Drosophila life span. One third of the 1,332 homozygous P–element insertion lines assessed had quantitative effects on life span; mutations reducing life span were twice as common as mutations increasing life span. We confirmed 58 mutations with increased longevity, only one of which is in a gene previously associated with life span. The effects of the mutations increasing life span were highly sex-specific, with a trend towards opposite effects in males and females. Mutations in the same gene were associated with both increased and decreased life span, depending on the location and orientation of the P–element insertion, and genetic background. We observed substantial—and sex-specific—epistasis among a sample of ten mutations with increased life span. All mutations increasing life span had at least one deleterious pleiotropic effect on stress resistance or general health, with different patterns of pleiotropy for males and females. Whole-genome transcript profiles of seven of the mutant lines and the wild type revealed 4,488 differentially expressed transcripts, 553 of which were common to four or more of the mutant lines, which include genes previously associated with life span and novel genes implicated by this study. Therefore longevity has a large mutational target size; genes affecting life span have variable allelic effects; alleles affecting life span exhibit antagonistic pleiotropy and form epistatic networks; and sex-specific mutational effects are ubiquitous. Comparison of transcript profiles of long-lived mutations and the control line reveals a transcriptional signature of increased life span.Understanding the genetic and environmental factors that affect variation in life span and senescence is of major interest for human health and evolutionary biology. Multiple mechanisms affect longevity, many of which are conserved across species, but the genetic networks underlying each mechanism and cross-talk between networks are unknown. We report the results of a screen for mutations affecting  Drosophila lines homozygous for a single P transposable element mutation. We found 58 mutations in novel loci that increase life span by up to 33%. Most mutations had different effects on male and female life span, and for some the effects were opposite between the sexes. Effects of these mutations on starvation resistance, chill coma recovery, and climbing ability varied, but all had a deleterious effect on at least one other trait. A sample of ten mutations with increased life span formed genetic interaction networks, but the genetic interactions were different, and sometimes in opposite directions, in males and females. Transcript profiles of seven long-lived mutations and the control line reveal a core transcriptional signature of increased life span involving novel candidate genes for future analysis.Recent advances in medical science as well as vastly improved living conditions have resulted in a steady increase in human life span, with a concomitant increase in health issues associated with aging. In addition, understanding life history evolution requires that we know why organisms age and why there is variation in aging and senescence. To identify genes involved in aging, we assessed longevity in a collection of over 1,300  Understanding the genetic and environmental factors affecting variation in life span and health span is of major interest for human health and evolutionary biology. As the world population ages, the incidence of age-related diseases, such as Alzheimer's disease, cancer, cardiovascular disease and Huntington's disease, is concomitantly increasing. From the evolutionary perspective, we seek to understand why aging occurs, and why there is variation in aging between and within species Multiple mechanisms affecting longevity have been documented, many of which are conserved across species. Dietary restriction Given the heterogeneity of mechanisms affecting life span and the need to understand the genetic networks underlying each mechanism as well as cross–talk between networks, there is a clear need for unbiased, genome–wide screens to identify genes and genetic networks affecting life span. Studies using microarray technology to observe changes in gene expression during normal aging or following exposure to conditions that extend or reduce life span have indeed confirmed that expression of a substantial fraction of the genome changes with age C. elegansDrosophila, and consequently there have been relatively few studies reporting mutations increasing life span in this organism Drosophila life span, utilizing a collection of over 1,000 single, homozygous P–element insertion lines that were constructed in isogenic backgrounds Genetic screens for mutations affecting life span give unambiguous insight regarding the genes and pathways required for normal aging, as elegantly demonstrated by mutagenesis and RNAi screens in the short-lived model organism, Drosophila life span, we quantified the life span of males and females of 1,332 homozygous P{GT1} insertion lines P–element insert lines (P<0.0001) as well as significant sex–specific effects on life span (P<0.0001). Our estimates of the broad–sense mutational heritability (MH2) and the cross–sex mutational genetic correlation (FMr±SE) of life span were MH2 = 0.557 and FMr = 0.555±0.025. Averaged over all mutations, the standardized effects  of deviations of line means from their corresponding controls , 55 (95) and 12 (49) lines had significantly increased (decreased) life span in at least one sex or averaged across sexes  (P≤0.0006)  . Both an≤0.0006) . Thus, 4Pa/σPa/σ is 0.27 pooled across sexes, 0.43 in males and 0.39 in females. Thus the average effects of P–element insertions on longevity, although statistically significant, are subtle, but effects range from 5% to as large as 33%.We quantified the magnitude of the mutational effects on life span for the 58 mutations with increased life span in terms of percentage increase over the control strain, and by computing their standardized mutational effects, P{GT1}–element insertions on longevity, as indicated by significant line by sex interaction terms in the ANOVAs pooled across sexes . Mutations associated with an increase in longevity have highly sex–specific effects, with a trend towards opposite effects in males and females.We observed substantial variation in sexual dimorphism for the effects of ss sexes . The croL) and line by sex (L×S) terms from ANOVAs comparing the life span of each long-lived mutant line to the control to infer whether the mutations affected both sexes, or were sex–specific, sex-biased, or sex antagonistic . The remaining 41 mutations (70.7%) affected males and females differently. We categorized the mutational effects as “sex–specific” if the L×S interaction from the analysis pooled across sexes was significant, and the L term from the separate sex analysis was significant only in one sex; “sex-biased” if the L and L×S terms from the analysis pooled across sexes were both significant, and the L term from the separate sex analysis was significant in both sexes; and “sex–antagonistic” if the L term from the analysis pooled across sexes was not significant, but the L×S interaction was significant, and the L term from the separate sex analysis was significant in both sexes. We found 22 male–specific, two male-biased, nine female–specific, two female-biased, and six sex–antagonistic mutations  or affect an un-annotated gene in the more immediate vicinity. The remaining 39 P–element inserts were <2 kb from the nearest gene. Of these, 27 were adjacent to or within the predicted transcript of the only gene in the region, and most parsimoniously affect these genes. A total of 13 inserts were located in an intergenic region, <2 kb from each flanking gene, and could affect either or both adjacent genes.To identify candidate genes affecting life span, we mapped the sequences flanking the e genome , Table 3P–element tagged candidate genes, forkhead box, sub–group O (foxo) has been previously implicated to affect adult life span Only one of the P–elements inserted into identical or nearly identical positions: five inserts in the first intron of mushroom–body expressed (mub), two inserts 500 base pairs upstream of polychaetoid (pyd), two inserts adjacent to CG9238, two inserts in the Tre1/Gr5a intergenic region, and two inserts between CG8418 and Gef64C. Since this screen is far from saturation, these sites likely represent hotspots for P{GT1} element insertion Several of the mub affected both sexes, two were male–specific, and one was female–specific. One insert near CG9238 was female–specific, while the other affected both sexes. One of the inserts in the CG18418/Gef64C intergenic region affected both sexes, while the other was strongly female-biased. On the other hand, both inserts in the Tre1/Gr5a intergenic region affected both sexes, and both inserts near polychaetoid were male–specific.The effects of multiple inserts in the same genomic region are often, but not always, heterogeneous. Two of the inserts in esg, pyd and mub associated with increased life span were all in the Canton S F genetic background. In addition, seven mutations in esg, two mutations in pyd, and two mutations in mub were associated with reduced life span in the initial screen. All of these mutations were in the Canton S A or B genetic backgrounds, and with one exception (mubBG02497) were in different locations from the mutations in these genes associated with increased life span  to create revertant alleles in which the P–element was precisely (or nearly precisely) excised, while maintaining the co–isogenic background. We measured the life span of the revertant alleles, the parental strains, and the P–element insert line simultaneously. If the disruption of the adjacent gene by the P–element insertion causes the increase in life span, we expect that the life span of the revertant alleles will not be significantly different from the control. This expectation was realized for each of the revertant alleles.We re-mobilized the mubBG00043 allele was associated with increased life span in both sexes. We obtained one precise revertant (mubrev1) and one imprecise revertant (mubrev3). Both revertant alleles had mean female life spans that fully reverted to the control, whereas the mean male life spans were intermediate between the control and mutant line  P<0.0001), between males and females (P<0.0001), and the genotype by sex interaction (P<0.0001) . Thus, the effects of the double heterozygous genotypes on life span are independent in the two sexes.Since all independently isolated long-lived <0.0001) . The eff<0.0001) ; howeverVariation among the double heterozygote genotypes can arise from two sources: variation in mean heterozygous effects of the different mutations, and variation from epistatic interactions.GCA) and specific (SCA) combining abilities. The GCA of each mutation estimates its average dominance in combination with all other mutations. The SCA of each double heterozygous genotype is the difference in the observed life span of the genotype from that expected given the GCAs of the two parental lines. Since alleles at all other loci are fixed and homozygous, any statistically significant SCA values must be due to dominance×dominance epistasis. We found significant variation in GCA and SCA values (P<0.0001) when pooled over both sexes, as well as significant GCA×Sex and SCA×Sex interaction terms (P<0.0001), indicating sex–specific GCA and SCA effects , and the latter when the life span of the double heterozygote genotype is greater than expected . We