based on higher-end pricing of USD$19.9 per dose , and USD$6.6 for costs of administration and adverse effects similar to a previous study We also assumed that everyone within a population (or selected population sub-group) would be given the vaccine (i.e. stockpiling and delivery costs for the entire population or population sub-group), but that only the proportion that sought vaccination early enough for sufficient antibody development would benefit.As pandemics are unpredictable with many uncertainties, we have modeled all uncertain input variables  as trianMultiple stockpiling scenarios were analyzed to observe the costs of different stockpile combinations. We explored scenarios where the pandemic arrives within the first stockpile (akin to vaccination for the current pandemic such as the H1N1-2009 without existing stockpiles), and after 10, 30, and 50 years, and we assumed that the stockpile would have a shelf-life of 3 and 5 years.Sensitivity analyses were performed to identify variables that have the largest influence on the outcome. In addition, one-way sensitivity analyses were performed across several key variables such as the efficacy and cost of the vaccine. Monte Carlo simulation analyses were also performed to determine the key outcomes under 1,000 scenarios each. We also explored key outcomes by the 6 sub-groups to determine if pandemic vaccines would be particularly beneficial in any population sub-group if resources only allow for selected sub-groups to be vaccinated.All costs were obtained and standardized to 2007 Singapore dollars, and represented in United States dollars using the following exchange rate . The model was run using Excel spreadsheets  and @Risk  simulation add-in. Details of the model and results are shown in the Technical Supplement.th and 95th percentiles of 350 and 1,122), with 78% of deaths occurring in the high-risk group. The mean number of hospital days is 13,950  with a total of 2.5 million workdays equivalent lost . The mean economic cost is USD$469.8 million .If treatment-only strategy were adopted during a pandemic, the mean number of simulated deaths in Singapore is 690  while the number of deaths was reduced by 41  for every 10% increase in vaccine efficacy. At vaccine efficacies of >55%, vaccination was cost-beneficial over treatment-only. The mean cost per life saved decreased with increasing vaccine efficacy, and treatment-only was less beneficial than vaccination when the vaccine efficacy exceeded 85%. However, the effectiveness changes once strain mismatches (lack of cross-reactivity) are taken into account. If the strain mismatch was 20%, vaccination was cost-beneficial only at vaccine efficacies of >65%; if the mismatch was 40%, vaccination was cost-beneficial at efficacies of >82%; while vaccination was not cost-beneficial if the mismatch was >50%.If the vaccine was to be used for an impending pandemic (within the first stockpile), the outcomes for a vaccine cost of USD$46.4, based on different levels of vaccine efficacy and cross-reactivity, are shown in th percentile with 100% vaccines efficacy and up to 20% strain mismatch.If vaccines were stockpiled for a future pandemic, the outcomes of the analyses are shown in Two-way sensitivity analyses were also conducted to determine the annual insurance premiums of the different strategies under different attack rates, case-fatality rates, and overall vaccine effectiveness; these are the key epidemic parameters which generate the greatest uncertainty in the outcome. Considering the maximum annual insurance premium based on vaccine stockpiling, the premium was higher with higher overall vaccine effectiveness and attack rates . For vacAt case-fatality rates of 0.1% [similar to inter-pandemic epidemics For long term stockpiling strategies with 1957 and 1968 pandemic case-fatality rates of 0.4 to 0.6%, stockpiling would be cost-beneficial only if the pandemic occurred within 10 years, the shelf-life was 5 years, and vaccine effectiveness was >80%. As the case-fatality increases, the maximal annual insurance premiums match the USD$46.4 estimated costs at a wider range of permutations. At a case-fatality of 5% [the higher end of the 1918 pandemic The population sub-group analysis considered the annual insurance premium when only a particular sub-group was vaccinated . ProtectWe have shown that pandemic vaccination is only cost effective in severe pandemics, high vaccine effectiveness, and low vaccine cost. The outcome of the model was very sensitive to the overall vaccine effectiveness (a combination of vaccine efficacy and strain matching); and the vaccine cost. Vaccine manufacturers and policy-makers should be aware of the importance of these key vaccine development parameters and the trade-off between effectiveness and price, while noting that the economic outcomes are different between countries and should be based on