identified eight statistically significant epistatic interactions in the analysis pooled across sexes; 10 significant interactions for females and 14 significant interactions for males . Thus, when examined separately the two sexes displayed vastly different epistatic interactions .We estimated the enotypes . Epistator males . The croractions . Of the We assessed whether mutations with significantly increased life span had pleiotropic effects on stress resistance (chill coma recovery and starvation resistance) as well as a general measure of health (climbing activity) at one week and six weeks of age. We observed substantial pleiotropy. Of the 50 lines tested for starvation resistance, 44 were significantly different from the control at one week (16 with increased starvation resistance and 28 with decreased starvation resistance in one or both sexes), and 46 were significantly different from the control at six weeks  . Of the H2 = 0.43–0.60 for starvation resistance and chill coma recovery, but were lower for climbing ability (H2 = 0.21 averaged over week 1 and week 6 measurements)  . Althougix weeks .r = 0.328±0.136, t48 = 2.41, P = 0.020) and six weeks  . None of the other correlations were significantly different from zero  and six weeks   ; i.e., mutations associated with increased stress resistance were less active  between longevity and chill coma recovery time was positive and significant at both one week ( 0.0025) . Thus, trom zero . In male<0.0001) . Further<0.0001) . Thus, ms active .P–insert lines associated with increased life span in at least one sex is depicted in The combination of significant pleiotropy but little directional correlation between longevity and other traits indicates that the pleiotropic effects are highly variable, as illustrated in pydBG00028, mubBG00043, crolBG00346, CG10990BG00495, CG9238BG00761, BG00817 and esgBG01042) affected whole genome transcriptional regulation. We performed these analyses at six weeks of age for all mutant lines and the Canton S F co–isogenic control – the age at which the control lines are beginning to die, but at which most of the P–element insert lines remain alive, and at which we assessed differences among these lines in senescence. The survival curves for this experiment are given in mubBG01042 females had reduced longevity, but in this assay, both males and females were long-lived.Genetic networks of mutations that affect a common phenotype can serve as focal points for the identification of additional candidate genes affecting that phenotype by transcript profiling q–value of 0.001 (0.0001), we found 11,111  sexually dimorphic transcripts. Remarkably, genotype was significant for 4,488 transcripts (35.5%) at q≤0.001, and 1,996 transcripts (15.8%) at q≤0.0001. The genotype by sex interaction was significant for 1,621 transcripts (12.8%) at q≤0.001, and 434 transcripts (3.4%) at q≤0.0001. We also ran reduced ANOVAs separately for each sex, and for each of the mutant lines compared to the control. A total of 619 and 561 transcripts were significant at q≤0.001 for females and males, respectively. The magnitude of transcriptional co–regulation varied among the mutant lines. At a significance level of q≤0.05, we observed 276 significant transcripts for CG10990BG00495; 313 for pydBG00028; 777 for CG9238BG00761; 1,815 for BG00814; 2,141 for crolBG00346; 2,193 for esgBG01042; and 3,969 for mubBG00043.Not all transcripts on the array are expressed in six week old adults. We eliminated all transcripts that were not considered present in both replicates of at least one line and sex, leaving 12,636 transcripts for analysis. We performed several analyses of variation of gene expression . First, We analyzed the Biological Process Gene Ontology (GO) categories represented by the significant transcripts to determine if particular categories are over-represented. In the separate sex analyses of all genotypes, there was over–representation of significant transcripts in the DNA integration, metabolism (particularly carbohydrate metabolism) and proteolysis categories in both sexes . Genes aAlthough all of the mutations assessed are long-lived, they have variable and sex–specific pleiotropic effects on longevity, resistance to starvation and chill coma stress, and climbing activity . Thus, wpyd affects the biological processes of the cell–cell adhesion, fusion cell fate specification and branch fusion in the open tracheal system  behaviors  (duction) .Since all seven mutant lines have increased life span relative to the control, we sought to define the transcriptional signature of increased life span from the probe sets with common patterns of co–regulation across multiple lines. A total of 553 transcripts were common to four or more of the mutant lines; of these, 187 probe sets were up-regulated relative to the control and 270 were down-regulated relative to the control . The up-P–element  are themselves significantly differentially expressed in the analysis considering all genotypes. Three of these genes  are also differentially expressed relative to the control in their own mutant backgrounds. Further, mub is differentially expressed in the pydBG00028 and esgBG01042 mutant lines, and esg is differentially expressed in the pydBG00028, crolBG00346 and CG9238BG00761 mutant lines. Six additional genes in which P–element mutations were associated with increased life span in our screen were differentially regulated among the seven mutations profiled in the array analysis . A further 13 genes in which P–element insertions were associated with decreased life span had differentially regulated transcripts in the mutant backgrounds . Finally, eight genes in which mutations have been described to negatively regulate life span were also differentially co-regulated in the mutant backgrounds .Genes that are co-regulated in the mutant backgrounds are themselves candidate genes affecting life span. Therefore, we tabulated variation in gene expression of known genes affecting life span in the mutant lines associated with increased life span . First, P{GT1} insertional mutations that were generated in one of six Canton S co–isogenic backgrounds for mutations affecting Drosophila longevity. In the initial screen, we identified 436 (32.7%) mutations with mean life spans that were significantly different from their co–isogenic control. Of these, 296 (67.89%) were associated with reduced life span, 135 (30.96%) were associated with increased life span, and 5 (1.15%) had sexually antagonistic effects on life span. The sample size per mutation in the initial screen was not large; therefore, many of the significant effects could be false positives. Nevertheless, if nearly one–third of mutations affect life span, the mutational target size for longevity must be large, consistent with the many mechanisms that are known to affect life span. We know the locations of the P–element inserts for 290 of the mutations associated with significant effects on longevity. Of these, 56 map to gene deserts  and likely define novel un-annotated genes. With the exception of foxoWe performed an unbiased, forward genetic screen of 1,332 Substantially more mutations were associated with decreased than increased life span. It is generally assumed that mutations decreasing life span are less interesting than mutations increasing life span, since the former category of mutations could be generally deleterious and affect all aspects of fitness, while the latter are more likely to have specific effects on life span. Thus, we concentrated on confirming the effects of mutations associated with increased life span with larger sample sizes in a secondary screen, and identified 58 mutations associated with increased life span. The mutations associated with significant increases in life span represent pathways known to affect life span , as well as novel pathways involving taste, the nervous system and embryonic development.P–elements to insert in genomic hot–spots generated many insertions in the same genes enabled us to observe directly the distribution of mutational effects in the same genes. While many mutations in the same genes did indeed have similar effects on life span, this was not always true. Mutations in the same gene can be associated with both increased and decreased life span, often in a sex–specific manner, depending on the location and orientation of the P–element insertion, and genetic background. Examples include insertions in the Tre1/Gr5a intergenic region mub, pyd and Defense repressor 1 (Dnr1)  . These oMutations in different locations in the same gene could have variable effects on longevity if they interfere with different aspects of gene regulation, or if some are in regulatory regions and others directly affect the protein. Different mutational effects could also arise due to variation in the amount of the vector inserted into the genome or by partial genomic excision during the insertion process. Variable effects of mutations in the same location and orientation but different genetic backgrounds may also be attributable to epistatic interactions with different alleles. Indeed, diallel crosses among just 10 of the mutations associated with increased life span revealed a surprisingly complex network of epistatic interactions involving all 10 mutations, suggesting pervasive epistasis between alleles affecting life span.Tre1/Gr5a intergenic regions interact epistatically with mutations in genes affecting insulin signaling Mutations in the Drosophila life span comes from observations that the effects of transgene over–expression and single mutations on longevity vary according to genetic background. The effect on increased life span of over-expressing Drosophila Superoxide dismutase was greater in the background of a relatively short-lived strain than in a long-lived strain background Indy mutation increased life span by 40–80% in the short-lived Shaker, Hyperkinetic and drop dead strains, but only by 15% in a strain selected for increased life span SOD in Drosophila motor neurons increases life span Further evidence for the importance of epistasis in the genetic architecture of MFr∼−0.3. Epistatic effects were similarly sex–specific, and in three cases the direction of the epistasis was opposite in males and females. This observation is consistent with previous studies documenting sex–specific effects on life span, beginning with Maynard Smith's D. subobscura males and females. More recently, QTLs affecting variation in life span between two laboratory strains, Oregon and 2b, have sex–specific effects Drosophila insulin–like receptor (InR) chicoDTS–3, a gene involved in ecdysone biosynthesis SOD in Drosophila motor neurons in different genetic backgrounds has sex–specific effects on life span D. melanogaster are greater in females than males p53 transgenes has sexually antagonistic effects on male and female life span that are in opposite directions depending on the developmental stage of over–expression Drosophila and other organisms A striking feature of our screen is that the effects of mutations increasing life span are highly sex–specific, with a low, but significant, negative cross sex–genetic correlation of Drosophila mutations affecting increased life span often exhibit antagonistic pleiotropy: mutations in chico and InR show a dwarf phenotype and have reduced fecundity Indy have decreased fecundity under adult caloric restriction The concept of trade–offs (antagonistic pleiotropy) is central to many evolutionary hypotheses for limited life span and senescence. Such trade–offs were historically envisioned to be governed by alleles with beneficial fitness effects early in life, when the force of natural selection is strong, but detrimental effects later in life, when natural selection is weak P–element insert lines associated with increased longevity were also associated with at least one deleterious pleiotropic effect on resistance to starvation stress, recovery after chill coma, and/or a general measure of health (climbing activity) at one week and/or six weeks of age  affected by the mutations. Taken at face value, our analysis of gene expression of six week old adults in seven mutant lines associated with increased life span and the control strain indicate that many genes affect life span. We identified 4,488 transcripts that were differentially expressed among all eight genotypes using a false discovery rate criterion of Drosophila and C. elegans. Pletcher et al.et al.Several other studies have reported whole genome changes in gene expression in aging P–element lines associated with increased life span with the analysis of normal aging in two Drosophila strains et al.q<0.0001, 50% (24) were significant between our genotypes at q<0.001.However, we were able to compare the genes that were co-regulated in the seven Drosophila mutations associated with increased life span with many of the C. elegans orthologs that were co-regulated in the long-lived daf–2 mutant background et al.Drosophila homologs. 30% (9) of the up-regulated genes were significant in our study at q<0.0001, and 63% (19) were significant at q<0.001. For the down-regulated genes, only 27% (3) were significant at q<0.001 (none were significant at q<0.0001). These numbers are slightly inflated as several heat shock genes in C. elegans are homologous to a single Drosophila gene, lethal (2) essential for life.There was also significant overlap of the genes that were co-expressed in InR was down-regulated in both the CG9238 and CG10990 backgrounds, consistent with the previously observed decrease of InR expression associated with increased life span Alcohol dehydrogenase (Adh) was up-regulated in the mutant pyd, mub and esg backgrounds. Adh expression has been shown to decrease with age Sirt2, a member of the Drosophila Sirtuin family mub, BG00817 and esg mutant backgrounds. The mub mutant displayed an increase in Sod expression and a decrease in chico expression which mirrors previous reports of changes of the expression of these genes in association with increased life span Many genes that have been previously shown to affect life span showed differential expression in the mutant lines associated with increased life span. For example, Drosophila longevity, and identified 58 mutations associated with increased life span. These mutations represent pathways known to affect life span , as well as novel pathways involving taste and nervous system and embryonic development. Mutations in the same gene can be associated with both increased and decreased life span, which could be caused by different insertion sites or epistatic interactions with different genetic backgrounds. Pervasive epistasis for mutations affecting life span is indicated by a diallel cross analysis of ten of the mutations associated with increased life span. A striking feature of our screen is that the main and epistatic effects of mutations increasing life span are highly sex–specific. Further, antagonistic pleiotropy of mutational effects is pervasive, in that all P–element insert lines associated with increased longevity were also associated with at least one deleterious pleiotropic effect on a component of fitness. However, the patterns of pleiotropy are also sex–specific and different for males and females. The 4,488 transcripts that are differentially expressed among all eight genotypes provide a glimpse into complex genetic networks affecting longevity, which include many genes previously shown to affect life span. Further studies are required to establish that P–element disruptions of all candidate genes cause the changes in longevity and to determine interactions of these novel mutations with mutations in genes of the insulin signaling pathway and other pathways known to affect life span. The causes of the sex–specific and background–dependent epistatic effects remain to be elucidated, as do any effects on early and late reproduction, and the contribution of the novel loci to naturally occurring variation in life span – in Drosophila, and other organisms.We performed an unbiased, forward genetic screen for mutations affecting P{GT1} insertion lines 1118w Canton–S backgrounds  as part of the Berkeley Drosophila Gene Disruption Project The P{GT1} insertion lines for changes in life span relative to their control line. The initial screen was conducted in blocks of 50–100 insert lines and the appropriate Canton S control line. Each block was initiated with virgin males and females that had eclosed within 48 hours of each other, with two replicate vials per sex per insert line, and ten replicate vials per sex of the control line. Each replicate vial contained five flies of the same sex and 5 ml cornmeal/molasses medium. We recorded deaths every two days until all flies were dead, and transferred the flies to fresh culture medium every 1–2 days.We screened 1,332 homozygous viable Y = μ+L+S+L×S+ε, and the reduced model for the analysis of sexes separately was Y = μ+L+ε, where μ is the overall mean, L is the line effect (random), S is the sex effect (fixed) and ε is the environmental variance between replicate vials. We computed the mutational broad sense heritability (MH2) from the full model as MH2 = (Lσ2+SLσ2)/(Lσ2+SLσ2+εσ2), where Lσ2, SLσ2 and εσ2 are, respectively, line, sex by line, and environmental variance components; and the cross–sex genetic correlation (MFr) as MFr = FMcov/LFσLMσ, where FMcov is the covariance between the mean life span of males and females, and LFσ and LMσ are the square roots of the variance components from the separate sex analyses of females and males, respectively.We evaluated mutational variation for life span using analyses of variance (ANOVAs) of the mean life span of replicate vials, expressed as deviations from the appropriate contemporaneous control means for each sex. The full two–factor mixed effects model for pooled sexes was α σz/(n)½. αz is the critical value for the normal distribution . σ is the phenotypic standard deviation, estimated as (Lσ2+SLσ2+εσ2)½ for the full model and (Lσ2+εσ2)½ for the reduced models. n is the number of replicate vials for each insert line (n = 4 in the full model and n = 2 in the reduced models). We also used the Dunnett's t–test, which corrects for multiple tests of different insert lines relative to a common control, to identify insert lines that were significantly different from the control within each block.We used two methods to identify insert lines with mean life spans that were significantly different from the control. We computed the 95%, 99% and 99.9% confidence intervals of the deviations from the control mean, assuming normality, as ±Y = μ+L+S+L×S+R(L×S)+ε (full model) and Y = μ+L+R(L)+ε (reduced model); where μ and S are defined above; L, the fixed effect of line, is the difference between the P–element insertion line and the co–isogenic control; R is the random effect of replicate vial; and ε is the environmental variance between individuals within each replicate vial. We computed the standardized effect of each mutation as a/Pσ, where a is one–half the difference in mean life span between the homozygous mutant and the corresponding control line, and Pσ is the phenotypic variation of the control line We re-assessed the life span of 83 lines with increased life span under the same conditions as the previous assay, but with larger sample sizes of at least 12 replicate vials per sex per line. We evaluated the significance of the difference in life span between each insert line and the control by ANOVAs pooled across sexes and for each sex separately, using the models et al.Hinp1I and ligated it to obtain circular fragments containing both genomic and P–element DNA from both ends of the insert. We used PCR to amplify the 5′ end with oligonucleotide primers pGT1.5 (CCGCACGTAAGGGTTAATG) and pGT1.d (GAAGTTAAGCGTCTCCAGG) and the 3′ end with primers Pry1 (CCTTAGCATGTCCGTG–GGGTTTGAAT) and Pry4 (CAATCATATCGCTGTCTCACTCA), at annealing temperatures of 55°C. We sequenced the resulting products using (5′) Sp1 (ACACAACCTTTCCTCTCAA–CAA) and (3′) Spep1 (GACACTCAGAATACTATTC). We aligned the flanking sequences to the D. melanogaster genome using BLAST in situ hybridization to polytene chromosomes. We generated biotin-labeled probes using the BioNick Labeling System (Invitrogen) protocol, and used the Vectastain ABC kit (Vector Laboratories) for signal detection.Bellen 2 insert lines  and one chromosome 3 insert line (BG00043), using crossing schemes that preserved the co–isogenic background of the revertant lines. To construct the chromosome 2 revertant lines, we crossed w1118; P; iso3 females to w1118; CyO/Sp; SbΔ2–3/TM6,Tb males. We mated male offspring of genotype w1118; CyO/P; SbΔ2–3/iso3 to w1118; CyO/Sp; iso3 females, and single male offspring of genotype w1118; CyO/P−; iso3 in which the P–element had excised were crossed to w1118; CyO/Sp; iso3 females. In the following generation, males and females of genotype w1118; CyO/P−; iso3 were mated inter se to make a homozygous revertant stock of genotype w1118; P−; iso3. To construct the chromosome 3 revertant lines, we crossed w1118; iso2; P females to w1118; CyO/Sp; SbΔ2–3/TM6,Tb males. We mated male offspring of genotype w1118; CyO/iso2; SbΔ2–3/P to w1118; iso2; TM3, Sb/H females, and single male offspring of genotype w1118; iso2; H/P− were crossed to w1118; iso2; TM3, Sb/H females. In the following generation, males and females of genotype w1118; iso2; TM3, Sb/P− were mated inter se to make a homozygous revertant stock of genotype w1118; iso2; P−. Here w1118, iso2 and iso3 are the three isogenic chromosomes of the Canton S F strain; P refers to the chromosome from the Canton S F strain with the P–element insertion associated with increased life span; and P− indicates a P–element excision allele.We generated revertant lines for two chromosome We assessed the life span of the revertant lines simultaneously with the corresponding insert and control lines, with 12 replicates for each line and sex. The analysis of the BG00043 revertants was done in Raleigh under the same conditions described for the previous tests. The analysis of the BG00346 and BG01042 revertants was done in Moscow, Russia. We used the same ANOVA models described above for the second analysis of life span to assess differences in life span among the lines, and Tukey tests to identify significant differences between mutant, revertant and control lines.1 genotypes following Griffing's 1 genotype as described above, with eight replicate vials per genotype per sex. The general combining ability (GCA) for each mutation is the difference between the mean life span of all genotypes containing that mutation and the overall mean GCA values as iGCA = [iT/(n−2)]−ΣT/n(n−2), where iT is the sum of mean life spans for all genotypes with the ith mutation, ΣT is twice the sum of mean life spans of all double–heterozygote genotypes, and n is the total number of mutant lines SCA) for any particular genotype is the difference between the mean life span of the genotype and the life span expected from the sum of the GCAs of the mutants involved in the cross SCA values as ijSCA = ijX−(iT+jT)/(n−2)+ΣT/(n−1)(n−2), where ijX is the mean life span of the offspring resulting from the cross of the ith and jth mutant