local data. Vaccine efficacy may vary widely as shown by the efficacy of existing H5N1 candidate vaccines against different H5N1 strains The differences in the outcomes where vaccination is deployed in the first stockpile cycle (immediate vaccination) versus some time during subsequent stockpile cycles will also have to be considered. As evident from Policy makers should consider funding research, development and local production of vaccines using annual insurance premiums as a guide to their willingness to pay for vaccine research and purchases to prevent a potential catastrophe. If vaccine stockpiling is to be considered, the cost of the vaccine is important. Other factors which must be considered are the cross-reactivity of the vaccine, context-specific political considerations , the potential benefit of reducing surge demand for healthcare services through vaccination, and the effect of rapid developments in vaccine technology which may favor stockpiling, since with each stockpile cycle progress in vaccine technology may make future vaccines cheaper or more efficacious or both. For example, for an impending pandemic occurring within the first stockpile cycle (influenza A (H1N1-2009) pandemic), USD$61.0 may be spent per capita to obtain a vaccine that has an overall effectiveness of 80% against a pandemic with 0.2% case-fatality. For Singapore, this amount of >USD$199.1 million may be used for novel virus vaccine development or purchase agreements. However, for future pandemics, the premium decreases to USD$10.0 and USD$3.3 if the pandemic is estimated to occur over the next 10 and 30 years respectively. Development of cheaper and more effective vaccines is therefore necessary for long-term stockpiling to be feasible.At high attack and case-fatality rates with low vaccine efficacy, the annual insurance premiums are much higher when compared to high vaccine efficacy but with low attack and case-fatality rates. If the perceived risk of disease severity is high (e.g. similar to the 1918 pandemic), the willingness to pay will be greater and even long term stockpiling will be cost-beneficial. The decision to purchase pandemic vaccines is therefore highly dependent on the perception of disease severity.It is also evident that high-risk subpopulations would benefit most from vaccination due to the high number of lives saved. High-risk children have high case-fatality rates and the highest economic value from future earnings, and hence command the highest annual insurance premiums. Similarly, high-risk elderly showed the lowest cost per life saved due to the largest reduction in deaths. Even if nation-wide vaccination or stockpiling are not viable options; or if vaccines are insufficient for the entire population, strategies which prioritize vaccination of high-risk groups could be considered independently, with children and adults favored for the highest economic effectiveness and elderly favored for more overall lives saved. Finally, our analysis also showed that the proportion who received early vaccination was also important, and underscores the point that stockpiles must be accompanied by well-planned programs to rapidly administer vaccinations. The current Influenza A H1N1 2009 pandemic has shown that additional high-risk sub-populations may be identified. These have included pregnant women and obese individuals and additional studies are required to determine the input and outcome parameters for these sub-populations Some limitations of this study include the exclusion of the societal value of health – although cost-utility analyses could address this, there are no available local indicators for Singapore. In addition, we have not included the pandemic's macro-economic impact which would likely favor interventions to a greater degree. While we have not considered recurring treatment stockpiles, any additional treatment stockpile costs will be borne equally by both strategies. For comparability, neither treatment nor vaccination was assumed to alter the pandemic's transmission dynamics. Vaccination, in particular, may reduce total population attack rates through increasing herd-immunity, but it is difficult to predict the impact of an imperfect pandemic vaccine on the transmission dynamics of a pandemic with characteristics that are still not fully defined Our study has shown that long-term vaccine stockpiling is expensive and may not be cost-effective at the population level for mild pandemics. However, stockpiling is cost-beneficial if insurance against a severe pandemic is the main priority, if specific high-risk groups are targeted, and if cheap and effective vaccines are rapidly available through research and development of novel vaccine technologies.Appendix S1Technical appendix with supporting information(0.47 MB DOC)Click here for additional data file.Figure S1(0.43 MB TIF)Click here for additional data file.