lines. We also estimated GCAs and SCAs separately for each sex. We used Diallel–SAS05 GCA and SCA effects and their standard errors; to perform ANOVAs to assess the significance of variation among the double heterozygous genotypes (G) for the full model pooled across sexes (Y = μ+G+S+G×S+R(G×S)+ε) and for the analyses of each sex separately (Y = μ+G+R(G)+ε); and to partition the G term into its GCA and SCA components, pooled across sexes (Y = GCA+SCA+S+GCA×S+SCA×S+R(S)+ε) and separately by sex (Y = GCA+SCA+R(S)+ε).We evaluated epistatic interactions among 10 mutations, generated in different genes in the F background, that had increased life span relative to the Canton S F strain . We crossed these lines in a half–diallel crossing scheme  to create all 45 possible double heterozygote FWe assessed pleiotropic effects of mutations with significantly increased life span on stress resistance (chill coma recovery and starvation resistance) as well as a general measure of health (climbing ability) for virgin flies at one week and six weeks of age. We tested the F lines in three blocks, the A lines in two blocks, and all B lines simultaneously. Each block included the appropriate control line. We measured chill coma recovery time and climbing ability for individuals within each block within 48 hours, and scored all individuals within a block for starvation resistance at the same time.Y = μ+L+S+L×S+ε) and for each sex separately(Y = μ+L+ε).We transferred 30 same–sex individuals without anesthesia into empty vials and placed the vials in chambers containing melting ice. After three hours, we transferred the vials to room temperature, and placed the flies from each vial on their backs. We scored the time it took for each individual to stand on its legs in one minute intervals, for a maximum of 30 minutes for one week old flies and 45 minutes for six week old flies. The total sample size was 30 males and 30 females per line per age. We evaluated the significance of the difference in chill coma recovery time between each insert line and the control using ANOVAs pooled across sexes (Y = μ+L+S+L×S+R(L×S)+ε) and for each sex separately (Y = μ+L+R(L)+ε).We assessed survival time of flies in vials containing non–nutritive medium (1.5% agar in 5 ml water). The sample size was 30 males and 30 females per line per age, with 10 same–sex flies in each of three replicate vials per sex per line. We recorded survival every eight hours until all flies were dead. We evaluated the significance of the difference in survival between each insert line and the control using ANOVAs pooled across sexes (Y = μ+L+S+L×S+ε) and for each sex separately(Y = μ+L+ε).We assessed the climbing ability of 30 flies per sex per line. We placed single flies in empty glass vials (15cm high×2.5cm diameter) without medium for one hour. We then tapped the flies to the bottom of the vial to stimulate a negative geotactic climbing response, and scored the height of the fly in the vial after 10 seconds (1 height unit = 5 mm). All climbing assays were conducted between 11:00 am and 12:30 pm. We evaluated the significance of the difference in climbing activity between each insert line and the control using ANOVAs pooled across sexes  for whole genome expression analysis. These seven lines were a subset of the lines used for the epistasis analysis: BG00028, BG00043, BG00346, BG00495, BG00761, BG00817 and BG01042. We collected over 100 virgin flies of each sex over a 4–day interval from each of the P–element insert lines and the co–isogenic Canton S F control line, and maintained them as described for the previous life span assays. We froze 42 day–old flies, and created two replicate pools of 25 flies per sex per line for RNA extraction. We used a TRIZOL (Gibco BRL)/chloroform protocol to extract RNA from whole flies, and prepared cRNA from 5 µg RNA following the recommended protocol for eukaryotic one–cycle target labeling. We hybridized fragmented cRNA to Affymetrix Drosophila Genome 2.0 GeneChip arrays.We chose seven th base. We used the weighted log(PM–MM) intensity of each probe set as the quantitative measure of expression, and scaled the expression scores to a median intensity of 500. Each probe set is identified as being present, marginal or absent. After excluding probe sets that did not have a present signal for both replicates for at least one sex and line, we retained 12,635 probe sets for analysis. We performed ANOVAs of gene expression on each probe set using the model Y = μ+L+S+L×S+ε. We considered probe sets for which the P–value for the L term exceeded a q–value threshold of q<0.0001 L×S term. To identify in which mutant lines gene expression was different from the control line, we used Tukey tests on probe sets for which the L or L×S terms were significant, and full model ANOVAs comparing each individual line with the control. We used χ2 tests to evaluate over– and under–representation of Gene Ontology (GO) biological process categories for probe sets with a significant L effect for all mutant lines together (q<0.0001) as well as individual mutant lines (q<0.05) and individual sexes (q<0.001). We based the expected values on the ratio of the biological process probe sets in the significant lists to the total number of biological process probe sets on the array.For each of the 18,800 probe sets on the array there are 14 25mer perfect match (PM) oligonucleotides and 14 25mer single nucleotide mis–match (MM) pairs, with the mis–match at the 13Data from all arrays used in the study are located at the Gene Expression Omnibus (GEO) public data repository .q–values.We performed all statistical tests using SAS V8.2, V9.1 and Microsoft Office Excel. We used QVALUE software Figure S1P-element insertions associated with increased life span on starvation resistance, chill coma recovery, and climbing activity, in males at one week of age. All values are expressed as deviations from the control. (A) Life span and starvation stress. (B) Life span and chill coma recovery. (C) Life span and climbing activity. (D) Starvation resistance and chill coma recovery. (E) Starvation resistance and climbing activity. (F) Chill coma recovery and climbing activity.Scatterplots of pleiotropic effects of (0.06 MB PDF)Click here for additional data file.Figure S2P-element insertions associated with increased life span on starvation resistance, chill coma recovery and climbing activity, in females at one week of age. All values are expressed as deviations from the control. (A) Life span and starvation stress. (B) Life span and chill coma recovery. (C) Life span and climbing activity. (D) Starvation resistance and chill coma recovery. (E) Starvation resistance and climbing activity. (F) Chill coma recovery and climbing activity.Scatterplots of pleiotropic effects of (0.06 MB PDF)Click here for additional data file.Figure S3P-element insertions associated with increased life span on starvation resistance, chill coma recovery and climbing activity, in males at six weeks of age. All values are expressed as deviations from the control. (A) Life span and starvation stress. (B) Life span and chill coma recovery. (C) Life span and climbing activity. (D) Starvation resistance and chill coma recovery. (E) Starvation resistance and climbing activity. (F) Chill coma recovery and climbing activity.Scatterplots of pleiotropic effects of (0.06 MB PDF)Click here for additional data file.Figure S4P-element insertions associated with increased life span on starvation resistance, chill coma recovery and climbing activity, in females at six weeks of age. All values are expressed as deviations from the control. (A) Life span and starvation stress. (B) Life span and chill coma recovery. (C) Life span and climbing activity. (D) Starvation resistance and chill coma recovery. (E) Starvation resistance and climbing activity. (F) Chill coma recovery and climbing activity.Scatterplots of pleiotropic effects of (0.06 MB PDF)Click here for additional data file.Table S1P-element insertions on life span. Cases in which the P-element insertion could possibly affect more than one gene (G1) are noted by listing the second (G2) and occasionally third (G3) gene in the region of the insertion. Mean life spans and life spans expressed as a deviation from the contemporaneous control are given for females, males and pooled across sexes. The 95, 99 and 99.9 confidence intervals (CI) are color coded for lines with increased (H) or decreased (L) life spans from the control line. The results from Dunnett's t-tests (D) are also color coded for lines with increased (H) or decreased (L) life spans from the control line.Results of initial screen for effects of (0.44 MB XLS)Click here for additional data file.Table S2P{GT1} insertion lines with increased life span. The table lists mean life span of double heterozygous genotypes and estimated GCA values for (A) sexes pooled; (B) females and (C) males. Parental homozygous P{GT1} insertion lines are indicated on the top row and first column of each panel. iT and GCA are defined in the text. Significant GCA values are indicated in bold font.Diallel cross between ten (0.07 MB DOC)Click here for additional data file.Table S3Analyses of variance of life span of (A) double heterozygote genotypes and (B) general and specific combining abilities.(0.05 MB DOC)Click here for additional data file.Table S4GCA) and (B) specific (SCA) combining abilities.Estimates of (A) general ((0.11 MB DOC)Click here for additional data file.Table S5Pleiotropic effects of mutations increasing life span. (A) Starvation resistance (week 1); (B) Starvation resistance (week 6); (C) Chill coma recovery (week 1); (D) Chill coma recovery (week 6); (E) Climbing ability (week 1); (F) Climbing ability (week 6).(0.54 MB DOC)Click here for additional data file.Table S6Analyses of variance of pleiotropic effects of mutations with increased life span. (A) Starvation resistance; (B) Chill coma recovery; (C) Climbing ability.(0.10 MB DOC)Click here for additional data file.Table S7P-values and q-values for the main effects of genotype (G), sex (S) and genotype by sex (GS). The first six columns refer to the analysis including all genotypes, pooled across sexes. The next four columns refer to the analysis including all genotypes, separately for males and females. The next 14 columns refer to the analysis comparing each mutant line to the control. The colors in each column highlight probe sets that are significant, with the darker colors denoting the more stringent significance threshold, accounting for multiple tests. For the analyses of each probe set compared to the control, blue denotes down-regulation and pink indicates up-regulation.Statistical analysis of gene expression data. The results of ANOVAs of gene expression are given for each probe set declared to be present, for three separate analyses. Entries in the columns are (4.49 MB XLS)Click here for additional data file.Table S8pyd; (D) mub; (E) crol; (F) CG10990; (G) CG9238; (H) BG00817; (I) esg.Over-represented Biological Process Gene Ontology (GO) categories. (A) Females; (B) Males; (C) (1.12 MB DOC)Click here for additional data file.Table S9Probe sets in common to four or more mutations affecting increased life span. Red cells indicate probe sets with increased expression relative to the control, blue cells indicate probe sets with decreased expression relative to the control, and grey cells indicate probe cells with levels of expression not significantly different from the control.(0.10 MB XLS)Click here for additional data file.Table S10Candidate genes on the array.(0.20 MB DOC)Click here for additional data file.


Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm–egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran  Diplosoma listerianum, cryptic female choice of sperm allows ova to avoid self-fertilization through discrimination against genetically similar sperm (Echinometra sp.), external fertilization is mediated by attachment to the egg of the sperm protein bindin , both under conditions that mimic the gamete microenvironment, we have demonstrated that relative sperm velocity is an important gametic predictor of sperm competitiveness  is a large cluster of genes involved in the acquired immune response and immunological self/non-self recognition. It includes the most variable genes known to date in vertebrates, with up to 349 alleles described for a single locus . MH geneA number of studies have documented mate choice in relation to MH genes, with females promoting offspring MHC diversity through preferences for MHC-dissimilar male mates , employing a split-family design, and although there was an MHC class II effect on disease resistance, no MHC influence was detected on relative fertility  Research Station, Ims, southwestern Norway. Sea-ranched anadromous adults were caught in a fish trap located 100 m above the outlet of the River Imsa, close to NINA Research Station. All fish had been tagged at release as smolts, and therefore known to be derived from the Imsa population. All animal experiments described were approved by the Norwegian Animal Research Authority.Forty-one males and 59 females were anaesthetized and passive integrated transponder tagged. Small fin clips were taken and used to type the fish for MH class I genes using neutral microsatellite markers that amplify simple repeats embedded in these MH genes of Atlantic salmon. The length of these repeats is diagnostic for allelic variation in MH class I genes of Atlantic salmon (Sasa-UBA) was PCR amplified using a fluorescently labelled forward primer (SasaUAgts2F 5′-AGGAAGGTGCTGAAGAGGAAC-3′) and a reverse primer (5′-CAATTACCACAAGCCCGCTC-3′). Genotyping was performed on an ABI prism 377 automated sequencer (Applied Biosystems).DNA was extracted using the Promega Wizard DNA extraction kit, following the manufacturer's instructions. Typing was done based on the genomic DNA extracted from the fin clips according to To confirm the MH class I type of the individuals included in the experimental design, sequencing of the expressed MHC class I alleles for six of the parents was conducted at the end of the experiment when the fish could be killed. Anterior kidney samples were stored on RNAlater and RNA extraction was performed using the Promega SV total RNA isolation system. The mRNA extracted was used to generate cDNA using RT-PCR  to amplify a region of approximately 500 bp (spanning the α1- and α2-domains) with a 50 : 50 mix of the following primers  such that female 1 was similar to male A but dissimilar to male B, and female 2 was dissimilar to male A but similar to male B, with respect to MHC class I alleles. For the similar crosses, males and females shared identical MH class I alleles; dissimilar crosses had different alleles.2 circular stream-section arenas designed to simulate natural breeding conditions  from the two males. The sperm/extender samples from each male were separately mixed, and gametes placed on the opposite sides of a dry 1 l plastic beaker. Sperm and eggs were activated and mixed in the turbulence when 500 ml Ims River water at the natural river temperature (3(±1)°C) was rapidly added.Sperm activity was recorded at 3(±1)°C within 30 min of fertilization using a Sony Hi8 tape deck connected to a JVC video camera (TK-1280E) fixed to an Olympus CK40 inverted stage microscope calibrated to a slide at 400× magnification under dark-field phase illumination. Subsamples of sperm in extender were activated by mixing milt in river water, and then 0.7 μl of the active diluent was immediately transferred onto a 12-well multi-test glass slide  (well depth=∼0.0116 mm) and a cover-slip carefully and quickly put in place. Milt (3–6 μl) and river water volumes (350–400 μl) used for activation were adjusted, so that 50–100 spermatozoa were visible on the field of view at 400× magnification, and all sperm are simultaneously and evenly activated. The time of sample activation was recorded, and sperm activity was video recorded until sample death.Sperm motion characteristics were analysed using a computer-aided sperm analysis system, the Hobson Sperm Tracker . The tracker was set to operate at a frame rate of 50 Hz and the ‘minimum track point’ setting was 50 frames. The ‘search radius’ used was 8.13–10.56 μm and the ‘threshold’ set to +30/−100 with the objective at 40×. Tracking was initiated 5 s after activation, allowing 3 s for activated sperm to be transferred to the slide, and 2 s for stabilization of the image. Tracking periods were set to 15 s, as this is the shortest time period in which the Hobson tracker can calculate the percentage of motile sperm. The most informative data come from the first tracking period, i.e. 5–20 s after activation  egg mortality during the incubation period, also suggesting no MHC-based effects on egg development.−1) and incubated at 50°C overnight. Fifteen microlitres of 6 M NaCl were added to each well before centrifugation at 3000g. Ten microlitres of supernatant from each well were transferred to a clean plate using a multi-channel pipette and 20 μl of 100 per cent cold ethanol added to each sample. The plate was then left at −80°C for at least 30 minutes before centrifugation at 3000g for 30 min. The supernatant was discarded and each DNA pellet washed with 150 μl of cold 70 per cent ethanol. The DNA pellets were then dried at 50°C for 20 min before resuspension in 100 μl water. The plates were left at 37°C for 30 min to ensure resuspension of the DNA, which was then stored at −20°C.Microsatellite genetic analysis was performed to determine parental genotypes and offspring paternity. Paternity of 30 offspring from each IVF competition was determined. A total of 36 adults and 540 embryos were screened. Tissue samples were placed in 96-well plates with 50 μl TEN buffer , 2 mM EDTA (pH 8) and 2% SDS (9 : 1 ratio)) and 5 μl proteinase K (10 mg ml2 (Abgene), 1 μl of BSA (10 mg ml−1), 0.5 μM of labelled forward primer, 0.5 μM of reverse primer and sterile distilled water to total volume. Forward PCR primers were fluorescently labelled with FAM (Ssa408), HEX (Ssa410) and NED (Ssa421) (Applied Biosystems). An initial 3 min denaturation at 94°C preceded 29 denaturing (94°C for 15 s), annealing (Χ°C for 15 s) and extension (72°C for 15 s) cycles. Annealing temperatures were 58°C for Ssa408 and Ssa421 and 53°C for Ssa410.Paternity was analysed by surveying microsatellite variation at two pure loci (Ssa408 and Ssa410) and one compound locus (Ssa421) (GeneScan and Genotyper software packages v. 3.7 (Applied Biosystems). Once the parental genotypes were known, only one, occasionally two, of the specified loci was needed for unambiguous paternity exclusion of the embryos in each two-male competition. Parentage was determined by comparing alleles at the locus or loci used, with alleles from the mother and both of the potential fathers.PCR products were run on an ABI3700 automated DNA sequencer with the Genescan-500 ROX-labelled size standard (Applied Biosystems). Fragment lengths were determined using the t-test. To determine that results from our random choice of focal males in the paired test was representative of the overall distribution of all possible data, we also performed Monte Carlo resampling (999 iterations with replacement) of all possible relative fertilization success differences for each of the nine 2-male/2-female combinations using the Excel add-in PopTools v. 3.0 (www.cse.csiro.au/poptools/).We employed a paired-male experimental design, so that relative fertilization success could be directly compared between different females when males were competing for fertilizations in both MH-similar and MH-dissimilar roles (in random order). Each two-male competition is therefore repeated, using a reciprocal MH genotype female (in random order), allowing comparison within one (randomly selected) male from each of the nine competing pairs using a paired p<0.1, randomizing male inclusion for each of the 100 runs.Having analysed for an effect of MH genotype using this controlled paired design, we then explored whether any MH-dependent relative fertilization success showed covariation with sperm traits known to be important in sperm competition using ANCOVA. Measurement of spermatozoal characteristics were repeated for each different fertilization trial, so that any within-male changes in sperm traits between trials  were individually controlled for. In the ANCOVA, we analysed across all 18 different females, so that our analytical unit of independence is an individual female's relative fertilization compatibility with one (randomly chosen) male in relation to MH similarity and the covariates of relative sperm velocity, density of motile sperm and sperm longevity  while revealing a significant covariance with relative sperm curvilinear velocity . There was no difference in average sperm velocity between MH treatments , p=0.24 (minimum p=0.15)). No significant covariation was found between motile sperm density  or sperm longevity  and sperm competition success, in consistence with previous work under similar conditions   . Lysin–Ve (VERL) .in vitro fertilization competitions having significant effects on the relative fertilization success of the delayed male's sperm (in vitro fertilizations using extreme sperm concentrations (50 : 50) show similar fertility and embryo survival as low dilutions . Given the large internal volume of a salmon egg (diameter 5000–6500 μm) relative to the average length of a sperm (30–40 μm) and the micropylar canal 30 μm (Less is known about the mechanisms of sperm–egg interaction at fertilization in salmon. Gamete association after release into turbulent water in salmonids is rapid , with a 's sperm , nothingal 30 μm , there mal 30 μm .


Purpose. This paper describes the clinical history and radiographic appearance of secondmalignancies in patients with bilateral retinoblastoma.					Subjects/methods. The imaging studies and clinical data of 14 patients with a history ofbilateral retinoblastoma who were treated for second malignancies were reviewed.					Results. A total of 17 tumors were identified in 14 patients during the period 1978–1996. The median age of occurrenceof the second malignancy was 17 years (range 10–32 years). Fourteen of the 17 malignancies occurred in the facialstructures and three developed in the lower extremities. The histologies included osteosarcoma (n = 5), malignant fibroushistiocytoma (n = 3), high-grade spindle cell sarcoma (n = 3), malignant mesenchymoma(n = 1), leiomyosarcoma (n = 4) and angiosarcoma(n = 1). The tumors were locally aggressive and had a similar appearance to those foundin nonretinoblastoma patients. Six of the 14 patients are alive and disease free.					Discussion. Most of the adolescent and young adult retinoblastoma survivors developed second malignancies in theirradiated facial structures but some occurred in distal sites. Radiologically, these tumors do not differ in appearance fromthose seen in non-retinoblastoma patients with the exception of their location.


In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg−1) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2α phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-κB (NF-κB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia.Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies  One of the primary substrates for PKR is eukaryotic initiation factor 2 (eIF2), which is phosphorylated on the α-subunit, leading to inhibition of translation initiation by blocking the action of the guanine exchange factor eIF2B  and reduced physical activity, terminating in impairment of respiratory muscle function and death of the patient through hypostatic pneumonia . In ordeor eIF2B . Both PIof eIF2α .κB (NF-κB) through activation of the upstream kinase, IκB kinase leading to degradation of the inhibitors IκBα and IκBβ and the concomitant release of NF-κB , hybond A nitrocellulose membranes and enhanced chemiluminescene (ECL) development kits were from Amersham Biosciences Ltd . Mouse monoclonal antibodies to 20S proteasome α-subunits and p42 were from Affiniti Research Products . Rabbit monoclonal antibodies to phospho-eIF2α (Ser 51) and to phospho-PKR (Thr 446) were purchased from Insight Biotechnology Ltd . Mouse monoclonal antibody to myosin heavy chain was from Novocastra , whereas polyclonal antisera to total PKR were from New England Biolabs Ltd . Rabbit polyclonal antisera to mouse β-actin were from Sigma Aldridge . Peroxidase-conjugated goat anti-rabbit antibody and peroxidase-conjugated rabbit anti-mouse antibody were purchased from Dako Ltd . The PKR inhibitor and PhosphoSafe™ Extraction Reagent were from Merck Eurolab Ltd  and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics .L- phenylalanine into acid insoluble material during a 2 h period in which soleus muscles were incubated at 37°C in RPMI 1640 without phenol red and saturated with O2 : CO2 (19 : 1). After incubation, muscles were rinsed in non-radioactive medium, blotted and homogenised in 4 ml 2% perchloric acid. The rate of protein synthesis was calculated by dividing the amount of protein-bound radioactivity by the amount of acid-soluble radioactivity.The method for the determination of protein synthesis in muscle has been previously described . ProteinM glucose and 0.5 mM cycloheximide. The protein degradation rate was determined by the release of tyrosine  Krebs–Henselit buffer (pH 7.4), containing 5 mtyrosine  over a 2μM). Only lactacystin-suppressible activity was considered to be proteasome specific. Activity was normalised to the protein content of the muscle determined by the Bradford assay (Sigma).The 20S proteasome functional activity was determined by measuring the ‘chymotrypsin-like’ activity by the method of After killing animals were heated to 80–90°C for 48 h, or until a constant weight was achieved. The water content was determined from the difference between the wet and dry weight. Lipids were extracted from the dried carcass with chloroform/methanol (1 : 1), ethanol/acetone (1 : 1) and diethyl ether, which was then allowed to evaporate. The fat content was determined from the weighed residue. The non-fat carcass mass was calculated as the difference between the initial weight of the carcass and the weight of water and fat.DNA-binding proteins were extracted from skeletal muscle using hypotonic lysis followed by high salt extraction of nuclei, as described . The EMSμl PhosphoSafe™ Extraction Reagent and centrifuged at 18 000 g for 5 min. Samples of cytosolic protein (10 μg) were resolved on 10% sodium dodecylsulphate–polyacrylamide gel electrophoresis (6% for eIF2α), and transferred to 0.45 μm nitrocellulose membranes, which had been blocked with 5% Marvel in Tris-buffered saline, pH 7.5, at 4°C for 1–2 h, and then washed for 15 min in 0.1% Tween buffered saline or PBS Tween before adding the primary antibodies. The primary antibodies were used at a dilution of 1 : 1000, except for phospho-eIF2α (1 : 500), actin (1 : 200) and myosin (1 : 100). The secondary antibodies were used at a dilution of 1 : 1000. Incubation was for 1 h at room temperature  or overnight, and development was by ECL. Blots were scanned by a densitometer to quantify differences.Samples (about 10 mg) of gastrocnemius muscle were homogenised in 500 P-values less than 0.05 were considered significant.Results are presented as mean±s.e.m. Differences in means between groups were determined by one-way analysis of variance followed by Tukey–Kramer multiple comparison test. −1) on change in body weight and tumour growth in mice bearing the MAC16 tumour over a 5-day period is shown in in vitro  that levels of phospho-PKR and -eIF2α were also significantly elevated in rectus abdominis muscle of weight losing cancer patients, when the weight loss exceeded 10%. There was a parabolic relationship between levels of phospho-PKR and -eIF2α and weight loss, with levels increasing with weight loss up to a maximum of 19–20%, and then decreasing. There was a linear relationship between expression of mRNA for the C2 proteasome subunit and levels of phospho-PKR, suggesting that phosphorylation of PKR was responsible for the induction of proteasome expression and degradation of myofibrillar proteins. Thus, if the in vitro results are also applicable in vivo this suggests that inhibition of PKR autophosphorylation may be useful for the treatment of muscle atrophy in cancer patients, particularly for those with weight losses between 10 and 20%.PKR is a serine/threonine protein kinase, which is normally inactive, but undergoes a conformational change upon binding of its activator, dsRNA, leading to autophosphorylation, with phosphorylation of substrates independent of dsRNA . The besynthesis . Studiesagonists  have shoin vitro study  that MAC16 cells have a higher requirement for isoleucine and tryptophan than a related tumour (MAC13), which does not induce cachexia. Previous studies have shown inhibition of the growth of the MAC16 tumour when cachexia was attenuated by More studies are needed on the antitumour activity of PKR inhibitors and the mechanism by which they exert their effect. Inhibitors of PKR may also be expected to be synergistic with DNA-damaging agents such as cisplatin and melphalan, as PKR-deficient mouse-embryonic fibroblasts have been shown to be hypersensitive to bulky adduct DNA damage (These results suggest that agents which inhibit autophosphorylation of PKR may be useful in the treatment of muscle atrophy associated with cancer cachexia and may have the added benefit of attenuating tumour growth.


These new antigenic determinant(s) akin to those associated with cancer basic protein appear rapidly (within 5.5 hours) in vitro. Cultures of “normal” cells are thus antigenically different from the same cells in context in vivo.Freshly excised human embryonic tissue used as antigen to test lymphocytes from a cancer bearing patient gives a “normal tissue” result of about 10% in the macrophage electrophoresis migration (MEM) test. When, however, it is grown


Plasmodium falciparum variant erythrocyte surface antigens known as PfEMP1, encoded by the var gene family, are thought to play a crucial role in malaria pathogenesis because they mediate adhesion to host cells and immuno-modulation. Var genes have been divided into three major groups  and two intermediate groups (B/A and B/C) on the basis of their genomic location and upstream sequence. We analysed expressed sequence tags of the var gene DBLα domain to investigate var gene transcription in relation to disease severity in Malian children. We found that P. falciparum isolates from children with cerebral malaria (unrousable coma) predominantly transcribe var genes with DBLα1-like domains that are characteristic of Group A or B/A var genes. In contrast, isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia patients) predominantly transcribe var genes with DBLα0-like domains that are characteristic of the B and C-related var gene groups. These results suggest that var genes with DBLα1-like domains (Group A or B/A) may be implicated in the pathogenesis of cerebral malaria, while var genes with DBLα0-like domains promote less virulent malaria infections.The  Our data show that P. falciparum isolates from cerebral malaria patients are significantly more likely to transcribe var genes with DBLα1-like domains characteristic of Group A and B/A var genes than isolates from patients with non-severe hyperparasitaemia, which predominantly transcribe var genes with DBLα0-like domains.We aimed to investigate the hypothesis that parasite virulence and clinical disease severity in African children are associated with the transcription of distinct subsets of 22.1var mRNA is present var transcription occurs The samples were collected as part of the Bandiagara Malaria Project case-control study of severe malaria that has been described in detail previously 2.2DNA was extracted from blood spots on filter paper using chelex-100 extraction 2.3RNA was treated for 30 min at room temperature with 1.5 units DNAase (Gibco) to remove any contaminating genomic (g) DNA. cDNA was prepared using Superscript First Strand Synthesis System (Invitrogen) with random hexamers according to manufacturers instructions.2.4var DBLα domain using unbiased degenerate primers αAF′ and αBR var gene repertoire of 3D7 apart from the strain-transcending var2CSA gene implicated in malaria in pregnancy var genes that are of unknown function. Amplification conditions were as described Reverse-transcriptase (RT)-PCR was used to amplify a region of 300–400 bp of the 2.5var gene from nine isolates was studied. The remaining upstream sequences were not studied due to lack of parasite material.Upstream PCRs were carried out on gDNA using upstream primers UpsA (5′-TAT TYH ATK TAT TAY ATT TGT TGT A) UpsB (5′-GTT AGA ACA TTT AAA ATT ATA) and UpsC (5′-AVA GAW ATA TGR TAG ATA YAG), based on sequences from 3D7, and a gene-specific DBLα primer for each isolate. Amplification conditions were 35 cycles of 94 °C, 5 s; 46 °C, 15 s; 60 °C, 2 min. The upstream sequence of the predominant 2.6PCR and RT-PCR products were run on agarose gels and extracted  then cloned , and used to transform One Shot TOP10F competent cells (Invitrogen). Transformed cells were grown overnight and individual white colonies were selected for culture. Plasmids were extracted from overnight cultures using a miniprep kit (QIAGEN), and were sequenced using BigDye terminator reaction mix (Applied Biosciences).2.7http://bioweb.pasteur.fr/docs/EMBOSS/tranalign.html)Sequences were analysed using Lasergene software (DNASTAR Inc). Contigs were created with a minimum percentage match of 95% to classify sequences for each isolate. Amino acid sequences were aligned using MUSCLE 2.8var genes are subject to recombination. The network allows visualisation of ambiguous and conflicting signals in the dataset due to recombination or other factors A phylogenetic network was generated rather than a traditional phylogenetic tree because 3P. falciparum field isolates were obtained from a vaccine trial site in Mali with intense seasonal transmission of P. falciparum . Hyperparasitaemia is generally considered to be an indication of severe malaria using World Health Organisation criteria Twenty-six There were no significant differences in patient age across the three clinical categories . The cervar gene sequences from each isolate were amplified by reverse transcriptase-PCR using unbiased degenerate primers var gene inserts were sequenced per isolate. Identical sequences from the same isolate were defined as isolate sequence a, b, c etc. in decreasing order of abundance (GenBank accession numbers DQ367086–DQ367226). The number of distinct DBLα var gene sequences detected per isolate varied between 1 and 14, and almost every isolate showed a predominant gene . There was no significant difference in the number of distinct DBLα sequences per isolate detected in each clinical category . Further experiments to validate the reproducibility of these data are shown in the The transcribed DBLα ant gene . Identicvar genes amplified that were DBLα1-like. 80.1% of the var gene sequences amplified from the cerebral malaria isolates were DBLα1-like, compared to only 25.7% of the sequences from the hyperparasitaemia isolates and 40.5% of the sequences from the uncomplicated malaria patients . There was a significant positive correlation between the proportion of DBLα1-like sequences and the rosette frequency .There was a highly significant difference between clinical categories in the percentage of var genes transcribed by the isolates from cerebral malaria patients compared to hyperparasitaemia patients was also demonstrated by mapping clinical categories across a phylogenetic network of DBLα sequence tags . Sequences from the cerebral malaria patients , while sequences from the hyperparasitaemia patients (blue) are significantly concentrated in a “DBLα0-like clade” . Sequences from the uncomplicated malaria patients (green) showed no bias in distribution between the two clades  had an UpsB sequence linked to a hybrid DBLα0/α1 domain and was therefore a Group B/A var gene  predominantly transcribe var genes with DBLα1-like domains that are characteristic of Group A or B/A var genes, whereas isolates from patients with hyperparasitaemia (a non-virulent form of disease) predominantly transcribe var genes with DBLα0-like domains. These results provide the first firm evidence from P. falciparum clinical isolates to support the hypothesis put forward previously that Group A var genes  could be implicated in the pathogenesis of severe malaria in African children The major finding of this study is that var gene transcription in clinical isolates have given conflicting results. A role for var genes with DBLα1-like domains in the multi-organ failure type of severe malaria that occurs in adults in low malaria transmission settings was suggested by a study of 10 Brazilian isolates var genes in clinical disease (severe and uncomplicated) compared to asymptomatic infections var genes in severe compared to uncomplicated and asymptomatic malaria patients var genes, but this was not statistically significant var gene group and disease manifestation, however only six isolates from assorted severe malaria patients were studied, only one of whom had cerebral malaria var gene transcription using isolates from non-severe hyperparasitaemia patients as a control group rather than uncomplicated malaria patients with low parasite burdens. Although it remains possible that there are genuine geographical differences in the relationship between var gene transcription and disease severity, we suggest that the reason why a clear pattern emerged from the current study, even though the sample numbers are not large, is because a strictly defined severe disease syndrome  was investigated and a non-severe malaria control group with equivalent parasite burdens (hyperparasitaemia) was used.Previous studies on var gene transcription in parasite isolates from children with uncomplicated malaria and lower parasite burdens that are equivalent to the control groups used in previous studies var gene transcription with four out of nine isolates transcribing mostly DBLα1-like var genes, while five out of nine isolates transcribed mostly DBLα0-like var genes  are sequestered in the microvasculature var gene transcription and particular disease syndromes would examine sequestered parasites, however, these are only accessible in post-mortem samples making such studies technically and ethically difficult. Two recent studies from Malawi suggest that the dominant parasite genotypes of sequestered parasites are usually the same as those in the peripheral blood var gene profile of peripheral blood parasites reflects that of sequestered ones, they are consistent with the possibility that the peripheral blood population could adequately reflect the sequestered parasite mass. Any difference in var gene transcription between peripheral and sequestered parasite populations would be likely to confound a clear picture of the relationship between var genes and disease syndromes. The fact that such a clear pattern of var gene transcription in isolates from cerebral malaria patients compared to hyperparasitaemia patients did emerge from this study suggests that such a confounding effect is not a major problem.This study and previous similar investigations var genes compared to the DBLα0-like var genes shown in this study be explained by the known functions of the different var gene groups? The only adhesion phenotype currently ascribed to the DBLα1 domain of some Group A or B/A var genes is binding to complement receptor 1 on uninfected erythrocytes to form rosettes , on the other hand, have been shown to bind to the endothelial receptor CD36 var genes with DBLα0-like domains are predominantly transcribed in non-virulent infections is consistent with this hypothesis. In addition, binding of infected erythrocytes to CD36 on dendritic cells leads to inhibition of dendritic cell maturation and impairment of host immune-responsiveness var genes could contribute to the lower virulence of malaria in the hyperparasitaemia patients if, as has been suggested previously, immuno-pathology contributes to the pathogenesis of severe malaria in vitro multiplication rates than parasites from patients in other disease categories var genes with DBLα1-like domains (characteristic of Group A and some B/A var genes) are predominantly transcribed in P. falciparum isolates from Malian cerebral malaria patients, whereas var genes with DBLα0-like domains are predominantly transcribed in isolates from patients with a non-virulent form of disease (hyperparasitaemia). These findings suggest fundamental differences in the roles played by the different var genes groups in host–parasite interaction that could be related to the contrasting cytoadhesive and immuno-modulatory functions of the var gene groups. Further research is required to examine the disease-associations, functions and diversity of var gene subsets in different geographical areas, and we emphasise the importance of strict clinical definitions and appropriate control groups in future work.In conclusion, we have shown that


The proposed novel one-piece scheme will have a significant potential of a paradigm shift from lab to fab in polarization metrology.This paper describes a new class of ultrafast dynamic spectro-polarimetry based on a specially designed one-piece polarizing interferometer. It provides spectral polarimetric parameters of an anisotropic object in milliseconds with high precision. The proposed ultrafast spectro-polarimetry has no moving parts and it is highly robust to external noises. The one-piece polarizing interferometric scheme enables the world fastest and simplest solution in spectroscopic polarimetry. The distinct simple concept on one-piece polarizing interferometer can extract spectroscopic polarimetric parameters Ψ Imaging polarimetry has been widely used to measure various information of target object in remote sensing7. Polarization also has been applied for engineering light–matter interactions in waveguide9 and telecommunication10. Recently, nanophotonics has opened a new possibility of ultracompact system based on meta-surfaces with a capability of engineering the polarization of light13. Spectral interferometry employing polarization technique has been playing a very important role in characterizing optical properties in various fields21. Especially, a variety of spectroscopic polarization measurement solutions have been widely used for material science fields associated with semiconductor and display industry22. Nowadays, spectroscopic ellipsometry (SE) which can provide topographic information relevant to subwavelength periodic structures and thin films has occupied a very important position in semiconductor manufacturing areas22. Various different types of spectroscopic ellipsometers have been demonstrated18. Most of them, however, require time-consuming and complex mechanically rotating polarizer or an electrically phase-modulated device to measure the state of polarization of the reflected wave from a measured object. Although the electrically phase-modulated techniques such as photo-elastic modulation, which employs a monochromator based wavelength scanning can provide a millisecond order high-speed ellipsometric parameter measurement capability, it is a speed for a single wavelength measurement and it takes seconds typically to obtain a wide range of ellipsometric parameters Ψ(k) and Δ(k) for a few hundreds of spectra18. To overcome such speed limitation, several snapshot spectroscopic polarimetry and ellipsometry have been proposed by replacing the mechanical or electrical modulation mechanism with polarimetric spectral interference based on thick wave-plates21, birefringent crystal23, and multiple spectrometers25. The channeled spectrum scheme employing multiple thick wave plates has inherent shortcomings such as high spectrum complexity of raw signal resulting in more time-consuming complicated signal processing, reduced spectral resolution, and limited spectral range depending on the availability of thick wave plates. The snapshot spectroscopic polarization measurements employing the dual-spectrum sensing scheme also have an inherent serious drawback of limited vibration robustness resulting in low-precision since it is based on conventional multi-piece interferometry. Moreover, the complicated and bulky schematic with dual spectrometers for snapshot measurement makes it impractical in real applications.Polarization characterizing the vectorial nature of electromagnetic wave reveals numerous information on the structure and composition of materials(k) and Δ(k) of an anisotropic object dynamically at over 200 Hz by employing only a single spectrometer. We envision that the uniqueness of the proposed distinct simple one-piece polarizing interferometric concept will stimulate numerous unprecedented novel attempts by which polarization measurement can be moved from lab to fab.This paper describes a specially designed one-piece polarizing interferometer which enables a new class of ultrafast dynamic spectro-polarimetry. The one-piece polarizing interferometric module needs to be fabricated with special care by mounting all optics in the interferometer tightly as a one-body so that the spectral interference signal with a carrier frequency remains the same all the time even in case the one-piece interferometric module has been shocked by an abrupt external vibration. A key task of the proposed one-piece interferometric scheme is in fixing the spectral carrier frequency created by the optical path difference between the two arms of the one-piece polarizing interferometric module almost perfectly same to extract highly precise spectroscopic polarimetric parameters ΨThe proposed ultrafast spectroscopic polarimeter based on a passive one-piece polarizing Michelson interferometric scheme depicted in Fig. The proposed system is comprised of a collimating part with a white light source and a linear polarizer, a specially designed one-piece polarizing interferometer part, and a single spectrum acquisition part including a linear polarizer. We use a 100 W tungsten-halogen lamp as a light source connected to an optical fiber with a diameter of 1,000 μm. The beam from the white light source passes through the collimating part and the linear polarizer set to 45-degree rotation angle. The linearly polarized beam enters the specially fabricated one-piece polarizing Michelson interferometer module employing a cube type non-polarizing beam splitter, two perpendicularly aligned linear polarizers, and two plane mirrors. The interfered wave modulated by the one-piece polarizing interferometer passes through the measured anisotropic transmissive object, and it enters the single spectrometer having 2047-pixel array with a spectral range of 393 to 647 nm.Ein, and a corresponding output field as Eout, respectively as follows.k is a wavenumber that equals to 2π/λ. j refers to a complex operator meaning j2 = −1. u and v denotes the amplitude of the incident wave. η and ξ correspond to the phase term of the propagating wave along x- and y-axis, respectively. E1(k) and E2(k) correspond to the complex waves travelled through the p- and s- polarization paths of the one-piece polarizing interferometer, respectively. E1(k) and E2(k) can be represented as follows.B is a Jones matrix of the non-polarizing beam splitter used in the one-piece polarizing interferometer. P(0), P(45), and P(90) signify the Jones matrices of the linear polarizers with rotation angles 0°, 45° and 90°, respectively. Note that the two polarizers in the one-piece interferometric module should be aligned to 0 and 90 degrees precisely. r1 and r2 denote the complex reflection coefficients of the mirrors M1 and M2 used in the interferometer, respectively. z1 and z2 indicate optical path length for p- and s- polarization modulation arms, respectively. Here, it is assumed that the optical path difference (OPD) between two paths in the non-polarizing beam splitter is zero. Notably, all components of the one-piece polarizing interferometer are fixed rigidly to maintain the OPD same. u′, ξ′, and v′, η′ denote newly defined unknown amplitude and phase terms of E1(k) and E2(k), respectively. Eventually, the inlet wave propagating to the spectrometer Esp(k) can be represented as in Eqs (5) and (6): Espref(k) means reference complex wave entering the spectrometer through the air where the air is used to get the interfered reference spectrum signal, and Espobj(k) corresponds to the object complex wave entering the spectrometer after passing through a transmissive anisotropic object as illustrated in Fig. |tp|, δp and |ts|, δs are the amplitude and the phase change terms of the measured transmissive anisotropic object along p- and s- direction, respectively. The OPD between the two arms of the one-piece polarizing interferometer generates a spectral carrier frequency needed for extracting the spectral polarimetric parameters Ψ(k) and Δ(k). Figure We define an input light wave entering the one-piece polarizing interferometer as entclass1pt{minimat = t2 (dark dotted line) from a spectrum at t = t1 (blue solid line) shown in Fig. The interfered spectrum measured by a single spectrometer as illustrated in Fig. entclass1pt{minimaγ represents a spectral coherence function which is systematic. It means that the systematic function is not varied by measured objects. The term z0 = z1 − z2 denotes the optical path difference (OPD) between z1 and z2. The OPD z0 = z1 − z2 between the two arms of the one-piece polarizing interferometer should be set to be around 30 to 50 μm for visible range. Φref(k) denotes the spectral phase function of the interfered reference spectrum Iref(k). α and β correspond to square root of the DC terms of the complex waves travelling through the p- and s- polarization paths of the one-piece polarizing interferometer, respectively.For dynamic measurement of a spectral Stokes vector of an anisotropic object, we first need a calibration step performed just one time as a pre-preparation procedure. We start the calibration step by measuring a spectrally interfered reference spectrum  and phase difference Δ(k) between p- and s-polarization using a single interfered spectrum of an anisotropic object, we need to extract the spectral coherence function γ(k) and the spectral phase function Φref(k) first. To extract them from the interfered reference spectrum Iref(k), we need to modify the interfered reference spectrum by subtracting α2 and β2, and then dividing it by 2αβ as in Eq.  and the spectral phase function Φref(k)) simultaneously as illustrated in Fig. In order to obtain the amplitude ratio Ψs in Eq. . α and βγ(k) and the spectral phase function Φref(k) are extracted as in Fig. 27.After getting the modified interfered reference spectrum depicted in Fig. γ(k) and the spectral phase function Φref(k) of the interfered reference spectrum through the calibration step, we are ready to measure the amplitude ratio Ψ(k) and the phase difference Δ(k) between p- and s-polarization by using a single interfered spectrum of an anisotropic object. In this experiment, we use a Quarter Wave Plate (QWP) as a test anisotropic object to show the feasibility of the proposed scheme. When the anisotropic transmissive object is placed at the object position, we acquire the interfered object spectrum as shown in Fig. Φobj(k) can be extracted by using the Fourier transform method in the same way we obtain the spectral reference phase function27. Eventually, the polarimetric phase difference Δ(k) created by the measured anisotropic object is obtained by subtracting Φref(k) from Φobj(k) since the unknown 2kz0 + [ξ′(k) − η′(k)] terms can be removed perfectly.(k) measurement results for the QWP designed at 632 nm. The solid lines depicted in Fig. (k) results extracted dynamically by rotating the QWP from −45° to 45° with an interval of 10° by using the proposed one-piece polarizing interferometer based spectroscopic polarimeter. The dotted lines in Fig. (k) measurement result. A highly good agreement has been achieved. The comparison results demonstrate that the proposed new concept works correctly and it provides a highly accurate spectral polarimetric phase measurement capability. The sinusoidal oscillation observed in Fig. (k) measurement capability of the QWP object is shown in Supplementary Video (k) measurement repeatability of around 0.1° while maintaining dynamic measurement capability. This is a dramatical precision enhancement compared with that of previously proposed complicated snapshot spectro-polarimetry25.Once we obtain the spectral coherence function (k) simultaneously as well as Δ(k) by using the same single object spectrum illustrated in Fig. To extract ΨAobjDC = (α|tp|)2 + (β|ts|)2 and AobjAC = αβ|tp||ts|, respectively. |tp| and |ts| can be extracted by using the two spectral variable functions AobjDC and AobjAC. We can obtain these two spectral variable functions from the object spectrum Iobj(k) by using the Fourier transform method27. After obtaining these two spectral variables AobjDC and AobjAC, we can calculate α|tp| and β|ts| by using the following equations.α and β are two known variables obtained at the calibration step, Ψ(k) of the anisotropic object can be extracted directly by using Eq. ((k) for the QWP optic axis angle of −15°. The solid line indicates Ψ(k) measured by the proposed snapshot spectro-polarimeter system and the dotted line is that obtained by using a commercial spectroscopic polarimeter. We can see that they are in good agreement for the entire visible range although there are some discrepancies due to limited precision of manual alignment of polarization optics consisting of the system. In summary, we have demonstrated that the spectral polarimetric parameters Ψ(k) and Δ(k) created by an anisotropic object can be extracted dynamically by using Eqs 16.(k) and the spectral phase difference Δ(k) between p- and s-polarization just by using a single interfered object spectrum of any anisotropic object, the spectral Stokes vector S1(k), S2(k), and S3(k) can be obtained dynamically by using Eq. ((k) and Δ(k) results for the QWP optics axis of −15°. We can see that the spectral Stokes vector measurement comparison results between the proposed ultrafast snapshot spectro-polarimeter and a commercial rotating analyzer type spectro-polarimeter are in good agreement. Notably, the proposed ultrafast dynamic spectro-polarimetry can measure and display a spectral Stokes vector with around 1,000 spectra at a speed of over 200 Hz with moderate precision and accuracy while the commercial spectro-polarimeter system takes at least seconds typically to obtain the similar level of spectral Stokes vector information.Stokes vector represents the polarization state of a propagating wave. Real time Stokes vector measurement for a monochromatic wavelength is well developed and commercially available. However, dynamic spectroscopic polarization measurement has not been demonstrated so far. This paper describes in detail how we can implement such dynamic spectroscopic polarization measurement capability by using a simple one-piece polarizing interferometer module. Note that the proposed scheme cannot be applied for generalized polarization analysis such as scattering in order to have a snapshot measurement capability described in the paper. In Jones matrix representation where we assume perfect polarization, the spectral Stokes vector can be directly calculated by using the following equations(k) and Δ(k) in milliseconds from which accurate spectral Stokes vector with over 1,000 spectra can be measured dynamically. We envision that the proposed distinct simple but powerful one-piece polarizing interferometer can be extended to numerous applications where ultrafast spectroscopic polarization measurement capability becomes critical and inevitable such large-scale R2R thin film coating and nano-patterning in-line monitoring for flexible electronics and optical film devices.A novel ultrafast spectro-polarimeter employing a specially designed highly compact and robust one-piece polarizing interferometric module has been described. In this paper, we have successfully extracted the spectral polarimetric parameters ΨSupplementary Video for ′One-piece polarizing interferometer for ultrafast spectroscopic polarimetry′Supplementary information for the Supplementary Video